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A Sensitive Medium-Throughput Method to Predict Intestinal Absorption in Humans Using Rat Intestinal Tissue Segments
2015; Elsevier BV; Volume: 104; Issue: 9 Linguagem: Inglês
10.1002/jps.24372
ISSN1520-6017
AutoresLais Da Silva, Taynara Lourenço Da Silva, Alisson Henrique Antunes, Kênnia Rocha Rezende,
Tópico(s)Neuropeptides and Animal Physiology
ResumoA range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A–B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10−6 cm/s) was determined for atenolol (10.7±1.2), caffeine (17.6±3.1), cimetidine (6.9±0.1), metoprolol (12.6±0.7), theophylline (15.3±1.6) and, ranitidine (3.8±0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r=0.89). These findings showed a high correlation with human data (r=0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association. A range of in vitro, ex vivo, and in vivo approaches are currently used for drug development. Highly predictive human intestinal absorption models remain lagging behind the times because of numerous variables concerning permeability through gastrointestinal tract in humans. However, there is a clear need for a drug permeability model early in the drug development process that can balance the requirements for high throughput and effective predictive potential. The present study developed a medium throughput screening Snapwell (MTS-Snapwell) ex vivo model to provide an alternative method to classify drug permeability. Rat small intestine tissue segments were mounted in commercial Snapwell™ inserts. Unidirectional drug transport (A–B) was measured by collecting samples at different time points. Viability of intestinal tissue segments was measured by examining transepithelial electric resistance (TEER) and phenol red and caffeine transport. As a result, the apparent permeability (Papp; ×10−6 cm/s) was determined for atenolol (10.7±1.2), caffeine (17.6±3.1), cimetidine (6.9±0.1), metoprolol (12.6±0.7), theophylline (15.3±1.6) and, ranitidine (3.8±0.4). All drugs were classified in high/low permeability according to Biopharmaceutics Classification System showing high correlation with human data (r=0.89). These findings showed a high correlation with human data (r=0.89), suggesting that this model has potential predictive capacity for paracellular and transcellular passively absorbed molecules. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association. INTRODUCTIONIt is widely acknowledged that highly attrition rate in the drug discovery process is mainly related to poor pharmacokinetic properties during absorption, distribution, metabolism, and excretion (ADME) phases.1.Li P. Screening for human ADME/Tox drug properties in drug discovery.Drug Discov Today. 2001; 6: 357-366Crossref Scopus (410) Google Scholar It would be more efficient if drug permeability performance could be assessed during the less costly, earlier stages of the drug discovery & development (DDD) process and, in parallel with bioactivity-guided assays. Although, several in silico, in vitro, in vivo and ex vivo methods are currently used, highly predictive human intestinal absorption models remain to be developed because of profusion of variables affecting permeability through gastrointestinal tract in humans.2.Larregieu C. Benet L.Z. Distinguishing between the permeability relationships with absorption and metabolism to improve BCS and BDDCS predictions in early drug discovery.Mol Pharm. 2014; 11: 1335-1344Crossref Scopus (51) Google ScholarFor the prediction of potential human intestinal absorption, great emphasis is currently placed on excised tissue permeability models such is Ussing chambers used to measure drug transport along rat intestinal segments.3.Tukker J.J. In vitro methods for the assessment of permeability.in: Dressman J.B. Lennernas H. Oral drug absorption: Prediction and assessment. Marcel Dekker, New York2000: 51-72Google Scholar Also, include original expression of influx and efflux transporters found in vivo tissues.3.Tukker J.J. In vitro methods for the assessment of permeability.in: Dressman J.B. Lennernas H. Oral drug absorption: Prediction and assessment. Marcel Dekker, New York2000: 51-72Google Scholar In addition, there is interplay between many absorption mechanisms such as passive and carried-mediated diffusion4.Lennernäs H. Regional intestinal drug permeation: Biopharmaceutics and drug development.Eur J Pharm Sci. 2014; 57: 333-341Crossref PubMed Scopus (69) Google Scholar allowing for high correlation between both process, i.e. the fraction of drug absorbed (Fa) in humans and the apparent permeability (Papp) in rat tissues models. Moreover, ex vivo permeability systems using tissue segments can be a valuable tool regarding morphological and physiological characteristics that closely mimics features of the in vivo intestinal epithelium. It is also recognized by the US Food and Drug Administration (FDA) as most ideal one for preclinical human intestinal permeability.5.FDA Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutical classification system. Center for Drug Evaluation and Research, 2000Google Scholar For instance, it provides both metabolism capacity and expression of a mucus layer on the luminal side working as a protective coating to reduce damage by sample cosolvents. This feature is often useful in DDD phases.Despite these advantages, maintenance of ex vivo tissue integrity and viability requires close attention and highly controlled experimental conditions, in order to avoid miscalculation of drug transport, membrane retention and low throughput.6.Deferme S. Annaert P. Augustijns P. In vitro screening models to assess intestinal drug absorption and metabolism.in: Ehrhardt C. Kim K. Drug absorption studies. In situ, in vitro and in silico models. Springer, New York2008: 182-215Crossref Google Scholar, 7.Yamashita S. Tanaka Y. Endoh Y. Taki Y. Sakane T. Nadai T. Sezaki H. Analysis of drug permeation across Caco-2 monolayer: Implication for predicting in vivo drug absorption.Pharm Res. 1997; 14: 486-491Crossref PubMed Scopus (118) Google Scholar, 8.Westerhout J. Steeg E.V. Grossouw D. Zeijdner E.E. Krul C.A.M. Verwei M. Woterlboer H.M. A new approach to predict human intestinal absorption using porcine intestinal tissue and biorelevant matrices.Eur J Pharm Sci. 2014; 63C: 167-177Crossref Scopus (76) Google Scholar Briefly, tissue segments should be continuously oxygenated with carbogen mixture (95%O2, 5% CO2) and kept under stirring in physiological buffer, at 37 °C, reducing the unstirred water layer and, at high-humidity level incubations to avoid sample evaporation.In this context, it is clear the need for drug permeability models that can be used early in DDD phases providing a reasonably high throught and predictive potential. Here, the medium throughput screening (MTS-Snapwell) model is showed as a new alternative ex vivo model to classify drugs in high and low permeability, using female rat small intestine tissue segments mounted in commercial Snapwell™ inserts, under stirring and sink conditions.MATERIALS AND METHODSChemicals and ReagentsMetoprolol tartrate, caffeine, theophylline, atenolol, cimetidine, ranitidine hydrochloride, sodium chloride, potassium chloride, monobasic sodium phosphate, magnesium sulfate heptahydrate, calcium chloride dihydrate and sodium bicarbonate were purchased from Sigma–Aldrich (St. Louis, Missouri). Sodium hydroxide and dibasic sodium phosphate anhydrous were purchased from J.T. Baker (Mexico City, Mexico), glacial acetic acid from Vetec (Rio de Janeiro, RJ, Brazil), and orthophosphoric acid from Scharlau (Barcelona, Spain), whereas HPLC chromatographic grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany).Rat Excised Tissue SegmentsThe study protocol for the use of rat intestine tissues were approved by the Ethics Committee on Animal Use at the Federal University of Goiás under number 013/2011. Female Wistar rats (200±25 g) were housed under controlled conditions at a temperature of 22 °C, 12-h light/dark cycle and access to standard food and tap water ad libitum for a minimum acclimatization period of one week. Prior to each experiment, animals were fasted for 12 h with free access to water.9.Salphati L. Childers K. Pan L. Tsutsui K. Takahashi L. Evaluation of a single-pass intestinal-perfusion method in rat for the prediction of absorption in man.J Pharm Pharmacol. 2001; 53: 1007-1013Crossref PubMed Scopus (115) Google Scholar, 10.Kim J.S. Mitchell S. Kijek P. Tsume Y. Hilfinger J. Amidon G.L. The suitability of an in situ perfusion model for permeability determinations: Utility for BCS class I biowaiver requests.Mol Pharm. 2006; 3: 686-694Crossref PubMed Scopus (146) Google ScholarFemale Wistar rats were anesthetized with ketamine/xilazyne (90 and 7.5 mg/kg). The small intestine was excised, washed and kept in an ice-cold oxygen-saturated Krebs–Ringer bicarbonate (KRB) solution. Proximal jejunal tissue (the first 12 cm from stomach) was excised and placed in beakers containing ice-cold KRB (10 °C), which was continuously gassed with an 95% O2 and 5% CO2. Next, the seromuscular layer was carefully stripped off from the membrane and cut into 20 mm segments. During this procedure, tissues were kept moist using KRB (10 °C). Rat jejunal segments were then mounted with the basolateral side set above the polycarbonate filter membrane (Isopore™ 0.4 µm, 13 mm; Millipore, Darmstadt, Germany). Using acrylic disks (02) with an internal diameter of 3 mm over and above the filter membrane (Fig. 1) and with the mucosal membrane facing upward, the permeability apparatus was set-up. Next, it was tightly mounted and mechanically sealed inside the donor chamber (Snapwell™ device). Then, it was checked for any leakage using 1 mL KRB solution. The MTS-Snapwell system was completed by filling the receiver (2 mL) and donor (0.4 mL) compartments with KRB solution.Viable intestinal segments (transepithelial resistance, TEER>30 Ω cm2), as described under Viability and Barrier Integrity, were mounted on the permeation apparatus and submitted to a preincubation period (30 min) under a controlled atmosphere (95% O2 and 5% CO2) inside a humidified incubator (MCO-18 AC; Sanyo Scientific, UK) at 37 °C and gentle shaking (60 rpm) for equilibration. KRB blank solution was then aspirated off from the donor compartment and replaced with drug solutions (400 μL) at variable concentrations (0.30–12.68 mM). Drug doses assayed were defined as the highest human commercial dose divided by 250 mL, as described in FDA guide.5.FDA Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutical classification system. Center for Drug Evaluation and Research, 2000Google Scholar During drug incubation, unidirectional drug transport from apical to basolateral side was measured by collecting samples at different time points (0, 20, 30, 40, 60, 90, and 120 min). Sampling volume was immediately replaced with the same volume of fresh prewarmed (37 °C) KRB solution. Samples were analyzed as described in Analytical Methods.For each drug, the apparent permeability coefficient (Papp) was calculated according to the following equation (Eq. (1)11.Zur M. Gasparini M. Wolk O. Amidon G.L. Dahan A. The low/high BCS permeability class boundary: Physicochemical comparison of metoprolol and labetalol.Mol Pharm. 2014; 11: 1707-1714Crossref PubMed Scopus (50) Google Scholar, 12.Sjöberg Å. Lutz M. Tannergren C. Wingolf C. Borde A. Ungell A.L. Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique.Eur J Pharm Sci. 2013; 48: 166-180Crossref PubMed Scopus (144) Google Scholar, 13.Li H. Jin H.E. Shim W.S. Shim C.K. An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.Drug Dev Ind Pharm. 2013; 39: 1515-1522Crossref Scopus (19) Google Scholar, 35.Zakelj S. Legen I. Veber M. Kristl A. The influence of buffer composition on tissue integrity during permeability experiments “in vitro”.Inter J Pharm. 2004; 272: 173-180Crossref Scopus (19) Google Scholar:Pappcm/s=dQdt1C0A(1) Where dQ/dt is the appearance rate of the drug in the receiver compartment, C0 is the initial concentration of the drug in the donor compartment, and A is the surface area (0.1 cm2) of the tissue segment.Viability and Barrier IntegrityViability of intestinal tissue segments mounted in the MTS-Snapwell system was assessed by measuring the transport of marker molecules (e.g., phenol red and mannitol) and TEER during incubation time (120 min) at 37 °C with 95% O2, and 5% CO2. TEER was measured at three different time points (0, 30, and 120 min). Additionally, potential damage to the barrier function for commonly used cosolvents (DMSO 1% and EtOH 1%) for poorly water-soluble drugs was evaluated.14.Krishna G. Chen K.J. Lin C.C. Nomeir A.A. Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2.Int J Pharm. 2001; 222: 77-89Crossref PubMed Scopus (116) Google Scholar, 15.Takahashi Y. Kondo H. Yasuda T. Watanabe T. Kobayashi S.I. Yokahama S. Common solubilizers to estimate the Caco-2 transport of poorly water-soluble drugs.Int J Pharm. 2002; 246: 85-94Crossref PubMed Scopus (75) Google Scholar, 16.Watanabe E. Takahashi M. Hayashi M. A possibility to predict the absorbability of poorly water-soluble drugs in humans based on rat intestinal permeability assessed by an in vitro chamber method.Eur J Pharm Sci. 2004; 58: 659-665Google ScholarThe integrity of the intestinal barrier function was measured by linearity of mucosal to serosal transport of permeability markers over 2 h. Caffeine was evaluated as a highly absorbed marker transported by passive transcellular mechanism, in addition to phenol red as a nonabsorbable one.Analytical MethodsDrug samples were analysed by HPLC–DAD (Infinity 1260; Agilent Technologies) with an ACE 5 C18 column (100×4.6 mm2, 5 µm), using compendial methods from the United States and Brazilian Pharmacopeia. The chromatographic conditions employed are described in Table 1.Table 1Chromatographic Conditions for Quantification of Tested DrugsCompoundLQ (μg/mL)Chromatographic Conditions on ACE 5 C18 (100×4.6 mm2, 5 μm)λ (nm)MetoprololaUnited States Pharmacopeia.1.0Acetate buffer pH 3.8: MeOH (58:42); 1.0 mL/min; 10 μL at 45 °C275CaffeineaUnited States Pharmacopeia.0.05Phosphate buffer pH 9.2: MeOH (55:45); 1.0 mL/min; 10 μL275TheophyllineaUnited States Pharmacopeia.1.2Acetate buffer pH 4.0: ACN (93:7); 1.0 mL/min; 10 μL280AtenololaUnited States Pharmacopeia.0.3Phosphate buffer pH 3.0: MeOH (80:20); 0.6 mL/min; 10 μL226RanitidineaUnited States Pharmacopeia.0.08Phosphate buffer pH 7.0: ACN (78:22) 1.0 mL/min; 10 μL at 35 °C230CimetidinebBrazilian Pharmacopeia.0.5Phosphoric acid 0.03%: MeOH (80:20); 1.0 mL/min; 20 μL220a United States Pharmacopeia.b Brazilian Pharmacopeia. Open table in a new tab Data AnalysisTEER values in the presence and absence of cosolvents were compared using Student’s t-test for two independent samples at the 5% significance level (Microsoft Excel 2010; Action®). Results were shown as arithmetic mean±standard deviation (SD).Potential correlation between Papp values obtained from the MTS-Snapwell model and Papp values from published data (Ussing chamber, rat perfusion, or % Fa in humans) were plotted. Linear regression was performed and expressed as a measurement of the goodness of fit (R2) on predicting human drug absorption.RESULTS AND DISCUSSIONViability and Barrier IntegrityIn order to assess tissue viability, reference TEER values were set at 30 Ω·cm2, as previously described in the literature (30 Ω·cm2; 34±6.2 Ω·cm2; 20–50 Ω·cm2).17.Narai A. Arai S. Shimizu M. Rapid decrease in transepithelial electrical resistance of human intestinal Caco-2 cell monolayers by cytotoxic membrane perturbents.Toxicol In Vitro. 1997; 11: 347-354Crossref Scopus (72) Google Scholar, 18.Pollentaruti B.I. Peterson A.L. Sjöberg A.K. Anderberg E.K.I. Utter L.M. Ungell A.L.B. Evaluation of viability of excised rat intestinal segments in Ussing chamber: Investigation of morphology, electrical parameters and permeability characteristics.Pharm Res. 1999; 16: 446-454Crossref Scopus (79) Google Scholar, 35.Zakelj S. Legen I. Veber M. Kristl A. The influence of buffer composition on tissue integrity during permeability experiments “in vitro”.Inter J Pharm. 2004; 272: 173-180Crossref Scopus (19) Google Scholar Using the present study protocol, the initial TEER value for rat jejunal tissue was found to be 53±8.0 Ω·cm2 (n=20) remaining above 20 Ω·cm2 through the incubation time (120 min). The maximum acceptable value for TEER reduction was 61.9±9.5%, consistent with the literature.19.Borchard G. Luegen L. Boer A.G. The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III?: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro.J Control Release. 1996; 39: 131-138Crossref Scopus (377) Google Scholar, 20.Petersen Nolan G. Maher S. Rahbek U.L. Gulddrandt M. Brayden D.J. Evaluation of alkylmaltosides as intestinal permeation enhancers: Comparison between rat intestinal mucosal sheets and Caco-2 monolayers.Eur J Pharm Sci. 2012; 47: 701-712Crossref PubMed Scopus (43) Google ScholarPotential cosolvents damage was also evaluated for 1% DMSO and 1% EtOH using TEER values. Starting values found were 48±9.2 Ω·cm2 (n=10) and 54±5.8 Ω·cm2 (n=10). After cosolvents addition, TEER values did not differ significantly (P>0.05) from blank samples, remaining above 20 Ω·cm2 through the incubation (2 h). Resistance to co-solvent damage may be explained by the presence of the protective mucus layer on the rat jejunal membrane during the incubation time. Additionally, integrity of intestinal barrier was assessed using nonabsorbable (phenol red) and high permeability (caffeine) markers (Fig. 2). Phenol red showed no permeation at all, as expected. For caffeine,21.Dixit P. Jain D.K. Dumbwani J. Standardization of an ex vivo method for determination of intestinal permeability of drugs using everted rat intestine apparatus.J Pharmacol Toxicol Methods. 2012; 65: 13-17Crossref PubMed Scopus (73) Google Scholar drug transport remained linear during the incubation time (30–120 min), after the estimated lag period (30 min), as seen for the jejunal membrane herein tested.22.Roy S.D. de Groot J.S. Percutaneous absorption of nafarelin acetate, an LHRH analog, through human cadaver skin and monkey skin.Int J Pharmaceutics. 1994; 110: 137-145Crossref Scopus (16) Google Scholar, 23.Rutherford S.W. Do D.D. Review of time lag permeation technique as a method for characterisation of porous media and membranes.Adsorption. 1997; 3: 283-312Crossref Scopus (179) Google Scholar Thus, epithelial integrity was confirmed both by monitoring TEER and drug markers, as recommended.12.Sjöberg Å. Lutz M. Tannergren C. Wingolf C. Borde A. Ungell A.L. Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique.Eur J Pharm Sci. 2013; 48: 166-180Crossref PubMed Scopus (144) Google Scholar For that, standardized experimental conditions used for the MTS-Snapwell model also included maintaining close control of the room temperature (18±2 °C) and using (10±2 °C) KRB saturated solution (5% CO2, 95% O2) as a tissue preservation bath.Figure 2Barrier integrity assessment by means of permeability markers as phenol red (nonabsorbable) and caffeine (high permeability) across rat jejunal tissue segments, expressed in percentage of drug dose versus incubation time (120 min). Data represented mean±SD (n=4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Permeability StudiesThe sampling interval and maximum time of exposure (120 min) were shown to be adequate, facilitating completion of the experiment before the system has reached steady-state concentration. The lag times were approximately 20–40 min, with a steady concentration gradient established across the tissue segments within 60 min. Stirring at 60 rpm was adequate to prevent the water layer stagnanting, ensuring sink conditions.24.Ingels F.M. Augustijns P.F. Biological, pharmaceutical, and analytical considerations with respect to the transport media used in the absorption screening system, Caco-2.J Pharm Sci. 2003; 92: 1545-1558Abstract Full Text Full Text PDF PubMed Scopus (86) Google ScholarPapp of six selected compounds of Biopharmaceutics Classification System (BCS) I and III, as recommended by the FDA guidance,5.FDA Guidance for industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage forms based on a biopharmaceutical classification system. Center for Drug Evaluation and Research, 2000Google Scholar were measured across rat jejunal tissue segments (apical to basolateral) mounted in the MTS-Snapwell system under sink conditions.The obtained Papp values ranged from 3.8±0.4 to 17.6±3.1 cm/s pointing to metoprolol as a low–high permeability class boundary marker as seen in Table 2. As a result, the same BCS classification was attained for all tested drugs. Drug permeability classification was obtained by calculating the Papptest/Pappmetoprolol ratio, as reported by Kim et al.10.Kim J.S. Mitchell S. Kijek P. Tsume Y. Hilfinger J. Amidon G.L. The suitability of an in situ perfusion model for permeability determinations: Utility for BCS class I biowaiver requests.Mol Pharm. 2006; 3: 686-694Crossref PubMed Scopus (146) Google Scholar Drugs showing a Papp ratio greater than 1.0 were considered to be highly permeable. Otherwise, they were classified as a low permeability drug (Table 2). Regarding the high permeability marker (metoprolol), one should be aware of its characteristic pH-dependent permeability. Zur et al.11.Zur M. Gasparini M. Wolk O. Amidon G.L. Dahan A. The low/high BCS permeability class boundary: Physicochemical comparison of metoprolol and labetalol.Mol Pharm. 2014; 11: 1707-1714Crossref PubMed Scopus (50) Google Scholar reported that its permeability at pH 7.5 can be 2-fold higher than that observed at pH 7.0. In the present study, metoprolol showed good interassay precision by means of relative standard deviation (RSD=0.52%; n=3) at pH 7.4. Additionally, MTS-Snapwell Papp values displayed the same ranking order (ranitidine<atenolol<metoprolol<theophylline<caffeine) as reported using an Ussing chamber,13.Li H. Jin H.E. Shim W.S. Shim C.K. An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.Drug Dev Ind Pharm. 2013; 39: 1515-1522Crossref Scopus (19) Google Scholar that is, a classical ex vivo assay in rat intestinal tissues. Previously reported Papp and Peff data from commonly used techniques such as Caco-2, intestinal perfusion, and the Fa were compared assuming linear regression. Overall, Papp values showed weaker linear goodness of fit for in vivo studies (R2=0.76–0.79) as compared with ex vivo data (R2=0.83) (Fig. 3).Table 2Drug Transport Mechanism Across Intestinal Barrier, Papp Values Measured in MTS-Snapwell Model is Compared with Reported Papp in Caco-2 and Ussing ChamberData are shown as mean±SD (N=3).CompoundPassive Transport mechanism25.Smetanova L. Stetinova V. Kholova D. Kvetina J. Smetana J. Svoboda Z. Caco-2 cells and biopharmaceutics classification system (BCS) for prediction of transepithelial transport of xenobiotics (model drug: caffeine).Neuro Endocrinol Lett. 2009; 30: 101-105Google Scholar, 26.Mols R. Brouwers J. Schinkel A.H. Annaert P. Augustijns P. Intestinal perfusion with mesenteric blood sampling in wild-type and knockout mice evaluation of a novel tool in biopharmaceutical drug profiling.Drug Metab Dispos. 2009; 37: 1334-1337Crossref Scopus (34) Google Scholar, 27.Gato-Peciña J.J. Ponz F. Use of the paracellular way for the intestinal absorption of sugars.Rev Esp Fisiol. 1990; 46: 343-352Google Scholar, 28.Zhou S.Y. Piyapolrungroj N. Pao L.H. Li C. Liu G. Zimmerman E. Fleisher D. Regulation of paracellular absorptionof cimetidine and aminosalicylate in rat intestine.Pharm Res. 1999; 16: 1781-1785Crossref Scopus (24) Google Scholar, 29.Mummaneni V. Dressman J.B. Intestinal uptake of cimetidine and ranitidine.Pharm Res. 1994; 11: 1599-1604Crossref Scopus (22) Google Scholar, 30.Gan L.S. Hsyu P.H. Pritchard J.F. Thakker D. Mechanism of intestinal absorption of ranitidine and ondansetron: Transport across Caco-2 cells monolayers.Pharm Res. 1993; 10: 1722-1725Crossref PubMed Scopus (84) Google ScholarBCSahttp://tsrlinc.com.Papp (×10−6 cm/s)Peff (×10−6 cm/s)%Fa9,10,32Drug Ratio Test/MetoprololMTS-SnapwellCaco-231.Castillo-Garit J.A. Ponce I.M. Torrens F. Domenech R.G. Estimation of ADME properties in drug discovery: Predicting Caco-2 cell permeability using atom-based stochastic and non-stochastic linear indices.J Pharm Sci. 2008; 97: 1946-1976Abstract Full Text Full Text PDF Scopus (77) Google ScholarUsing Chamber4.Lennernäs H. Regional intestinal drug permeation: Biopharmaceutics and drug development.Eur J Pharm Sci. 2014; 57: 333-341Crossref PubMed Scopus (69) Google Scholar, 13.Li H. Jin H.E. Shim W.S. Shim C.K. An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.Drug Dev Ind Pharm. 2013; 39: 1515-1522Crossref Scopus (19) Google ScholarRat9.Salphati L. Childers K. Pan L. Tsutsui K. Takahashi L. Evaluation of a single-pass intestinal-perfusion method in rat for the prediction of absorption in man.J Pharm Pharmacol. 2001; 53: 1007-1013Crossref PubMed Scopus (115) Google Scholar, 10.Kim J.S. Mitchell S. Kijek P. Tsume Y. Hilfinger J. Amidon G.L. The suitability of an in situ perfusion model for permeability determinations: Utility for BCS class I biowaiver requests.Mol Pharm. 2006; 3: 686-694Crossref PubMed Scopus (146) Google Scholar, 32.Pratap S.S. Wahajuddin Raju K.S.R. Nafis A. Jain G.K. Simultaneous determination of nine model compounds in permeability samples using RP-HPLC: Application to prove the cassette administration principle in single pass intestinal perfusion study in rats.J Pharm Biomed Anal. 2012; 68: 71-76Google ScholarHuman13.Li H. Jin H.E. Shim W.S. Shim C.K. An improved prediction of the human in vivo intestinal permeability and BCS class of drugs using the in vitro permeability ratio obtained for rat intestine using an Ussing chamber system.Drug Dev Ind Pharm. 2013; 39: 1515-1522Crossref Scopus (19) Google Scholar, 37.Lennernäs H. Animal data: the contributions of the using chamber and perfusion systems to predicting human oral drug delivery in vivo.Advanced Drug Delivery Reviews. 2007; 59: 1103-1120Crossref Scopus (128) Google ScholarMetoprololTranscellularI12.6±0.725.716.1±6.859.0±13.013496–CaffeineTranscellularI17.6±3.138.937.2±3.254.0±17.02931001.4TheophyllineParacellularI15.3±1.644.727.7±8.268.0±19.02061001.2AtenololParacellularIII10.7±1.20.3210.7±1.818.0±9.020500.8RanitidineParacellularIII3.8±0.40.496.1±1.57.3±6.027500.3CimetidineParacellularIII6.9±0.11.29N.A.10.5±6.026600.5Reported Peff in rat and human perfusions plus absorbed fraction (%Fa) in human is also included. BCS, Biopharmaceutical Classification System; N.A., not available.a http://tsrlinc.com. Open table in a new tab Figure 3Relationship between apparent permeability coefficients (Papp) in rat jejunal tissue mounted in MTS-SNAPWELL system and (a) Peff rat perfusion;9.Salphati L. Childers K. Pan L. Tsutsui K. Takahashi L. Evaluation of a single-pass intestinal-perfusion method in rat for the prediction of absorption in man.J Pharm Pharmacol. 2001; 53: 1007-1013Crossref PubMed Scopus (115) Google Scholar, 10.Kim J.S. Mitchell S. Kijek P. Tsume Y. Hilfinger J. Amidon G.L. The suitability of an in situ perfusion model for permeability determinations: Utility for BCS class I biowaiver requests.Mol Pharm. 2006; 3: 686-694Crossref PubMed Scopus (146) Google Scholar, 32.Pratap S.S. Wahajuddin Raju K.S.R. Nafis A. Jain G.K. Simultaneous determination of nine model compounds in permeability samples using RP-HPLC: Application to prove the cassette administration principle in single pass intestinal perfusion study in rats.J Pharm Biomed Anal. 2012; 68: 71-76Google Scholar (b) Peff human perfusion;10.Kim J.S. Mitchell S. Kijek P. Tsume Y. Hilfinger J. Amidon G.L. The suitability of an in situ perfusion model for permeab
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