Ex vivo and in situ approaches used to study intestinal absorption

Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

Review

Ex vivo and in situ approaches used to study intestinal absorption Zhiqiang Luo, Yang Liu ⁎, Baosheng Zhao, Mingmin Tang, Honghuan Dong, Lei Zhang, Beiran Lv, Li Wei School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, PR China

a r t i c l e

i n f o

Article history: Received 31 May 2013 Accepted 25 June 2013 Keywords: Animal model Drug intestinal absorption Ex vivo In situ Methods

a b s t r a c t Over the recent years, intestinal absorption has been recognized as a critical factor affecting the bioavailability of oral drugs. Intestinal absorption is affected by many factors including the physicochemical property of the drug, the absorption mechanisms, and the need for absorption enhancers. Ex vivo and in situ methods have been used extensively to evaluate the intestinal absorption of new drugs. Biological performance can be obtained rapidly and reliably using these techniques. However, these approaches have many inadequacies which need to be recognized so that appropriate adjustments can be made to the methodology. These shortcomings also need to be accounted for during the interpretation and application of the results in vivo situations. This review describes ex vivo and in situ models of drug absorption, and compares their relative advantages and drawbacks to assist researchers in selecting appropriate models for different drug and therapeutic situations. © 2013 Elsevier Inc. All rights reserved.

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . Ex vivo methods . . . . . . . . . . . . . . . . 2.1. Intestinal perfusion . . . . . . . . . . . 2.2. Everted gut sac experiments . . . . . . . 2.3. Ussing chamber . . . . . . . . . . . . . 3. In situ methods . . . . . . . . . . . . . . . . 3.1. Closed-loop method . . . . . . . . . . . 3.2. Thiry–Vella fistula . . . . . . . . . . . . 3.3. Intestinal single-pass perfusion (SPIP) . . . 3.4. Intestinal recirculating perfusion . . . . . 3.5. Intestinal perfusion with venous sampling . 3.6. Vascularly perfused intestine-liver (IPIL) . 3.7. Loc-I-Gut . . . . . . . . . . . . . . . . 4. Conclusions . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . Author contribution . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

1. Introduction Bioavailability is commonly defined as the extent and rate at which a drug becomes available in the general circulation (Mannhold, Kubinyi, Folkers, Waterbeemd, & Testa, 2009). It is one of the most important aspects of the drug development process, especially for natural compounds or new molecular entities with significant biological activity (Smith & O'Donnell, 2006). Bioavailability following oral administration ⁎ Corresponding author. Tel.: +86 13810283092; fax: +86 10 61740475. E-mail address: [email protected] (Y. Liu). 1056-8719/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.vascn.2013.06.001

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . .

208 209 209 210 211 211 211 212 212 212 213 213 214 214 214 214 214

is affected by factors such as dissolution, transit time, enzymatic transformation in digestive juice, intestinal permeability, biotransformation by intestinal flora tract, and gastrointestinal and hepatic metabolism (Testa & Waterbeemd, 2008). Among these factors, intestinal permeability is a principal determinant of drug absorption following oral administration. Indeed, transport across the intestinal barrier is a prerequisite for the clinical effect of most drugs (Amidon, Hussain et al., 2002), and, intestinal permeability is used for classifying drugs in the Biopharmaceutics Classification System (BCS) (CDER, 2000). Numerous methods have been used to evaluate intestinal absorption early in the process of drug development in order to exclude new

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

compounds with poor absorption properties (Holmstock, Annaert, & Augustijns, 2012). The perfusion technique of Loc-I-Gut is regarded as the ‘gold standard’ for evaluating human intestinal absorption. However, experimental models in small animals are routinely used as they are easy to undertake. Ex vivo and in situ models in small animals are also widely used when there is a good agreement between intestinal permeability results in rats and human subjects. Transcellular in vivo absorption properties of new chemical entities are usually determined early during the drug discovery process, together with the evaluation of the new drug's physico-chemical properties. In vitro techniques include simple artificial or biological membrane systems, or assays based on biological cell layers (e.g., Caco-2 cells, IAM, and PAMPA) (Clere, Desangle-Gouty, Genty, González, & Legendre, 2001; Gubernator, Kansy, & Senner, 1998). These techniques enable rapid determination of artificial or biological membrane permeability of drugs, making them suitable for high-throughput drug screening (Holmstock et al., 2012). However, differences between predicted passive permeability and observed permeability emphasize the need to assess absorption in vivo in order to obtain more complete knowledge of the transport mechanism(s) involved in the absorption process (Amidon, Hussain et al., 2002). This review describes and evaluates ex vivo and in situ methods which are used to replicate the in vivo situation and allow more accurate prediction of human intestinal drug absorption (Table 1). Our objective is to present scholars with an overview of methods available for estimating intestinal drug absorption, and to facilitate the selection of the optimal model for drug investigation in late discovery and early development stages.

209

2. Ex vivo methods Ex vivo methods provide a theoretical means of estimating human intestine absorption. They include intestinal perfusion, everted gut sac (Fig. 1), and Ussing chambers. Ex vivo models have a number of distinctive features that separate them from the in vitro Caco-2 model. Adequate paracellular permeability is provided by the small intestinal epithelium, a mucus layer is present in the model, and there is also expression of transport proteins and drug metabolism. However, results from ex vivo sometimes inappropriately estimate the degree of oral absorption, due to the interruption of normal blood flow and the lack of a nervous system. For example, investigation of an ester prodrug using ex vivo methods, failed to provide evidence of significant transport enhancement (Annaert et al., 2000). Despite these shortfalls, ex vivo methods are simple, and widely used in the design and testing of potential new drugs. 2.1. Intestinal perfusion Since the experiments of Salvioli, numerous procedures have been devised to study the intestinal absorption and metabolism using isolated perfused intestinal segments (Parsons & Prichard, 1966). The surviving intestine preparation is set up apart from the body and has a viable mucosa. Pig intestine was used in early experiments and carbohydrate absorption has been studied from viable specimens of human intestine obtained at operation (Mansford, 1965). Krebs's bicarbonate saline, Krebs's phosphate saline, Tyrode's solution or Ringer–Locke's solution was used as perfusates, and was sampled at predefined time intervals.

Table 1 Strengths and limitations of each method, along with the earliest date of use. Strengths

Limitations

Earliest date of use

Ex vivo intestinal perfusion

The model has a viable mucosa, and it is a quick, simple technique for estimating of intestinal drug transport.

R. Fisher and Parsons (1949)

Ex vivo everted gut sac experiments

The model is simple, and it is very useful for predicting the extent of transfer and intestinal metabolism of drugs. The method is well validated and it can be used to study the permeability of drugs that are poorly absorbed, the absorption mechanisms of different compounds, the drug–drug interactions, and drug transport processes. It enables intestinal absorption to occur at body temperature for an appointed time. The model also allows absorption to be measured separately at different regions of rat intestine, jejunum, ileum and colon. And it avoids the uncertainty of gastric emptying time. It enables intestinal absorption to be studied in conscious animals with an intestine maintained at near normal physiological conditions. It significantly reduces the number of animals utilized and animals act as their own controls for analyzing segmental-dependent membrane permeability. It can magnify the concentration changes, which is suitable for studying drugs which are absorbed slowly. It is a useful method for obtaining realistic drug absorption rates, and it allows the determination of intestinal metabolism without interference by the confounding effects of hepatic first-pass metabolism. It allows the investigation of the hemodynamics and metabolism for each organ, as well as the interrelationships between the small intestine and liver. It is an accurate method that provides direct estimates of local drug absorption in human subjects. And it is not influenced by other gastrointestinal factors such as transit time and regional pH-differences.

Tissue viability and integrity of the intestinal respiratory system have a marked effect on the results. And the barrier imposed by the intestinal wall and serosa may result in slower absorption rates than those obtained in intact animals. The everted intestinal sacs gradually lose structural integrity.

Ex vivo Ussing chamber

In situ closed-loop method

In situ Thiry–Vella fistula

In situ intestinal single-pass perfusion In situ intestinal recirculating perfusion In situ intestinal perfusion with venous sampling

In situ vascularly perfused intestine–liver In situ Loc-I-Gut

The Ussing chamber method appears to be unsuitable for evaluating ester prodrugs. And it is also not suitable for use with rabbit tissues as the duodenal and jejunal sections are too thick for the diffusion chambers and leaks are observed. The procedure does not allow estimation of absorption at steady state. It is also necessary to undertake a large number of experiments before statistically significant results can be obtained. And the operative procedure is complex.

Wilson and Wiseman (1953) Ussing and Zerahn (1951)

Gibson and Wiseman (1951)

It requires sophisticated surgical procedures and instrumentations.

Clarke et al. (1951)

It requires sophisticated surgical procedures and instrumentations.

Brodie et al. (1958)

It requires sophisticated surgical procedures and instrumentations. It requires sophisticated surgical procedures and instrumentations.

Brodie et al. (1958) Kavin et al. (1967)

It requires sophisticated surgical procedures and instrumentations.

Cherry et al. (1985)

It requires sophisticated surgical procedures and instrumentations.

Knutson et al. (1989)

210

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

Fig. 1. Schematic drawing of ex vivo everted gut sac experiments.

The experiments provided estimates of the amount of drug in the ‘mucosal fluid’ circulating through the intestinal lumen and in the ‘serosal fluid’ that bathed the exterior of the intestine (Smyth & Taylor, 1957). The results were obtained by comparing the changes of mucosal fluid before and after perfusion. Early experiments used tied segments or sheets, and obvious limits were imposed by the deficiency of available oxygen. The first preparation which satisfactorily solved this problem was described by Fisher & Parsons. The segmented rat small intestine preparation was obtained by cannulation at both ends and was provided with a closed circulation in the form of oxygen-saturated, CO2−bicarbonate-buffered fluid which circulated through the intestinal wall. The segment was suspended in a bath of oxygenated Ringer's solution, and absorption was estimated by sampling the mucosal and serosal fluid (Fisher & Parsons, 1949). The circulation of fluid through the intestinal lumen was maintained by a gas mixture of 5% carbon dioxide and 95% oxygen, when the fluid being circulated was bicarbonate saline. Pure oxygen was used for other types of solution and nitrogen was used instead of oxygen for anaerobic experiments (Smyth & Taylor, 1957). Fisher's experimental system was based on the assumption that the mucosal cells were not deprived of oxygen at any time. However, Michael argued that it was not possible to maintain continuous oxygenation in ex vivo preparations. He investigated the absorptive viability of isolated intestine prepared from dead animals and showed that suboptimal absorption occurred during hypoxia. It was, therefore, suggested that oxygen perfusion may not accurately reflect physiological processes and may result in experimental variability (Gardner, 1978). Despite this possible shortfall, the method proposed by Fisher & Parsons was found to provide a feasible means of studying absorption from the rat small intestine (Fisher & Parsons, 1950, 1953). Various simplified techniques were subsequently developed. Agar used a segment of approximately 40 cm of small intestine obtained just distal to the duodenum. The blood supply was preserved until the last moment to facilitate accurate measurement of the initial length of the intestine. It is generally considered unnecessary to begin the circulation through the lumen while there is still an intact intestinal blood supply. The exact length of the loop of intestine used was measured at the end of the experiment, and absorption was expressed in terms of amount per cm length (Agar, Hird, & Sidhu, 1953). In order to study the absorption of sugars from the intestine, Darlington and Quastel refined the technique by providing two independent circulatory systems, one running through the lumen of the intestine and the other external to it (Darlington & Quastel, 1953). Wiseman's method further developed the technique and provided a

relatively large ratio of intestine to outer fluid, together with a low distention pressure, and less interrupted perfusion. Stereo-chemically specific methods were subsequently employed to investigate the ability of the small intestine to transfer amino-acids against a concentration gradient. However, the length of intestine used was extremely critical in these experiments (Wiseman, 1953). A later model was developed with only one loop of intestine, instead of the three loops that had been previously used. Other workers refined Wiseman's method of oxygenating the mucosal fluid, and were able to control the rate of circulation by direct observation. This system provided metabolic CO2 collection in a closed circuit (Smyth & Whaler, 1953). In order to obtain more accurate results, labeled compounds were used to analyze the sample for total radioactivity. In addition, fluid was routinely replenished to avoid volume changes to the circulation after withdrawal of samples (Chain, Mansford, & Pocchiari, 1960). Phenol red, inulin, or 14C-PEG-4000 has been used as inner circuit indicator of leakage. However, these markers may interact with the drug being tested and have the potential to alter membrane function (Ehrhardt & Kim, 2008). Furthermore, other adaptations of the method of Fisher and Parsons found that estimating volume change by weighing provided more accurate results than that could be achieved using volume indicators (Parsons & Wingate, 1961). Ex vivo methods of intestinal perfusion technique are quick, simple techniques for estimating of intestinal drug transport. However, as all ex vivo models, tissue viability and integrity of the intestinal respiratory system have a marked effect on the results. In addition, the barrier imposed by the intestinal wall and serosa may result in slower absorption rates than those obtained in intact animals (Darlington & Quastel, 1953).

2.2. Everted gut sac experiments The everted gut sac preparation was reported for the first time by Wilson and Wiseman (1953). The model has since been used to predict the extent of transfer and intestinal metabolism of drugs. After excision, the prepared rat intestine is everted on a glass rod (Carmona, 1998), and filled with test drug solution (typically 1.0 mL). Absorption is estimated by the differences in weight before and after draining fluid in the intestine. Samples of luminal medium are taken at the beginning and end of each run. The increase in the initial volume of serosal fluid during the experiment represents the volume transferred from mucosal to the serosal side of the preparation (Wiseman, 1957).

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

An entire length rat intestine, 5 to 10 cm of intestine, or a jejunum segment (at about 15 cm below pylorus) (Horie & Tomimatsu, 2005) is most frequently used for these experiments. However, duodenum and ileum can also be used to explore absorption through these segments (Fang & Sha, 2004; Li et al., 2011). Hamster intestine was used in the original experiments as rat intestine was found to be more fragile (Wilson & Vincent, 1955; Wiseman, 1955, 1956). The thickness of the wall of rat intestine was about 0.30 mm in the upper region of the jejunum but only 0.15 mm in the lower ileum. The tissue was preserved in ice cold saline during the preparation of the sacs (Wilson, 1954). Prior to incubation, an isolated intestinal segment was removed and placed quickly in a beaker containing ice (L. Chen et al., 2010). The segment was washed with chilled saline (Ma et al., 2011) or ice-cold phosphate (Freedman et al., 1999) and everted. At the end of the experiment, the sacs were washed several times and the excised intestine was rinsed with cold buffer. The incubation media has a significant influence on the results and progressive modifications have been made to the procedure to account for this. Initially, Warburg flasks were used for incubation in most aerobic and all anaerobic experiments. A few aerobic experiments used continuously gassed Krebs flasks. Phosphorus was placed in the center well of the Warburg flask in anaerobic experiments (Wilson, 1954), and the difficulty of adequate oxygenation was overcome by everting a piece of intestine. In order to maintain high activation of the mucosa in the incubation medium, a sac containing sufficient fluid and oxygen was tied at both ends (Wiseman & Wilson, 1954). Later experiments used a gas mixture of 5% CO2 and 95% O2 (Kershaw, Neame, & Wiseman, 1960). Attempts were also made at incubating intestine in culture medium, so that the tissue was metabolically active and viable for a long period of time (Barthe, Houin, Kenworthy, & Woodley, 1998). The medium was then replaced by TC 199 (concentrated 10 times with Earle's salts) and oxygenated with 95% O2 and 5% CO2 (Chan et al., 2006). The tissue was everted, cut into pieces and bathed in medium. It was then perfused continuously through the serosal side with oxygen. This avoided solute accumulation in the tissue phase of the everted sacs. After incubation, the intestinal segment is rinsed, and its dry weight determined. The precise determination of tissue absorption was made at the end of incubation (Carmona, 1998). As with all in vitro techniques, tissue viability is an acute issue in everted gut sac experiments. Histological investigations have been undertaken to determine whether there were any significant changes in tissue morphology during the course of the experiments. It was found that the everted intestinal sacs gradually lost structural integrity. The sacs remained morphologically unchanged immediately after eversion and before incubation, but changes became noticeable 5 min after incubation at 37 °C in oxygenated buffer. With the passage of time, there was disappearance of normal epithelium and total disruption of the epithelial border. Everted intestinal sacs from golden hamster maintained integrity longer than tissues from rats when both were maintained at 23 °C. Sacs of hamster intestine showed only moderate loss of structural detail at villi tips 30 to 60 min after being everted. Other workers have shown that tissue disintegration is slower in tissues from animals sacrificed under anesthesia or at 23 °C compared with those sacrificed at 37 °C (Kornguth, LeBlanc, Levine, & McNary, 1970). In recent years, procedures have been gradually modified. Tchercansky modified the procedure for incubating tissue at 37 °C by constant shaking in aerated buffer (Acevedo, Rubio, & Tchercansky, 1994). Wilson and Wiseman reported that an optimal agitation frequency of 90 oscillations/min (4 cm amplitude) minimized tissue disintegration in the sac. Other workers fixed and tied a 1.0 or 2.0 g glass weight to the end of the everted gut segment to secure the tissue and prevent the sac septum from thinning (Gurib-Fakim, Mahomoodally, & Subratty, 2004). In other studies, the protein of the sacs was measured by digested tissue in NaOH at 37 °C for 1 h, or homogenized in ultrapure water using an Ultra-turrax blender (Arellano, Houin, Philibert, Vachoux,

211

& Woodley, 2007). It has also been found to be important to replace solution taken from the mucosal side (outside) with the same volume of blank buffer at each sampling interval (Ma et al., 2012). 2.3. Ussing chamber According to Bader et al., isolated mucosa particles are unsuitable for investigating the transport of metabolites into the intestinal lumen or the portal vein, and isolated intestinal loop methods have shortcomings in terms of stability (Bader, Christians, Gonschior, Hackbarth, Lampen, et al., 1996). The Ussing chamber was first described in 1951 for the direct measurement of unidirectional uptake of sodium from an external solution into frog skin (Ussing & Zerahn, 1951). The technique has since been used as an ex vivo model to study the permeability of intestinal tissue to various molecules (Boudry, 2005). A cut is made at the junction between the duodenum and the jejunum of male rats, and a section of small intestine approximately 30 cm in length is removed. Sections with 3 cm length, containing no Peyer's patches, were isolated from the segment, opened along the mesenteric border and immediately mounted in Ussing chambers. The experiment is started by replacing the blank Krebs' solution with the test solutions: the test solutions act as donors and Krebs' solution as a receiver. Samples are withdrawn from the receiver side at intervals, and the same volume of Krebs' solution at 37 °C is immediately replaced (Abaut, Chevanne, & Le Corre, 2007). Samples are analyzed by conventional advanced analytical instruments (Berggren, Fagerholm, Hoogstraate, & Lennernäs, 2004). According to Artursson, paracellular absorption in humans can be studied mechanistically using this ex vivo process (Artursson, Löfroth, & Ungell, 1993). The method is well validated and has been adapted to study transport and permeability of epithelial tissues in laboratories throughout academia. Scientists have used the Ussing chamber method to study the permeability of drugs that are poorly absorbed (Cao et al., 2011). The Ussing chamber has also been used in experiments designed to study the absorption mechanisms of different compounds, to evaluate the drug–drug interactions, and drug transport processes (Fischer et al., 2012; Grass & Sweetana, 1988). The Ussing chamber method also appears to be unsuitable for evaluating ester prodrugs (Annaert et al., 2000). However, the method is not suitable for use with rabbit tissues as the duodenal and jejunal sections are too thick for the diffusion chambers and leaks are observed (Abaut, Chevanne, & Le Corre, 2007). 3. In situ methods These methods offer many advantages in that they provide intact intestinal mucosa, nerve system and blood flow, together with expression of enzymes and transporters (Holmstock et al., 2012). However, they require sophisticated surgical procedures and instrumentations that make them unsuitable for some laboratories. In direct in situ studies, absorption is measured by the disappearance of the absorbed drug from the gut. However, many studies involve indirect measurements, appraising intestinal absorption by the rate of appearance of drugs in plasma, excretion in urine, or by the speed of onset or degree of pharmacologic action (Brodie, Hogben, Schanker, & Tocco, 1958). The efficiency of four in situ perfusion techniques including singlepass perfusion, recirculating perfusion, closed-loop method and the oscillating perfusion has been evaluated in rats by comparing effective permeability constants (ka) (Bijdendijk, Crommelin, Schurgers, & Tukker, 1986). 3.1. Closed-loop method The closed intestinal loop method avoids the uncertainty of gastric emptying time that is associated with the method of Cori. The method

212

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

makes it possible to use the same region of the intestine in every case and to measure the length of loop filled with solution. However, the operative procedure is complex. A loop of gut between the chosen ligatures is removed from the abdomen of an anesthetized animal, washed and filled with prepared solution. It is then returned to the abdomen to enable absorption to occur at body temperature for an appointed time. Measured portions of the solution are taken for analysis and compared with standards prepared from stock solution used for injection (Gibson & Wiseman, 1951). The method is widely used to study the rate of drug absorption. Fadi detailed a method of intravenously administering drugs via the femoral vein after the ligatured intestine had been returned to the body. In this procedure, the isolated intestinal segment is perfused at steady state with the effluent collected from the distal cannula. The absorption of the sample is determined as the difference between the quantity of substance introduced in the beginning and recovered at the end of the experiment (Mourad, 2004). The model also allows absorption to be measured separately at different regions of rat intestine, jejunum, ileum and colon (Kataoka, Masaoka, Tanaka, Sakuma, & Yamashita, 2006). Unfortunately, the procedure does not allow estimation of absorption at steady state. It is also necessary to undertake a large number of experiments before statistically significant results can be obtained. This is due to the wide degree of variability between rates of absorption in different rats. However, variations due to differences between rats could be eliminated by using paired loops in a single animal (Agar, Hird, & Sidhu, 1956). 3.2. Thiry–Vella fistula The Thiry–Vella loop is perfused using the apparatus described by Clarke & Smyth. The method has been adapted for use in dogs and has been used to study absorption in unanesthetized animals with an intestine maintained at near normal physiological conditions (Clarke, Gibson, Smyth, & Wiseman, 1951). Fluid can be perfused through a completely closed circuit making the preparation suitable for both absorption or motility studies (Smyth & Clarke, 1951). The method has also been used to assess prodrug stability measured by collecting fasting small intestinal fluids from four male Labrador dogs who had a chronic fistula at mid-jejunum (approx 76 cm below the pylorus) (Andersson, Abrahamsson, Borde, Bjorhall, Karlsson, & Lennernas, 2012). The main advantage of this model is that it measures fluid transport in conscious animals and in a segment of bowel in vivo that has an intact blood supply (Mourad, 2004). 3.3. Intestinal single-pass perfusion (SPIP) This in situ method was first described by Brodie et al. in 1958 (Brodie et al., 1958). Sections of intestine from anesthetized rats are isolated, cannulated, perfused with drug solution and sampled for analysis. To maintain normal body temperature (between 36 °C and 37 °C), the rats are placed on a heated slide warmer under a heating lamp. Breathing is facilitated by the introduction of a plastic tube into the trachea during surgery. It is important to maintain adequate circulation during surgery and to keep the exposed segment moist with the saline maintained at body temperature (Fagerholm, Lennernäs, & Johansson, 1996). Reference substances have been added to the solution to estimate the viability of the intestinal membrane. 14C-Labeled PEG-4000 and phenol red can be used as non-absorbable markers for fluid loss or membrane leakage, and 3H-D-glucose has been used as a marker for active transport (Fredholt, Friis, Lepist, Lennernäs, & Østergaard, 1999). However, it has subsequently found that phenol red may interfere with the procedure and analysis of results, and radiolabeled isotopes are associated with safety concerns.

A gravimetric method, involving the determination of the weight of intestinal perfusate collected over time and its conversion to a volumetric parameter, has been used as a simple alternative that overcomes these concerns (Bansal, Gupta, & Issa, 2003). It has been shown that the gravimetric method is as accurate as using nonabsorbed markers (radiolabeled (14C) polyethylene glycols (14C-PEG), phenol red), and is especially suited to the determination of net water flux (NWF) (Sutton, Rinaldi, & Vukovinsky, 2001). Modified Hank's balanced salt solution (HBSS), supplemented with antibiotics such as penicillin and streptomycin, have been used to reduce experimental variability without significantly changing mean intestinal wall permeability (Pw) (Chen et al., 1994). Chawla attempted to explore changes in absorption parameters in the intestinal perfusion model using co-perfusion with sodium deoxy cholate sodium caprate and piperine. These additives increased intestinal permeability (Papp) and absorption rate constant (Ka) by as much as two and four fold, respectively (Chawla, Panchagnula, Sharma, & Varma, 2005). Effective permeation enhancers (AE) have also been used to enhance the absorption of low intestinal permeability drugs (Fredholt et al., 1999). Amidon et al. used the technique to simultaneously perfused three distinct segments of intestine: the proximal jejunum, mid-small intestine and distal ileum. In this way, the model can be used to diminish the number of animals used for segmental-dependent permeability research without compromising the quality of data obtained (Amidon, Dahan, & West, 2009). A modification of the original rat model has been adapted for use in mice. In these experiments, the upper small intestine and colon of mice were perfused simultaneously and samples were collected. An internal standard (IS) was added to the perfusate prior to centrifugation and analysis (Dong et al., 2012). The major advantage of all these new approaches is that they significantly reduce the number of animals utilized and animals act as their own controls for analyzing segmental-dependent membrane permeability. Some scholars have combined single-pass perfusion and Ussing chamber experiments, with drug efficacy and toxicology data in order to define molecular structure and predict the dynamic characteristics of a drug in terms of its intestinal permeability (Lennernäs, 2007). In the past decade, success in predicting absorption in human using in situ single-pass perfusion in rat intestine has been widely reported (Amidon, Ibuki, Ohike, Tamura, & Yamashita, 2002; Fleisher et al., 1999). The obvious advantages of this model are that it provides a functional intestinal barrier and conditions that closely mimic the normal physiological state in terms of providing an intact blood supply following oral administration. Quantitative differences between permeability in human and rat models have been identified using model drugs with a broad range of physicochemical properties that confer both high and low permeability. A high correlation has been reported between rat and human jejunum estimates of passively absorbed solutes (Fagerholm et al., 1996). A high correlation between rat and human Peff values has also been found (Azarmi et al., 2007). These findings suggest that the SPIP technique is a reliable technique for predicting fractional dose absorption in human subjects. 3.4. Intestinal recirculating perfusion The single-pass perfusion technique facilitates the study of rapid absorption and is suitable for drugs that are rapidly absorbed. In the recirculating perfusion model, the probability of absorption is considerably increased because of the longer retention time of the solution in the intestine (Cadelli & Grassi, 2001). Thus, it is recommended that drugs which are absorbed comparatively slowly as shown by single-pass perfusion techniques are reexamined using recirculating perfusion to magnify the concentration changes (Brodie et al., 1958). Rats or rabbits are anesthetized, and the intestine is incubated and infused with normal saline (37 °C) or the test solution. Intestine at

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

both ends of the glass tube is connected to a peristaltic pump to form a loop, and the peristaltic pump is started. Samples are taken at different time periods and the system is replenished with same volume of blank circulating fluid. During the perfusion, approximately 10 cm of inlet tubing is placed inside the abdominal cavity to provide an inlet for the perfusion of solution at 37 °C (Ashton, Carlborg, Lennernäs, Sandström, & Svensson, 1999). All perfusion syringes and sample cups are weighed before and after perfusion. The perfusate samples are collected on ice and prepared for analysis. Phenolsulfonpthalein (PSP), PEG-4000 or inulin can be added as impermeable reference markers. Ferrocyanide, a nontoxic, quadruple-charged anion that is not absorbed can be added to the perfusion fluid as an osmotically active solute (Pappenheimer & Reiss, 1987).

3.5. Intestinal perfusion with venous sampling Intestinal perfusion with venous sampling has been widely reported as a useful method for obtaining realistic drug absorption rates (Fig. 2). In the early years, the methodology involved perfusion of an isolated intestinal segment with regional mesenteric venous cannulation being used to follow the disappearance kinetics of drug or prodrug from the intestinal lumen and its concurrent appearance in blood (Anderson, Ho, & Singhal, 1998). The experiments were performed using either open-loop or closed-loop (perfusate was recirculated) systems (Annaert et al., 2008). The absorbed drug was collected before it reaches other body tissues, which prevented contamination of the sample with circulating hepatic metabolites (Blanchard, Earle, & Tang, 1990). The quantity of drug absorption is usually based on its disappearance rate from the intestinal lumen. This approximates to the absorption rate in most but not all cases. In other experiments, mesenteric blood was collected in heparinized tubes to evaluate drug absorption at different intervals and donor blood was obtained from two to three animals by cardiac puncture (Chan et al., 2008). Freshly drawn heparinized rat blood was then infused into the jugular vein using a constant rate infusion pump to replenish the blood lost (Borchardt, Burton, & Kirn, 1993). Claude and coworkers described a new model of chronic catheterization of the portal vein which did not involve ligating a branch of the portal system (Ferré, Girard, Morin, & Smadja, 1988). Samples were taken from the perfusion fluid at the end of the cannulated segment

213

and at the inlet of perfused segment, and blood was drawn from the portal veins (Pithavala, Soria, & Zimmerman, 1997). To obtain more complete pharmacokinetic analysis of drug absorption, the rat intestinal preparation was modified to include portal and jugular blood collection techniques with sampling from the intestinal lumen. A catheter was inserted and secured in the gastrosplenic vein with the tip protruding into the portal vein for sampling (Dvorchik, Pritchard, Renzi, & Yorgey, 1986; Hu & Liu, 2002). The jejunal cannula was connected to a short cannula (1.5 to 2 cm long) which could be easily disconnected or reconnected to the main tube. In Dharmendra Singhal's study, two additional sites (the ileum and mesenteric vein) were cannulated for perfusion, in addition to the jugular vein (Anderson et al., 1998). Yunn-Fang Ho, used the same surgical procedures, and withdrew venous samples through implanted intravenous catheters (Ho et al., 2009). The section of mesenteric vein used for collecting blood is normally obtained from a specified segment of intestine. The perfusion buffer is isotonic, and in some experiments, phenol red is added to the perfusate as a nonabsorbable marker. In experiments by Kavin et al., a procedure for surgically isolating and artificially perfusing the small intestine was described. Blood was sampled from the arterial and venous channels (Kavin, Levin, & Stanley, 1967). All samples were analyzed by fluorescent spectrophotometry, validated HPLC, or other advanced techniques. Intestinal perfusion with venous sampling allows the determination of intestinal metabolism without interference by the confounding effects of hepatic first-pass metabolism. It, therefore, provides a model for future in depth research (Benet, Cummins, Reid, & Salphati, 2003).

3.6. Vascularly perfused intestine-liver (IPIL) The intestine and liver both play a crucial role in the systematic availability of orally administrated compounds. Owing to the fact that the relative role of the intestine and the liver is poorly defined, a simple method for portal vein infusion using the pyloric vein cannulation in the rat has been used to study first-pass effects. This method overcomes several disadvantages inherent in the use of direct portal vein cannulation by using delicate surgery to prevent excessive engorgement of the intestinal veins and acute loss of blood from the liver (Ishida, Isozaki, Saitoh, & Suzuki, 1973). In preliminary experiments, substances which failed to be glucogenic in slices also gave

Fig. 2. Schematic drawing of in situ intestinal perfusion with venous sampling.

214

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

negative results in the perfused organ (Berry, Hems, Krebs, & Ross, 1966). This led to a systematic investigation of the perfusion technique. The model of vascularly perfused intestine–liver was first described by Pang et al. In this model, a metal catheter was inserted into the right renal artery, through the aorta, and the superior mesenteric artery, through which drugs can be administered at amounts that are not usually well tolerated in vivo (Cherry, Pang, & Ulm, 1985). A further modification is that the intestine is perfused via the superior mesenteric artery and portal venous outflow, which together with hepatic arterial flow, constitutes the total hepatic blood flow (Gardemann, Grosse, Hesse, Jungermann, & Watanabe, 1992; Hirayama, Pang, & Xu, 1989; Remmel, Wen, & Zimmerman, 1999). Human red cells can be added to the perfusate (Hirayama et al., 1989). The main advantage of this model is that it allows the investigation of the hemodynamics and metabolism for each organ, as well as the inter-relationships between the small intestine and liver (Gardemann et al., 1992).

3.7. Loc-I-Gut Loc-I-Gut is an accurate method that provides direct estimates of local drug absorption in human subjects. The Loc-I-Gut instrument (Synectics, Sweden) is a six-channel, 175 cm long, sterile, disposable polyvinyl perfusion tube which is used for intestinal perfusion in humans (Nilsson, Fagerholm, & Lennernäs, 1994). A more detailed description of the procedures has been described by Knutson (Knutson, Odlind, & Hällgren, 1989) or Lennernäs (Lennernäs et al., 1992). The Loc-I-Gut method has been used to study the effects of induced net fluid absorption on the small intestinal absorption of levodopa in healthy volunteers (Nilsson et al., 1994). It is also suitable for assessing first-pass effects of drugs in the liver. Drug metabolism in intestinal tissue can be evaluated by measuring the metabolite levels in the outlet perfusate. In vivo drug dissolution, local pharmacological studies, nutrient absorption, and biological mechanisms involved in gastrointestinal diseases, food–drug interactions, and intestinal secretion of drugs and endogenous compounds have also been examined using this technique (Lennernäs, 1998). The main advantage of the perfusion technique is that it is not influenced by other gastrointestinal factors such as transit time and regional pH-differences (Amidon, Hussain et al., 2002).

4. Conclusions The ex vivo and in situ methods described above provide vital information which contributes to our understanding of the pathophysiology of intestinal absorption, and assists in the development and assessment of new drugs with potential medical applications. The four fluid compartments (blood vessels, lymphatics, interstitial space, and lumen) are all maintained in the in situ rat small intestinal model, which thereby overcomes the principal limitations of ex vivo methods such as everted gut sac method or Ussing chambers. In situ models can also be used to investigate elementary processes such as intestinal barrier functions, fluid homeostasis, immune responses and transport mechanisms. They, therefore, provide important insights into the physiology of the small intestine (Bade, Dombrowsky, Frerichs, Kuchenbecker, Lautenschlager, Schultz, et al., 2010). An in situ rat small intestinal model with inhibitors added to the perfusate has been developed in recent years to study intestinal gut metabolism. The role of in situ rat small intestinal models in metabolic studies will become increasingly prominent in future years. However, it should be noted that extrapolation of ex vivo or in situ data to in vivo situations is far from straightforward. Thus, model systems which are much closer to in vivo physiology are urgently needed for investing intestinal absorption.

Acknowledgments This work was supported by the National Natural Science Foundation of China (30801510 and 81274042). Author contribution Yang Liu designed the review; Zhiqiang Luo, Mingmin Tang, Honghuan Dong, Lei Zhang, and Beiran Lv collected the literature and summarized the results; Baosheng Zhao reviewed the paper and gave us some useful suggestions; Li Wei did the artwork; Zhiqiang Luo and Mingmin Tang wrote the paper and all authors reviewed all; Zhiqiang Luo takes responsibility for the manuscript. References Abaut, A. Y., Chevanne, F., & Le Corre, P. (2007). Oral bioavailability and intestinal secretion of amitriptyline: Role of P-glycoprotein? International Journal of Pharmaceutics, 330, 121–128. Acevedo, C., Rubio, M. C., & Tchercansky, D. M. (1994). Studies of tyramine transfer and metabolism using an in vitro intestinal preparation. Journal of Pharmaceutical Sciences, 83, 549–552. Agar, W. T., Hird, F., & Sidhu, G. (1953). The active absorption of amino-acids by the intestine. The Journal of Physiology, 121, 255–263. Agar, W., Hird, F., & Sidhu, G. (1956). The absorption, transfer and uptake of amino acids by intestinal tissue. Biochimica et Biophysica Acta, 22, 21–30. Amidon, G. L., Dahan, A., & West, B. T. (2009). Segmental-dependent membrane permeability along the intestine following oral drug administration: Evaluation of a triple single-pass intestinal perfusion (TSPIP) approach in the rat. European Journal of Pharmaceutical Sciences, 36, 320–329. Amidon, G., Hussain, A., Knutson, L., Knutson, T., Lennernäs, H., Lesko, L., et al. (2002a). The effect of amiloride on the in vivo effective permeability of amoxicillin in human jejunum: Experience from a regional perfusion technique. European Journal of Pharmaceutical Sciences, 15, 271–277. Amidon, G. L., Ibuki, R., Ohike, A., Tamura, S., & Yamashita, S. (2002b). Tacrolimus is a class II low-solubility high-permeability drug: The effect of P-glycoprotein efflux on regional permeability of tacrolimus in rats. Journal of Pharmaceutical Sciences, 91, 719–729. Anderson, B. D., Ho, N. F., & Singhal, D. (1998). Absorption and intestinal metabolism of purine dideoxynucleosides and an adenosine deaminase-activated prodrug of 2′, 3′dideoxyinosine in the mesenteric vein cannulated rat ileum. Journal of Pharmaceutical Sciences, 87, 569–577. Andersson, K., Abrahamsson, B., Borde, A. S., Bjorhall, K., Karlsson, E. M., & Lennernas, H. (2012). Assessment of enzymatic prodrug stability in human, dog and simulated intestinal fluids. European Journal of Pharmaceutics and Biopharmaceutics, 80, 630–637. Annaert, P., Augustijns, P., De Clercq, E., Kinget, R., Naesens, L., Tukker, J. J., et al. (2000). In vitro, ex vivo, and in situ intestinal absorption characteristics of the antiviral ester prodrug adefovir dipivoxil. Journal of Pharmaceutical Sciences, 89, 1054–1062. Annaert, P., Augustijns, P., Martens, J. A., Mellaerts, R., Mols, R., Kayaert, P., et al. (2008). Ordered mesoporous silica induces pH-independent supersaturation of the basic low solubility compound itraconazole resulting in enhanced transepithelial transport. International Journal of Pharmaceutics, 357, 169–179. Arellano, C., Houin, G., Philibert, C., Vachoux, C., & Woodley, J. (2007). The metabolism of midazolam and comparison with other CYP enzyme substrates during intestinal absorption: In vitro studies with rat everted gut sacs. Journal of Pharmacy and Pharmaceutical Sciences, 10, 26–36. Artursson, P., Löfroth, J. E., & Ungell, A. L. (1993). Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments. Pharmaceutical Research, 10, 1123–1129. Ashton, M., Carlborg, Ö., Lennernäs, H., Sandström, R., & Svensson, U. S. (1999). High in situ rat intestinal permeability of artemisinin unaffected by multiple dosing and with no evidence of P-glycoprotein involvement. Drug Metabolism and Disposition, 27, 227–232. Azarmi, Y., Barzegar, S., Barzegar-Jalali, M., Islambolchilar, Z., Valizadeh, H., Tajerzadeh, H., et al. (2007). Predicting human intestinal permeability using single-pass intestinal perfusion in rat. Journal of Pharmacy and Pharmaceutical Sciences, 10, 368–379. Bade, S., Dombrowsky, H., Frerichs, I., Kuchenbecker, S. C., Lautenschlager, I., Schultz, H., Scholz, J., Uhlig, S., Weiler, N., & Zabel, P. (2010). A model of the isolated perfused rat small intestine. American Journal of Physiology. Gastrointestinal and Liver Physiology, 298, 304–313. Bader, A., Christians, U., Gonschior, A. K., Hackbarth, I., Lampen, A., Sewing, K. F., & von Engelhardt, W. (1996). Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber. British Journal of Pharmacology, 117, 1730–1744. Bansal, A. K., Gupta, P., & Issa, C. (2003). Implications of density correction in gravimetric method for water flux determination using rat single-pass intestinal perfusion technique: A technical note. AAPS PharmSciTech, 4, 44–49. Barthe, L., Houin, G., Kenworthy, S., & Woodley, J. (1998). An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine. European Journal of Drug Metabolism and Pharmacokinetics, 23, 313–323.

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216 Benet, L. Z., Cummins, C. L., Reid, M. J., & Salphati, L. (2003). In vivo modulation of intestinal CYP3A metabolism by P-glycoprotein: Studies using the rat single-pass intestinal perfusion model. Journal of Pharmacology and Experimental Therapeutics, 305, 306–314. Berggren, S., Fagerholm, U., Hoogstraate, J., & Lennernäs, H. (2004). Characterization of jejunal absorption and apical efflux of ropivacaine, lidocaine and bupivacaine in the rat using in situ and in vitro absorption models. European Journal of Pharmaceutical Sciences, 21, 553–560. Berry, M., Hems, R., Krebs, H., & Ross, B. (1966). Gluconeogenesis in the perfused rat liver. Biochemical Journal, 101, 284–292. Bijdendijk, J., Crommelin, D. J., Schurgers, N., & Tukker, J. J. (1986). Comparison of four experimental techniques for studying drug absorption kinetics in the anesthetized rat in situ. Journal of Pharmaceutical Sciences, 75, 117–119. Blanchard, J., Earle, M. E., & Tang, L. M. (1990). Reevaluation of the absorption of carbenoxolone using an in situ rat intestinal technique. Journal of Pharmaceutical Sciences, 79, 411–414. Borchardt, R. T., Burton, P. S., & Kirn, D. C. (1993). A correlation between the permeability characteristics of a series of peptides using an in vitro cell culture model (Caco-2) and those using an in situ perfused rat ileum model of the intestinal mucosa. Pharmaceutical Research, 10, 1710–1714. Boudry, G. (2005). The Ussing chamber technique to evaluate alternatives to in-feed antibiotics for young pigs. Animal Research, 54, 219. Brodie, B. B., Hogben, C. A. M., Schanker, L. S., & Tocco, D. J. (1958). Absorption of drugs from the rat small intestine. Journal of Pharmacology and Experimental Therapeutics, 123, 81–88. Cadelli, G., & Grassi, M. (2001). Theoretical considerations on the in vivo intestinal permeability determination by means of the single pass and recirculating techniques. International Journal of Pharmaceutics, 229, 95–105. Cao, Y. C., Liang, X. L., Liao, Z. G., Yang, M., Yin, R. L., Zhao, G. W., et al. (2011). The absorption characterization effects and mechanism of Radix Angelica dahurica extracts on baicalin in Radix Scutellariae using in vivo and in vitro absorption models. Journal of Ethnopharmacology, 139, 52–57. Carmona, A. (1998). A simple in vitro perfusion system to measure intestinal nutrient uptake. The Journal of Nutritional Biochemistry, 9, 52–57. CDER (2000). Guidance for Industry: Waiver of in vivo bioavailability and bioequivalence studies for immediate-release solid oral dosage. Forms based on a Biopharmaceutics Classification System. In (pp. 13): FDA Maryland. Chain, E., Mansford, K., & Pocchiari, F. (1960). The absorption of sucrose, maltose and higher oligosaccharides from the isolated rat small intestine. The Journal of Physiology, 154, 39–51. Chan, K., Cheung, M. C., Gu, Z. M., Han, Y. F., Li, W. M., Pang, Y. P., et al. (2008). Preclinical characterization of intestinal absorption and metabolism of promising antiAlzheimer's dimer bis (7)-tacrine. International Journal of Pharmaceutics, 357, 85–94. Chan, K., Jiang, Z. H., Liu, L., Liu, Z. Q., Wong, Y. F., Xu, H. -X., et al. (2006). The effects of sinomenine on intestinal absorption of paeoniflorin by the everted rat gut sac model. Journal of Ethnopharmacology, 103, 425–432. Chawla, H. P., Panchagnula, R., Sharma, P., & Varma, M. V. (2005). In situ and in vivo efficacy of peroral absorption enhancers in rats and correlation to in vitro mechanistic studies. Il Farmaco, 60, 874–883. Chen, J., Elmquist, W., Hu, M., Yang, H., Zheng, L., & Zhu, Y. (1994). Comparison of the transport characteristics of D-and L-methionine in a human intestinal epithelial model (Caco-2) and in a perfused rat intestinal model. Pharmaceutical Research, 11, 1771–1776. Chen, L., Li, W., Ma, S., Ping, L., Yang, Z., & Zhou, Y. (2010). Enhancement of intestinal absorption of akebia saponin D by borneol and probenecid in situ and in vitro. Environmental Toxicology and Pharmacology, 29, 229–234. Cherry, W., Pang, K. S., & Ulm, E. (1985). Disposition of enalapril in the perfused rat intestine–liver preparation: absorption, metabolism and first-pass effect. Journal of Pharmacology and Experimental Therapeutics, 233, 788–795. Clarke, E., Gibson, Q. H., Smyth, D., & Wiseman, G. (1951). Selective absorption of amino-acids from Thiry–Vella loops. Journal of Physiology (London), 112, 46P. Clere, C., Desangle-Gouty, V., Genty, M., González, G., & Legendre, J. -Y. (2001). Determination of the passive absorption through the rat intestine using chromatographic indices and molar volume. European Journal of Pharmaceutical Sciences, 12, 223–229. Darlington, W., & Quastel, J. (1953). Absorption of sugars from isolated surviving intestine. Archives of Biochemistry and Biophysics, 43, 194–207. Dong, L., Feng, Q., Liu, W., Liu, Z., Tang, L., Yang, C., et al. (2012). Involvement of UDPglucuronosyltranferases and sulfotransferases in the liver and intestinal first-pass metabolism of seven flavones in C57 mice and humans in vitro. Food and Chemical Toxicology, 1460–1467. Dvorchik, B. H., Pritchard, J. F., Renzi, N. L., & Yorgey, K. A. (1986). Evaluation of drug absorption and presystemic metabolism using an in situ intestinal preparation. Journal of Pharmaceutical Sciences, 75, 869–872. Ehrhardt, C., & Kim, K. -J. (2008). Drug Absorption Studies: In Situ, In Vitro and In Silico Models. Vol. 7, New York: Springer (Chapter 2). Fagerholm, U., Lennernäs, H., & Johansson, M. (1996). Comparison between permeability coefficients in rat and human jejunum. Pharmaceutical Research, 13, 1336–1342. Fang, X., & Sha, X. (2004). Transport characteristics of 9-nitrocamptothecin in the human intestinal cell line Caco-2 and everted gut sacs. International Journal of Pharmaceutics, 272, 161–171. Ferré, P., Girard, J., Morin, J., & Smadja, C. (1988). Metabolic fate of a gastric glucose load in unrestrained rats bearing a portal vein catheter. American Journal of Physiology — Endocrinology and Metabolism, 254, 407–413. Fischer, T., Hoepner, U., Kamiyama, E., Mueller, J., Nakai, D., Rozehnal, V., et al. (2012). Human small intestinal and colonic tissue mounted in the Ussing chamber as a tool for characterizing the intestinal absorption of drugs. European Journal of Pharmaceutical Sciences, 46, 367–373.

215

Fisher, R., & Parsons, D. (1949). A preparation of surviving rat small intestine for the study of absorption. The Journal of Physiology, 110, 36–46. Fisher, R. B., & Parsons, D. S. (1950). Glucose absorption across the wall of the rat small intestine. Journal de Physiologie, 110, 281–293. Fisher, R., & Parsons, D. (1953). Galactose absorption from the surviving small intestine of the rat. The Journal of Physiology, 119, 224–232. Fleisher, D., Li, C., Winward, B., Pao, L., Zhou, S., & Zimmermann, E. (1999). Intestinal metabolism and transport of 5-aminosalicylate. Drug Metabolism and Disposition, 27, 479–485. Fredholt, K., Friis, G. J., Lepist, E. I., Lennernäs, H., & Østergaard, J. (1999). Stability and perfusion studies of Desmopressin (dDAVP) and prodrugs in the rat jejunum. Experimental and Toxicologic Pathology, 51, 363–368. Freedman, B. D., Harnpicharnchai, P., Hertzog, B. A., Jacob, J. S., Mathiowitz, E., Press, D. L., et al. (1999). Correlation of two bioadhesion assays: The everted sac technique and the CAHN microbalance. Journal of Controlled Release, 61, 113–122. Gardemann, A., Grosse, V., Hesse, S., Jungermann, K., & Watanabe, Y. (1992). Increases in intestinal glucose absorption and hepatic glucose uptake elicited by luminal but not vascular glutamine in the jointly perfused small intestine and liver of the rat. Biochemical Journal, 283, 759–765. Gardner, M. L. (1978). The absorptive viability of isolated intestine prepared from dead animals. Experimental Physiology, 63, 93–95. Gibson, Q. H., & Wiseman, G. (1951). Selective absorption of stereo-isomers of aminoacids from loops of the small intestine of the rat. Biochemical Journal, 48, 426–429. Grass, G. M., & Sweetana, S. A. (1988). In vitro measurement of gastrointestinal tissue permeability using a new diffusion cell. Pharmaceutical Research, 5, 372–376. Gubernator, K., Kansy, M., & Senner, F. (1998). Physicochemical high throughput screening: Parallel artificial membrane permeation assay in the description of passive absorption processes. Journal of Medicinal Chemistry, 41, 1007–1010. Gurib-Fakim, A., Mahomoodally, M. F., & Subratty, A. H. (2004). A kinetic model for in vitro intestinal uptake of L-tyrosine and D (+) glucose across rat everted gut sacs in the presence of Momordica charantia, a medicinal plant used in traditional medicine against diabetes mellitus. Journal of Cell and Molecular Biology, 3, 39–44. Hirayama, H., Pang, K. S., & Xu, X. (1989). Viability of the vascularly perfused, recirculating rat intestine and intestine–liver preparations. American Journal of Physiology — Gastrointestinal and Liver Physiology, 257, 249–258. Ho, Y. F., Huang, D. K., Hsueh, W. C., Lai, M. Y., Tsai, T. H., & Yu, H. Y. (2009). Effects of St. John's wort extract on indinavir pharmacokinetics in rats: Differentiation of intestinal and hepatic impacts. Life Sciences, 85, 296–302. Holmstock, N., Annaert, P., & Augustijns, P. (2012). Boosting of HIV protease inhibitors by ritonavir in the intestine: The relative role of Cyp and P-gp inhibition based on Caco-2 monolayers versus in situ intestinal perfusion in mice. Drug Metabolism and Disposition, 40, 1473–1477. Horie, T., & Tomimatsu, T. (2005). Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil. Chemico-Biological Interactions, 155, 129–139. Hu, M., & Liu, Y. (2002). Absorption and metabolism of flavonoids in the caco-2 cell culture model and a perused rat intestinal model. Drug Metabolism and Disposition, 30, 370–377. Ishida, R., Isozaki, S., Saitoh, Y., & Suzuki, T. (1973). Simple method for portal vein infusion in the rat. Journal of Pharmaceutical Sciences, 62, 345–347. Kataoka, M., Masaoka, Y., Tanaka, Y., Sakuma, S., & Yamashita, S. (2006). Site of drug absorption after oral administration: Assessment of membrane permeability and luminal concentration of drugs in each segment of gastrointestinal tract. European Journal of Pharmaceutical Sciences, 29, 240–250. Kavin, H., Levin, N. W., & Stanley, M. M. (1967). Isolated perfused rat small bowel— Technic, studies of viability, glucose absorption. Journal of Applied Physiology, 22, 604–611. Kershaw, T. G., Neame, K. D., & Wiseman, G. (1960). The effect of semistarvation on absorption by the rat small intestine in vitro and in vivo. The Journal of Physiology, 152, 182–190. Knutson, L., Odlind, B., & Hällgren, R. (1989). A new technique for segmental jejunal perfusion in man. The American Journal of Gastroenterology, 84, 1278–1284. Kornguth, P. J., LeBlanc, R., Levine, R. R., & McNary, W. F. (1970). Histological reevaluation of everted gut technique for studying intestinal absorption. European Journal of Pharmacology, 9, 211–219. Lennernäs, H. (1998). Human intestinal permeability. Journal of Pharmaceutical Sciences, 87, 403–410. Lennernäs, H. (2007). Animal data: The contributions of the Ussing Chamber and perfusion systems to predicting human oral drug delivery in vivo. Advanced Drug Delivery Reviews, 59, 1103–1120. Lennernäs, H., Ahrenstedt, Ö., Hällgren, R., Knutson, L., Ryde, M., & Paalzow, L. K. (1992). Regional jejunal perfusion, a new in vivo approach to study oral drug absorption in man. Pharmaceutical Research, 9, 1243–1251. Li, M., Li, G., Pan, H., Qiu, J., Rabba, A. K., Si, L., et al. (2011). Excipients enhance intestinal absorption of ganciclovir by P-gp inhibition: Assessed in vitro by everted gut sac and in situ by improved intestinal perfusion. International Journal of Pharmaceutics, 403, 37–45. Ma, B. L., Wang, C. H., Gao, C. L., Qiu, F. R., Wu, J. S., Zhong, J., et al. (2011). Increased systemic exposure to Rhizoma coptidis alkaloids in lipopolysaccharide-pretreated rats due to enhanced intestinal absorption. Drug Metabolism and Disposition, 40, 381–388. Ma, B. L., Yao, M. K., Zhong, J., Ma, Y. M., Gao, C. L., Wu, J. S., et al. (2012). Increased systemic exposure to rhizoma coptidis alkaloids in lipopolysaccharide-pretreated rats attributable to enhanced intestinal absorption. Drug Metabolism and Disposition, 40, 381–388. Mannhold, R., Kubinyi, H., Folkers, G., Waterbeemd, H., & Testa, B. (2009). Drug bioavailability (2nd ed.): Wiley-VCH (Chapter 1).

216

Z. Luo et al. / Journal of Pharmacological and Toxicological Methods 68 (2013) 208–216

Mansford, K. (1965). A preparation of surviving small intestine for the study of absorption in mammalian species. The Journal of Physiology, 180, 722–734. Mourad, F. H. (2004). Animal and human models for studying effects of drugs on intestinal fluid transport in vivo. Journal of Pharmacological and Toxicological Methods, 50, 3–12. Nilsson, D., Fagerholm, U., & Lennernäs, H. (1994). The influence of net water absorption on the permeability of antipyrine and levodopa in the human jejunum. Pharmaceutical Research, 11, 1540–1544. Pappenheimer, J., & Reiss, K. (1987). Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat. Journal of Membrane Biology, 100, 123–136. Parsons, D., & Prichard, J. (1966). Oxford Meeting 4 June 1966 Demonstrations. Parsons, D., & Wingate, D. (1961). The effect of osmotic gradients on fluid transfer across rat intestine in vitro. Biochimica et Biophysica Acta, 46, 170–183. Pithavala, Y. K., Soria, I., & Zimmerman, C. L. (1997). Use of the deconvolution principle in the estimation of absorption and pre-systemic intestinal elimination of drugs. Drug Metabolism and Disposition, 25, 1260–1265. Remmel, R. P., Wen, Y., & Zimmerman, C. L. (1999). First-pass disposition of (−)-6-aminocarbovir in rats. I. Prodrug activation may be limited by access to enzyme. Drug Metabolism and Disposition, 27, 113–121. Smith, C. G., & O'Donnell, J. T. (2006). The Process of New Drug Discovery and Development (2nd ed.)New York: Informa Healthcare (Chapter 1). Smyth, D. H., & Clarke, E. W. (1951). Technique for perfusion of the Thiry–Vella loop in dogs. Journal of Physiology (London), 112, 45–46. Smyth, D., & Taylor, C. (1957). Transfer of water and solutes by an in vitro intestinal preparation. The Journal of Physiology, 136, 632–648. Smyth, D., & Whaler, B. (1953). Apparatus for the in vitro study of intestinal absorption. The Journal of Physiology, 121, 2P.

Sutton, S. C., Rinaldi, M. T., & Vukovinsky, K. (2001). Comparison of the gravimetric, phenol red, and 14C-PEG-3350 methods to determine water absorption in the rat single-pass intestinal perfusion model. The AAPS Journal, 3, 93–97. Testa, B., & Waterbeemd, H. v. d (2008). Drug Bioavailability: Estimation of Solubility, Permeability, Absorption and Bioavailability (2nd ed.)New York: John Wiley & Sons (Chapter 1). Ussing, H. H., & Zerahn, K. (1951). Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiologica Scandinavica, 23, 110–127. Wilson, T. (1954). Concentration gradients of lactate, hydrogen and some other ions across the intestine in vitro. Biochemical Journal, 56, 521–527. Wilson, T. H., & Vincent, T. N. (1955). Absorption of sugars in vitro by the intestine of the golden hamster. Journal of Biological Chemistry, 216, 851–866. Wilson, T., & Wiseman, G. (1953). A method for studying intestinal metabolism and absorption. The Journal of Physiology, 121, 45P. Wiseman, G. (1953). Absorption of amino-acids using an in vitro technique. The Journal of Physiology, 120, 63–72. Wiseman, G. (1955). Preferential transference of amino-acids from amino-acid mixtures by sacs of everted small intestine of the golden hamster (Mesocricetus auratus). The Journal of Physiology, 127, 414–422. Wiseman, G. (1956). Active transport of amino acids by sacs of everted small intestine of the golden hamster (Mesocricetus auratus). The Journal of Physiology, 133, 626–630. Wiseman, G. (1957). Effect of pyridoxal on the active transport of amino acids by sacs of everted intestine of the golden hamster (Mesocricetus auratus). The Journal of Physiology, 136, 203–207. Wiseman, G., & Wilson, T. H. (1954). The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. The Journal of Physiology, 123, 116–125.