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Regional intestinal permeability in rats: a comparison of methods Carl Roos, David Dahlgren, E. Sjögren, Christer Tannergren, Bertil Abrahamsson, and H. Lennernas Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00279 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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Molecular Pharmaceutics Roos, C.

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Regional intestinal permeability in rats: a comparison between methods.

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Abstract

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Currently, the screening of new drug candidates for intestinal permeation is typically based on in vitro

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models which give no information regarding regional differences along the gut. When evaluation of

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intestinal permeability by region is undertaken, two preclinical rat models are commonly used, the

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Ussing chamber method and single-pass intestinal perfusion (SPIP). To investigate the robustness of in

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vivo predictions of human intestinal permeability, a set of four model compounds was systematically

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investigated in both these models, using tissue specimens and segments from the jejunum, ileum, and

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colon of rats from the same genetic strain. The influence of luminal pH was also determined at two pH

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levels. Ketoprofen had high and enalaprilat had low effective (Peff) and apparent (Papp) permeability in

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all three regions and at both pH levels. Metoprolol had high Peff in all regions and at both pHs and high

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Papp at both pHs and in all regions except the jejunum, where Papp was low. Atenolol had low Peff in all

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regions and at both pHs, but had high Papp at pH 6.5 and low Papp at pH 7.4. There were good

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correlations between these rat in situ Peff (SPIP) and human in vivo Peff determined previously for the

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same compounds by both intestinal perfusion of the jejunum and regional intestinal dosing. The results

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of this study indicate that both investigated models are suitable for determining the regional

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permeability of the intestine; however, the SPIP model seems to be the more robust and accurate

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regional permeability model.

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Introduction

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It is common to administer pharmaceutical products orally once daily as it is safe, convenient, and

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associated with improved patient compliance 1, 2. To accommodate this, many active pharmaceutical

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ingredients (APIs) are formulated as oral modified-release (MR) formulations. Currently, the majority

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of candidate drugs permeate well through the intestinal membranes but are poorly water soluble (class

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II according to the biopharmaceutical classification system; BCS) 3-5. Several of these APIs have been

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formulated by various approaches into innovative pharmaceutical products and are consequently

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protected by intellectual proprietary rights (IPR). Drugs designed to be MR orally administered

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products need biopharmaceutical properties that will result in sufficiently high absorption in both the

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small and large intestines to cause the desired therapeutic result 6, 7. In general, more innovative

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formulation work and longer periods of development are required to turn BCS II drugs into successful

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commercial MR pharmaceutical products with adequate bioavailability 4, 8, 9. In addition, a better

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understanding of the dynamic and variable physiology of the gastrointestinal (GI) tract, especially the

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permeability to APIs at different intestinal sites, is needed to define the rate-controlling GI absorption

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processes and to predict the performance of novel MR oral formulations 10. Drug permeation is

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currently commonly evaluated in in vitro screening models (e.g. Caco-2 cell studies and artificial

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membrane assays such as PAMPA), which give no information regarding regional intestinal

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absorption. In order to predict the regional intestinal absorption of candidate drugs, site-specific

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permeability measurements performed in pre-clinical animal models are needed. Furthermore, at an

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industrial level, there is a need to switch from an empirical development approach to more rational

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strategies 7. In particular, it is important to explore the in vivo assessment of regional intestinal

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permeability in humans compared to common in vitro and in vivo pre-clinical models, which will

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subsequently form a basis for robust, reliable translation approaches and predictive models 6, 11-14.

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Identifying the rate-limiting step for absorption along the intestine may also be crucial from an IPR

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perspective as it may justify the patent claims for a specialized, patent-protected release mechanism

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that governs the absorption rate of an API.

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Two commonly used rat models for evaluating the regional intestinal permeability to drugs, nutrients,

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disease markers, and formulations are the Ussing chamber system and the in situ single-pass intestinal

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perfusion (SPIP) model. The Ussing chamber method has been used to assess regional differences in

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transport rates in several species 15-20. This in vitro transport model has been described in detail

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elsewhere but, briefly, it is based on a vessel with two chambers filled with media (e.g. buffer with and

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without the API), which are separated by a membrane (e.g. excised intestine or a synthetic membrane)

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investigation is determined over a specified time and an apparent intestinal permeability (Papp) value is

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calculated for the API from its appearance rate in the receiver chamber.

. The absorptive and/or efflux transport from the donor chamber across the barrier under

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The in situ SPIP method is more complex and physiologically relevant than the Ussing chamber

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method, because intact blood flow, local endocrine conditions and membrane integrity are maintained.

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In addition, blood sampling enables accurate and more sensitive pharmacokinetic assessment of the

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absorption rate, permeability, and first-pass extraction 21, 22. In this method, a segment of the rat

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intestine is perfused (single-pass) with a drug solution and the effective permeability (Peff) is

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determined based on the difference between the drug concentration entering and that leaving the

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segment, by applying the parallel tube model 22, 23. It is common to add established absorptive marker

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compounds, such as metoprolol, to account for intra- and interlaboratory variability 24-26. These two

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methods have previously been compared with respect to human Peff, but only for jejunal segments and

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jejunal tissue specimens, which means that further investigations are warranted with regard to the

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other intestinal regions 27.

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Four model drugs known to be transported across the intestinal membranes by passive lipoidal

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diffusion were investigated in this study: atenolol, enalaprilat, ketoprofen, and metoprolol; their

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physicochemical properties are described in Table 1. Atenolol and metoprolol are both basic drugs

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(pKa 9.6 and 9.7, respectively) and β1-receptor antagonists that are used to treat e.g. hypertension 28, 29.

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Metoprolol is considered by some to be a marker for the cut-off point between BCS high- and low-

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permeability drugs 30. This has, however, been challenged, as the permeability may change along the



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small intestine for some drugs, even if metoprolol is not affected specifically 3, 31, 32. Enalaprilat, the

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active metabolite of the ACE inhibitor enalapril, which is used for the treatment of hypertension, is an

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ampholyte (basic pKa 3.17 and acidic pKa 7.84) 29, 33. Ketoprofen is a nonsteriodal anti-inflammatory

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drug (NSAID) with acidic properties (pKa 3.89) 29. Atenolol and enalaprilat are classified as BCS

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class III drugs while ketoprofen and metoprolol are classified as BCS class II and BCS class I drugs,

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respectively 3, 34. Ketoprofen is, however, readily soluble in the intestinal lumen as it is completely

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charged at all normal intestinal pH values.

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The primary objective of this study was to determine the permeability of the jejunum, ileum and colon

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to a set of model drugs, using two intestinal transport methods, in the same laboratory, and to compare

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the results with corresponding human regional intestinal permeability data. A unique aspect of this

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study was that all the animals used, in both the Ussing chamber and the SPIP model, were of the same

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genetic rat strain (male Wistar Han rats, strain 273), were from the same breeder, and had the same

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diet. Furthermore all experiments were conducted in the same laboratory. The secondary objective was

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to investigate the influence of different luminal (donor side) buffers with different pHs on the rate of

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intestinal transport in bothmethods.

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Methods

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Drugs and other chemicals

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Atenolol and metoprolol tartrate were provided by AstraZeneca AB (Mölndal, Sweden). Enalaprilat,

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ketoprofen, phenol red, sodium pyruvate, sodium fumarate dibasic, L-glutamic acid, D-glucose,

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sodium bicarbonate (NaHCO3), magnesium sulfate heptahydrate (MgSO4 • 7H2O), potassium chloride

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(KCl), and calcium chloride (CaCl2) were purchased from Sigma-Aldrich (St. Louis, MO, US).

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Sodium phosphate dibasic dihydrate (Na2HPO4 • 2H2O), potassium dihydrogen phosphate (KH2PO4)

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and sodium chloride (NaCl) were purchased from Merck KGaA (Darmstadt, Germany). Water used in

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the buffer was purified in an ELGA Maxima Prima USF system (Elga Labwater, Lane End,

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Buckinghamshire, UK) in the Ussing experiments and a Millipore Milli-Q Advantage A10 system

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(Millipore Corporation, Billerica, MA) in the SPIP experiments.

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Buffer and perfusate preparations

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Ussing chamber experiments

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Buffers with pHs of 6.5 (buffer A) and 7.4 (buffer B) were prepared for use as donor chamber media.

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Buffer B was used as receiver chamber medium in all experiments. Buffer A consisted of 6.3 g NaCl,

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0.35 g KCl, 0.30 g MgSO4 • 7H2O, 0.32 g Na2HPO4 • 2H2O, 0.082 g KH2PO4, 1.3 g NaHCO3, 0.86 g

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sodium fumarate dibasic, 0.83 g L-glutamic acid, 0.54 g sodium pyruvate, 2.1 g D-glucose and 0.69

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mg CaCl2 per liter. Buffer B consisted of 6.3 g NaCl, 0.35 g KCl, 0.30 g MgSO4 • 7H2O, 0.19 g

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Na2HPO4 • 2H2O, 0.25 g KH2PO4, 1.3 g NaHCO3, 0.86 g sodium fumarate dibasic, 0.83 g L-glutamic

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acid, 0.54 g sodium pyruvate, 2.1 g D-glucose, 5.25 ml 2M HCl and 0.69 mg CaCl2 per liter.

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Atenolol, metoprolol, ketoprofen and enalaprilat were added from DMSO stock solutions to the donor

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chamber medium (mucosal side) to achieve a final concentration of 25 µM for each drug. The final

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concentration of DMSO never exceeded 0.5% (w/w), which has been validated previously with respect

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to intestinal tissue integrity 35.

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Single-pass intestinal perfusion (SPIP)



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Three buffers, I, II, and III, were prepared for the rat SPIP experiments. Buffer I was of low buffer

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strength (6.7 mM) at pH 6.5, buffer II was of high buffer strength (67 mM) at pH 6.5, and buffer III

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was of high buffer strength (100 mM) at pH 7.4. Buffer I consisted of 0.59 g KH2PO4, 0.41 g

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Na2HPO4 • H2O, and 8 g NaCl per liter. Buffer II consisted of 6.35 g KH2PO4, 3.56 g Na2HPO4 • H2O,

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and 5.00 g NaCl per liter. Buffer III consisted of 1.81 g KH2PO4, 9.50 g Na2HPO4 • H2O, and 4 g NaCl

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per liter. Prior to the single-pass perfusion of each segment, atenolol, metoprolol, ketoprofen, or

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enalaprilat were added from DMSO stocks to each buffer to achieve a final perfusate concentration of

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100 µM. Phenol red was added as a fluid flux marker, to a final perfusate concentration of 25 µM. The

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final DMSO concentration in the buffer never exceeded 0.5% (w/w), which has been validated

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previously with respect to intestinal tissue integrity 35.

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Animals and study design

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This study was approved by the local ethics committee for animal research (no: 66-2014 for SPIP and

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no: 48-2013 for Ussing chamber) in Gothenburg, Sweden. Male Wistar Han rats from Charles River

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(strain 273), aged 8-10 weeks, were used in all experiments. The animals arrived at the animal lab

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facility at least one week prior to the experiment and were allowed water and food ad libitum prior to

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the experiments. They were kept in a 12-hour light/dark cycle, at 21°C and 50% relative humidity.

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Animals of the same genetic strain were used in both intestinal transport models.

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Ussing method

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Male Wistar Han rats weighing 250-350g were anesthetized using Attane isoflurane (Piramal

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Healthcare, Hallbergmoos, Germany). The abdominal cavity was opened and specimens from the

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jejunum, ileum and colon were identified and carefully removed. Any debris was removed by rinsing

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with ice-cold Krebs-Ringer bicarbonate buffer (KRB). The intestinal specimens were placed in a

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beaker containing ice-cold KRB, which was bubbled continuously with carbogen gas (95% O2/5%

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CO2). The specimens used for the jejunum were excised 10-30 cm distal to the ligament of Treitz, the


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ileal specimens were excised 5-25 cm proximal to the ileocecal junction, and the colonic specimens

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were excised 2-7 cm proximal to the rectum. The specimens were cut into 2 cm pieces and placed in a

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preparation basin containing cold KRB which was bubbled continuously with carbogen gas. Before

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mounting the specimens in the Ussing chamber, they were cut open along the mesenteric border. The

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exposed tissue area for drug permeation in the Ussing chamber setup was 1.14 cm2 36. Specimens

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containing Peyer’s patches were identified by visual inspection and avoided. 25°C KRB was added

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simultaneously to both the mucosal and serosal sides and the intestinal specimen was allowed to

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equilibrate for 20 minutes while being warmed to 37°C by heating jackets. The viability of the tissue

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during the experiment was monitored by measuring the potential difference (PD) and electrical

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resistance (R) across the specimen. Small intestinal specimens with initial PD and R values below 4

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mV and 30 Ohm • cm2, respectively, were replaced with another specimen prior to the start of the

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experiment. The corresponding PD and R cut-off values for large intestinal specimens were 6 mV and

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70 Ohm • cm2, respectively. At time zero, the KRB in the chambers was replaced with 37°C KRB

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containing the drug mix on the mucosal (donor) side and 37°C KRB on the serosal (receiver) side.

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Throughout the course of the experiment there was stirring present in both the mucosal and the serosal

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chambers, by form of rotors operating at approximately 300 rpm. 200 µl samples were taken from the

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mucosal side at 0 minutes and 150 minutes, and from the serosal side at 0, 30, 60, 90, 120 and 150

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minutes for drug quantification. Equal volumes of fresh KRB at 37°C was added to compensate for the

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sample volume taken. Samples were immediately frozen and stored at -20 °C awaiting analysis.

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Male Wistar Han rats weighing approximately 270-370g were anesthetized using Attane isoflurane

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(Piramal Healthcare, Hallbergmoos, Germany) and placed on a heating table, to maintain their body

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temperature at 37°C. The abdomen was opened by a 3-5 cm longitudinal incision along the midline.

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An intestinal segment of 6-12 cm was located and cannulated with polypropylene tubing (O.D. 4 mm,

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I.D. 2mm). The jejunal segments were located approximately 10 cm distal to the ligament of Treitz,

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the ileal segments approximately 10 cm proximal to the ileocecal junction, and the colonic segments

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approximately 1 cm distal to the cecum. Each segment was carefully rinsed with 20-30 ml 25°C saline



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solution for 1-2 minutes to remove nonadherent mucus and debris, until a clear outlet perfusate was

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attained. The intestinal segments were placed into the abdominal cavity along with a section of ~10 cm

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of the tubing to ensure that the perfusate was at body temperature before entering the cannulated

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intestinal segment. The animals were sutured, to minimize heat and fluid loss, leaving the inlet and

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outlet tubing accessible from the outside. A 50 ml syringe (Becton Dickinson, Franklin Lakes, NJ)

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containing the perfusate drug mix was attached to the inlet tubing and mounted in a syringe pump

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(Perfusor® Space, Braun Melsungen AG, Germany). At time 0 a 5 ml bolus dose of 25 °C perfusate

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was infused (10 ml/min) to fill the entire segment. The subsequent perfusion rate was set at 0.2

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ml/min. The experiment lasted for 105 minutes, with perfusate outlet samples quantitatively collected

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at 45, 60, 75, 90 and 105 minutes. The samples were immediately frozen and stored at -20 °C awaiting

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analysis.

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Analytical method

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Sample preparation

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Ussing experiments

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The samples were thawed at 25°C while being shaken at 500 rpm. When completely thawed, the

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Ussing donor-side samples were diluted 10-fold with KBR. 30 µl of the diluted donor-side samples

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were then transferred to a 96-well plate and mixed with 420 µl KBR and 150 µl internal standard

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solution [acetonitrile (ACN):H2O 60:40, 200 nM warfarin]. 60 µl of the receiver-side samples were

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transferred to a 96-well plate and mixed with 20 µl internal standard solution. The plate was shaken

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for 20 minutes at 1000 rpm, and then centrifuged at 3500 rpm at 4°C for 20 min prior to analysis on

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UPLC-MS.

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Perfusate samples were thawed at 25°C, while being shaken at 500 rpm. When the samples were

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completely thawed, 30 µl of sample was taken to a 96-well plate and mixed with 420 µl KBR and 150

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µl internal standard solution (ACN:H2O 60:40, 200 nM warfarin). The 96-well plate was shaken for 20


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minutes at 1000 rpm, and then centrifuged at 3500 rpm at 4°C for 20 min prior to analysis with the

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UPLC-MS.

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UPLC-MS

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Samples from the Ussing chamber and SPIP experiments were analyzed on a UPLC I-Class binary

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solvent manager, with a Waters Acquity flow through a needle sample manager (Waters Corporation,

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Milford, MA). Chromatographic separation was achieved using a Waters BEH-C18 column (I.D. 2.1

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mm, length: 50 mm, particle size 1.7 µm) and a pre-column (VanGuard™ HSS T3 2.1 x 5 mm id,

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particle size 1.8µm, Waters Corporation, Milford, MA) kept at 60 ˚C. The mobile phase consisted of

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(A) 95% water, 5% ACN, 0.1% formic acid (FA) and (B) ACN, 0.1% FA. A gradient was run as

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follows: initially 5% B, 5-95% B for 1.5 min, 95% B for 0.5 min, and 95-5% B for 0.2 min. The total

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run time was 2.2 min, the flow rate was 600 µL/min, and the injection volume was 5 µL for all

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samples. All compounds were quantified simultaneously in the same chromatographic run, on a QDa

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Mass Detector (Waters Corporation, Milford, MA) in selected ion recording (SIR) positive

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electrospray ionization mode. The cone voltage was 10 kV, the source block temperature was 120°C,

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and the source probe temperature was 600°C. The ratio of analyte to internal standard as a function of

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the concentration of the analyte was calibrated using a linear curve fit model, with a weighing of 1/x2

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for all compounds. The calibration curve was linear in the range 10-300 nM for all compounds. The

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limit of quantification (LOQ) for all compounds was 10 nM, with a relative standard deviation of

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