Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal

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Preclinical effect of absorption modifying excipients on rat intestinal transport of five model compounds and the intestinal barrier marker Cr-EDTA 51

David Dahlgren, Carl Roos, Anders Lundqvist, Peter Langguth, Christer Tannergren, Markus Sjöblom, E. Sjögren, and H. Lennernas Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00353 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Molecular Pharmaceutics Dahlgren, D

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Absorption Modifiers

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Preclinical effect of absorption modifying

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excipients on rat intestinal transport of model

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compounds and the mucosal barrier marker

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Cr-EDTA

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Dahlgren, David1; Roos, Carl1; Lundqvist, Anders2; Tannergren, Christer2; Langguth, Peter3; Sjöblom,

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Markus4; Sjögren, Erik1; Lennernäs, Hans1*

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1

Department of Pharmacy, Uppsala University, Uppsala, Sweden

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AstraZeneca R&D, Gothenburg, Sweden

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3

School of Pharmacy, Johannes Gutenberg-University, Mainz, Germany

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Department of Neuroscience, Division of Physiology, Uppsala University, Uppsala, Sweden

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*Address correspondence to:

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Hans Lennernäs, PhD

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Professor in Biopharmaceutics

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Department of Pharmacy

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Uppsala University

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Box 580

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SE-751 23 Uppsala, Sweden

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Email: [email protected]

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Phone: +46 – 18 471 4317

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Fax: +46 – 18 471 4223

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ACS Paragon Plus Environment


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Abstract

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There is a renewed interest from the pharmaceutical field to develop oral formulations of compounds

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such as peptides, oligonucleotides, and polar drugs. However, these often suffer from insufficient

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absorption across the intestinal mucosal barrier. One approach to circumvent this problem is the use of

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absorption modifying excipient(s) (AME). This study determined the absorption enhancing effect of

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four AMEs (sodium dodecyl sulfate, caprate, chitosan, N-acetylcysteine) on five model compounds in

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a rat jejunal perfusion model. The aim was to correlate the model compound absorption to the blood-

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to-lumen clearance of the mucosal marker for barrier integrity, 51Cr-EDTA. Sodium dodecyl sulfate

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and chitosan increased the absorption of the low permeation compounds but had no effect on the high

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permeation compound, ketoprofen. Caprate and N-acetylcysteine did not affect the absorption of any

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of the model compounds. The increase in absorption of the model compounds was highly correlated to

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an increased blood-to-lumen clearance of

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could be used as a general, sensitive, and validated marker molecule for absorption enhancement when

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developing novel formulations.

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Cr-EDTA, independent of the AME. Thus,

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Cr-EDTA

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………

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Keywords: absorption modifiers, permeation enhancers, intestinal perfusion, bioequivalence,

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pharmaceutical development.

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Abbreviations: AME – absorption modifying excipient, CL – clearance, IEC - intestinal epithelial

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cells, NAC - N-acetylcysteine, SDS – sodium dodecyl sulfate,


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Introduction

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There is a renewed interest from the pharmaceutical field to develop oral formulations of drugs with

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low intestinal membrane permeability, such as peptides, oligonucleotides, and polar drugs (polar

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surface area >100Å2) with a molecular mass between 250-1200 g/mol.1 For drug molecules to be

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absorbed following oral administration, they have to cross the single layer of columnar intestinal

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epithelial cells (IECs). These IECs form a semi-permeable mucosal barrier that is under strict

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physiological regulation by inflammatory factors and luminal content.2 The IECs, together with a thin

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mucus layer, form a barrier that restricts translocation of large, luminal constituents, such as proteins

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and bacteria, while allowing a rapid and high uptake of nutrients, water, electrolytes, and some

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xenobiotics (including drugs and antioxidants).3 Oral formulations with absorption modifying

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excipients(s) (AME) affect the IEC barrier integrity to increase transport of dissolved drug from the

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intestinal lumen to the blood. AMEs can be used alone or in combination to increase drug uptake in at

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least four ways. The AME can: i) adhere to the IEC and/or mucus to increase drug concentration at the

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mucosal membrane; ii) degrade the intestinal mucus to increase contact between the IEC and drug; iii)

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increase drug permeation by altering the integrity of the IEC lipid bilayer; and iv) increase drug

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permeation by altering the paracellular tight junction complexes between IECs.4,5 The use of AMEs

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has not reached clinical breakthrough, but there are several formulations in various stages of clinical

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development.4

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However, there are several unresolved issues related to the development and use of AME in oral

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pharmaceutical products. The safety of AMEs are a major concern for patients, pharmaceutical

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companies, and regulatory agencies, and consequently most novel formulation strategies are based on

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AMEs shown to be safe in humans.1 For instance, luminal pH, osmolarity, bile acids, nutrients, and

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microbiota, are all shown to induce various physiological responses affecting the function of the

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epithelial barrier.6-8 An alteration of the mucosal barrier function facilitating epithelial permeation of

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drugs might also increase translocation of harmful entities, such as toxins, bacteria, and viruses.

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Further, many pharmaceutical excipients with a suspected absorption enhancing effect are also used in



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oral enabling formulations, e.g., surfactants as a wetting/solubilizing agent.9 Whether these excipients

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affect the bioequivalence (BE) of drug products – or drug product-drug interactions – is another

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important issue.10

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To enable early and more reliable assessment of AMEs, their effects in pre-clinical models need to be

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better understood. The promising effects in in vitro (e.g. Caco-2 and/or excised intestinal segments in

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Ussing models) as well as in animal models (e.g. rat intestinal perfusion) are notoriously difficult to

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replicate in humans. Often they do not take into account the designed formulation features, such as

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release, effects of local solubilization, and co-exposure of AME(s) and drug to the mucus and/or

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epithelial barrier.1,11

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The main objective of this study was to improve the mechanistic understanding of four commonly

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used AMEs on the small intestinal absorption of model compounds (atenolol, acyclovir, enalaprilat,

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ketoprofen, and phenol red). The rat was used as model animal as it is shown to be most accurate in

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predicting human intestinal absorption.12-14 The model compounds were single-pass perfused in a

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segment of the rat jejunum, with an intact blood supply, as a cocktail solution in two consecutive 75

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min periods. The first period was the control (no AME) and the second period contained one of the

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following AME: sodium dodecyl sulfate (SDS), sodium caprate, chitosan, N-acetylcysteine (NAC),

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and chitosan+NAC (ChNAC). The AMEs were tested at two concentrations. A second important

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objective was to study the effect of the AME on the blood-to-lumen clearance (CL) of a common

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biologically inert marker for mucosal integrity, 51Cr-EDTA, which is transported across the rat IEC by

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the paracellular route when intravenously administered.15 An increased intestinal CL of 51Cr-EDTA is

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highly correlated with an altered IEC barrier function in both animal and human experiments.16,17

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Methods

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Active pharmaceutical ingredients, pharmaceutical excipients and other chemicals

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Six model compounds were selected: acyclovir, atenolol, enalaprilat, ketoprofen, phenol red, and

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metoprolol. All metoprolol plasma samples were below the lower limit of quantification in the

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analytical assay and therefore no results for metoprolol were obtained. The model compounds belong

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to classes II and III according to the biopharmaceutics classification system (BCS).18 BCS class and

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physicochemical properties are summarized in Table 1. The four AMEs were: SDS (an anionic

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surfactant), sodium caprate (a fatty acid), chitosan (a polysaccharide), and NAC (a mucolytic

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agent)(Table 2). Atenolol and metoprolol tartrate were provided by AstraZeneca AB (Mölndal,

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Sweden). Acyclovir, enalaprilat, ketoprofen, phenol red, sodium caprate, sodium dodecyl sulfate

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(SDS), N-acetyl-L-cysteine (NAC), bovine albumin (A2153), and Inactin were purchased from Sigma-

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Aldrich (St. Louis, MO, US). Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O), potassium

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dihydrogen phosphate (KH2PO4), sodium hydroxide (NaOH), and sodium chloride (NaCl) were

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purchased

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ethylenediaminetetraacetate (51Cr-EDTA) was purchased from PerkinElmer Life Sciences (Boston,

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MA). Chitosan hydrochloride (molecular mass 40-300 kDa, degree of acetylation 8.8%) was

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purchased from Kraeber & Co GmbH (Ellerbek, Germany).

from

Merck

KGaA

(Darmstadt,

Germany).

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Chromium-labeled

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Study solutions

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The intravenous (iv) cassette solution (312.5 µM) was prepared by dissolving all of the model

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compounds in 100 mL saline and adjusting pH to 6.5 (0.1 M NaOH solution).

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Ten perfusion solutions were prepared, all containing 50 µM of all model compounds. One contained

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no AME (control) and nine test solutions contained AME. Eight test solutions contained one of the

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following AME (w/v): SDS 0.1% (3.5 mM), SDS 0.5% (17.3 mM), caprate 0.04% (2.1 mM), caprate

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0.2% (10.3 mM), chitosan 0.1%, chitosan 0.5%, NAC 0.1% (6.1 mM), and NAC 0.5% (30.6 mM).

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One test solution contained NAC (30.6 mM) and chitosan (ChNAC), at 0.5% w/v each. The highest



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concentration of AME was always below a clinically acceptable dose to swallow (1 g), according to

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the BCS (dissolved in 250 mL water). The two caprate concentrations (2.1 and 10.3 mM) were both

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below the reported critical micelle concentration (CMC) of caprate in saline (25 mM).19 The highest

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caprate concentration was chosen to be below its solubility limit at pH 6.5.20 For SDS, one solution

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(3.5 mM) was close to the reported CMC (between 2-6 mM depending on salt concentration) and one

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above (17.3 mM).21,22

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Each perfusion solution (100 mL) was prepared on the day of experiment by mixing 16 mL of a 312.5

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µM stock solution containing a cocktail of all model compounds and one of the AME (pre-dissolved in

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water at a concentration of 1g/100 mL), yielding the wanted concentrations mentioned above. Stock

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solutions (67 mM) of Na2HPO4 and KH2PO4 were added at volumes giving a final pH of 6.5 at a

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phosphate concentration of 5-8 mM. The solutions containing chitosan and NAC required addition of

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1 M NaOH to obtain a final pH of 6.5. Deionized water was added to a final volume of 100 mL and

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isotonicity (290 mOsm) was adjusted using NaCl. No incompatibility or degradation of the study

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compounds in solution (pH 6.5, 37 °C) was observed during 4 h.

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Animals and study design

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The study was approved by the local ethics committee for animal research (no: C64/16) in Uppsala,

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Sweden. Male Wistar Han rats (strain 273) from Charles River (Germany) weight 300-440 g were

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used. The animals arrived at the animal lab facility at least one week before the experiment and were

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allowed water and food ad libitum during this period. Housing conditions were 12:12 h light-dark

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cycle and 21-22 °C. On the study day, the rats were anesthetized using an intraperitoneal injection of a

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10% w/v Inactin solution (160 mg/kg) then laid down on a pre-warmed heating pad. Body temperature

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was maintained at 37.5 ± 0.5°C by means of an IR-lamp and a temperature regulator controlling the

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heating pad. A cannula was inserted into the trachea to ensure a free airway. The femoral vein and

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artery were thereafter cannulated with PE-50 polyethylene catheters (Becton, Dickinson, Franklin

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Lakes, NJ). For continuous recordings of the systemic arterial blood pressure (BP), the arterial catheter


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containing 20 IU/ml heparin isotonic saline was connected to a transducer operating a PowerLab

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system (AD Instruments, Hastings, UK), in order validate the condition of the animal.

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At the iv administration, 1 mL of the 312.5 µM stock solution containing the model compounds was

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bolus administered through the femoral vein (dose: acyclovir 70 µg, atenolol 83 µg, enalaprilat 109

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µg, ketoprofen 80 µg, metoprolol 83 µg, and phenol red 111 µg) followed by a 0.5 mL saline flush to

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ensure complete administration of the doses.

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The single- pass perfusion experiment in rat was performed as follows. The abdomen was opened with

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a 3-5 cm longitudinal incision along the midline. A jejunal segment of 9-14 cm, approximately 5 cm

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distal to the ligament of Treitz, was located and cannulated with a silicone tube (Silastic, 1.0 mm ID

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Dow Corning, Midland, MI). The chosen segment was placed on the abdomen of the rat to avoid

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intestinal folding and to visually check intestinal motor activity during the experiment. The intestine

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was covered with Saran wrap to maintain body temperature and to avoid fluid loss. The bile duct was

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cannulated with a PE-10 polyethylene tube close to its entrance into the duodenum (2-3 mm) to avoid

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pancreaticobiliary secretion into the duodenum. After completion of surgery,

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administered iv as a bolus of 75 µCi (0.4 mL) followed by a continuous infusion at a rate of 50 µCi

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per hour (1 ml/h) for the duration of the experiment. Each intestinal segment was single-passed

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perfused at 0.2 ml/min (peristaltic pump, Gilson Minipuls 3, Le Bel, France) with 37° C, phosphate

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buffered saline (6 mM, pH 6.5), during the first 30 minutes following surgery. This was to allow

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cardiovascular, respiratory, and intestinal functions to stabilize, and to achieve stable

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activity in blood plasma. The length of the intestinal segment was measured after the jejunal

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cannulation and the weight of the segment was measured after the experiment.

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Cr-EDTA was

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Cr-EDTA

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Following the equilibration, each experiment was divided into two parts: i) in the first part, the

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segment was perfused with the control solution (containing model compounds but no AME) for 75

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min; ii) this was directly followed by a 75 min perfusion of a solution containing one of the nine AME

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containing test solutions (containing model compounds and AME). In one experiment the same



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control solution was perfused during both 75 min periods to evaluate possible time-dependent effects

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on compound flux. The experimental design enabled each rat to serve as its own control. Each

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experiment was started with a rapid filling of the whole segment with the solution being perfused. The

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intestinal segment and perfusion solutions were kept at 37 °C and all outgoing perfusate was collected

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quantitatively at 15 min intervals, giving five samples during each of the two consecutive 75 min

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perfusion periods, for a total of 10 samples.

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Blood samples of

Preclinical Effect of Absorption Modifying Excipients on Rat Intestinal

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