Regional Intestinal Permeability in Dogs: Biopharmaceutical Aspects

Aug 8, 2016 - Based on plasma data from two separate study occasions for each dog, regional Peff values were calculated using a validated intestinal ...
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Regional intestinal permeability in dogs: biopharmaceutical aspects for development of oral modified-release dosage forms David Dahlgren, Carl Roos, Pernilla Johansson, Anders Lundqvist, Christer Tannergren, Bertil Abrahamsson, Erik Sjögren, and Hans Lennernas Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00515 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 10, 2016

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Regional

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biopharmaceutical aspects for development of

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oral modified-release dosage forms

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Dahlgren, David1; Roos, Carl1; Johansson, Pernilla2; Lundqvist, Anders2; Tannergren, Christer2;

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Abrahamsson, Bertil2; Sjögren, Erik1; Lennernäs, Hans1*

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Department of Pharmacy, Uppsala University, Uppsala, Sweden

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AstraZeneca R&D, Gothenburg, 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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dogs:

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Abstract

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The development of oral modified-release (MR) dosage forms requires an active pharmaceutical

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ingredient (API) with a sufficiently high absorption rate in both the small and large intestine. Dogs are

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commonly used in preclinical evaluation of regional intestinal absorption and in the development of

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novel MR dosage forms. This study determined regional intestinal effective permeability (Peff) in

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dogs with the aim to improve regional Peff prediction in humans.. Four model drugs—atenolol,

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enalaprilat, metoprolol, and ketoprofen—were intravenously and regionally dosed twice as a solution

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into the proximal small intestine (P-SI) and large intestine (LI) of three dogs with intestinal stomas.

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Based on plasma data from two separate study occasions for each dog, regional Peff values were

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calculated using a validated intestinal deconvolution method. The determined mean Peff values were

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0.62, 0.14, 1.06, and 3.66 × 10-4 cm/s in the P-SI, and 0.13, 0.02, 1.03, and 2.20 × 10-4 cm/s in the LI,

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for atenolol, enalaprilat, metoprolol, and ketoprofen, respectively. The determined P-SI Peff values in

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dog were highly correlated (R2=0.98) to the historically directly determined human jejunal Peff after a

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single-pass perfusion. The determined dog P-SI Peff values were also successfully implemented in GI-

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Sim software to predict the risk for overestimation of LI absorption of low permeability drugs. We

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conclude that the dog intestinal stoma model is a useful preclinical tool for determination of regional

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intestinal permeability. Still, further studies are recommended to evaluate additional APIs, sources of

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variability, and formulation types, for more accurate determination of the dog model in the drug

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development process.

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………

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Keywords: dog intestinal permeability, regional intestinal drug absorption, bioavailability, effective

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permeability, pharmacokinetics, intestinal perfusion, pharmaceutical development.

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Abbreviations: fabs – fraction oral dose absorbed, Papp – apparent permeability, Peff – effective

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permeability, P-SI – proximal small intestine, LI – Large intestine

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Introduction

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Oral drug treatment is the most common route of administration for pharmaceutical products. Based

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on units sold and total revenues, the top 50 prescription drugs in the US that are oral products account

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for 58% of all units sold and 44% of total revenues (IMS Health 2013). The fraction of the oral dose

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absorbed (fabs) across the apical membrane of the intestine is primarily governed by the drug's

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solubility, its dissolution rate within the gastrointestinal (GI) lumen, and its effective intestinal

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permeability (Peff). These biopharmaceutical parameters are the cornerstones of the Biopharmaceutics

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Classification System (BCS).1 The transport of drugs across the intestinal barrier at various sites

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occurs by a coexistence of passive and carrier-mediated (CM) trans-epithelial processes.2 The

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intestinal permeability of BCS class I and II drugs is sufficiently high in both the small and large

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intestine to not be rate-limiting in drug absorption.3 Still, regional permeability differences for BCS

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class I and II drugs are important to consider when adjusting the release rates to optimize the

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pharmacodynamic properties of any modified-release (MR) product. Given that the solubility and

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dissolution are sufficient, development of a MR dosage form is feasible. This is illustrated by the BCS

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class I and II drugs metoprolol and nifedipine that have absorption times as MR products up to 20 h

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post oral dosing.4,5 However, there is a gap in the understanding as to which molecular characteristics

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that determine regional intestinal absorption rate, regardless of the compound's BCS classification. For

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instance, it has been reported that the development of MR dosage forms was considered very difficult

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(59%) or challenging (6%) for novel drugs, due to low absorption in the distal intestinal tract.6 The

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IMI project (OrBiTo) states that improved understanding of regional intestinal absorption for drugs

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with different physicochemical properties is a primary research objective.7

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To further improve the accuracy of the development process, there is a need to increase the

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understanding of the preclinical models used to characterize regional intestinal absorption rate and in

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vivo formulation performance. The dog absorption model, together with predictive in silico software,

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are commonly used for this purpose in industrial settings.8 The size of the dog (around 10-40 kg)

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enables administration of clinical formulations, and dogs have GI anatomy, biochemistry, and

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physiology comparable to that of humans, although diet, age and breed can affect the absorption

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assessment in dogs.9,10 The similarities and differences need to be better understood, especially

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regional permeability, for accurate trans-species extrapolation of intestinal permeability data from

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different intestinal sites in the dog. These considerations are fundamental for accurate trans-species

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extrapolation of intestinal permeability data from different sites in the dog. To date, absorption rate in

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dog is mainly based on PK data following oral and intraluminal administrations, while direct in vivo

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determination of intestinal permeability is limited.11-13 Hence, there is a need to improve the

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understanding of the factors determining regional Peff of drugs in dogs, as well as the experimental

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design of the dog preclinical absorption model.14 Understanding the dog absorption model and

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subsequent implementation of dog Peff data into in silico tools is crucial for more accurate use of the

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model. This is especially important as several of these theoretical in silico models are not fully

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validated regarding the contribution of the large intestine (LI) to the overall absorption and their

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potential for MR product development.15,16

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The main objective of this study was to assess the usefulness of dog transabdominal stomas in drug

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permeability studies. The Peff was measured in the proximal small intestine (P-SI) and LI for four

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physicochemically diverse model drugs (atenolol, enalaprilat, metoprolol, and ketoprofen). These

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model drugs are classified as BCS class III, III, I, and II, respectively, and are transported mainly by

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passive lipoidal and/or paracellular diffusion in all intestinal regions. The regional Peff values were also

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evaluated regarding their predictive value for simulation of human intestinal absorption by a

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mechanistic absorption in silico model. The predictability of the dog Peff was evaluated by simple

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molecular descriptors. Finally, the Peff values were compared to directly determined human jejunal in

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vivo Peff (single-pass perfusion) and regional in vivo Peff with a novel and validated deconvolution

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method.17,18

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Methods

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Chemicals

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

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and ketoprofen were purchased from Sigma-Aldrich (St. Louis, MO, US). Sodium phosphate dibasic

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dihydrate (Na2HPO4·2H2O) and sodium chloride (NaCl) were purchased from Merck KGaA

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(Darmstadt, Germany). Sterile saline sodium chloride (0.9%) used for injection and dilution was

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purchased from B. Braun Melsungen (Melsungen, Germany). Water used in the buffer was purified

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using a Milli-Q water purification system (Millipore Corporation, Billerica, MA). Rifen vet®,

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(ketoprofen) 100 mg/mL solution for injection was purchased from Salfarm Scandinavia AB

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(Helsingborg, Sweden). Seloken® (metoprolol tartrate), 1 mg/mL solution for injection was purchased

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from AstraZeneca AB (Södertälje, Sweden).

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Model drugs

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Some physicochemical properties of the four model drugs are summarized in Table 1. The doses used

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in this study were chosen based on the safety considerations in the cassette dosing.

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The four intravenous (iv) solutions of the drugs were prepared separately with a volume of 2 mL and

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were administered sequentially. The iv solutions for atenolol (0.25 mg/mL, 470 µM) and enalaprilat

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(0.25 mg/mL, 359 µM) were prepared by dissolving the drugs in saline for injection. The iv solutions

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of metoprolol (0.19 mg/mL, 468 µM - 0.25 mg/mL as metoprolol tartrate) and ketoprofen (1.25

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mg/mL, 4.92 mM) were prepared by diluting the commercially available products Seloken®

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(metoprolol tartrate 1 mg/mL) and Rifen vet® (100 mg/mL), respectively, with saline for injection.

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The isotonic (294 mOsm) drug solution for intraluminal administration in the P-SI and LI was

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prepared as a cassette dose as follows: atenolol 5 mg (1.88 mM), enalaprilat 20 mg (5.75 mM),

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metoprolol 12.5 mg (16.25 mg as tartrate salt, 4.68 mM), and ketoprofen 2.5 mg (0.98 mM). All drugs

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were dissolved in 10 mL isotonic phosphate buffer containing 118 mg Na2HPO4·2H2O (67 mM), 27

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mg NaCl (46.2 mM) and 2M HCl to pH 6.8. No incompatibility or degradation of the four study drugs

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in solution (pH 6.8, 37 °C) was observed during 4 h.

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Animals and study design

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This preclinical study was approved by the local ethics committee for animal research (no: 34-2015) in

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Göteborg, Sweden. The study included 3 male Labrador dogs (age: 2-3 years; weight: 37-40 kg)

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supplied from Jonas Falck, Norway. At 12 months of age, the dogs had two permanent nipple-valve

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stomas surgically inserted into the abdomen wall, creating direct connections to the P-SI and LI

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lumen.19 During the first study period, each dog received one iv and two intraluminal administrations

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(P-SI and LI). The intraluminal administrations were repeated at a second study period, four months

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later. There was at least one week washout between all drug administrations in each study period. The

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repeated intraluminal dosing into the same site was performed to better understand intra-dog

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variability. The dogs were fasted 20 h prior and 4 h post drug dosing. The dogs had access to water ad

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lib until the administration and from 3 h post dosing. No adverse events were observed during

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continuous monitoring by the animal staff.

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The four model drugs were administered separately and sequentially on the same day. The

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administration order and doses were as follows: metoprolol 0.39 mg, atenolol 0.5 mg, ketoprofen 2.5

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mg, and enalaprilat 0.5 mg. The drugs were administered as bolus doses within 2 min of each other

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through the same catheter in a peripheral vein in the back leg (vena saphena) to establish individual

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references. The catheter was rinsed with 2 mL saline after each of the four iv administrations. The

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administration and rinsing time for each drug was 30 s in total.

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The intraluminal administration and doses were: atenolol 5 mg, enalaprilat 20 mg, metoprolol 12.5

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mg, and ketoprofen 2.5 mg. These were administered as 10-mL cassette dose solutions with a thin,

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prefilled plastic tube directly into the P-SI or LI through the transabdominal stoma. The administration

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time was 2 min.

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Blood samples of 1 mL were taken from a peripheral vein (routinely rotated) in the front leg, back leg,

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or neck (vena cephalica, vena saphena or vena jugularis), for a total volume of 12 and 11 mL during

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the iv and the intestinal administrations, respectively. Blood was sampled immediately before the iv

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administration and over 24 h (at 5, 10, 20, 30, 40, 50, 60 min, and 2, 4, 6, 8 and 24 h). For the

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intraluminal administration, blood was sampled immediately before administration and during 6 h

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after (at 5, 10, 20, 30, 40, 50, 60 min, and 2, 4, and 6 h). The blood samples were put on ice and

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centrifuged (3000 × g, 10 min at 4 °C) within 20 min. 50 µL of the plasma was transferred to a 1-mL

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96-well plate (Thermo Scientific) and the remaining plasma was transferred to 2 mL tubes (Sarstedt)

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for storage. The plasma samples were frozen and stored at -20 °C until analysis.

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Plasma pharmacokinetic (PK) calculations

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Descriptive plasma PK parameters were calculated following the iv and intraluminal administrations

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using non-compartmental analysis in WinNonlin software version 6.3 (Certara, L.P.,St. Louis,

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Missouri). Calculations of the iv PK data were corrected for the difference in injection times for each

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of the four model drugs. These PK parameters included: area under the plasma concentration-time

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curve (AUC), maximum plasma concentration (Cmax), apparent volume of distribution at steady state

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(VSS), and plasma clearance (CL). PK parameters derived from the intraluminal doses included: AUC,

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Cmax and time to reach maximum concentration (Tmax). The AUC from 0 to 24 h (AUC0-24) and infinity

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(AUC0-∞) were calculated following the iv administration, and from 0 to 6 h (AUC0-6) following P-SI

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and LI administration. The AUC0-6, Cmax, and Tmax following the P-SI and LI administrations for each

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dog used in the statistical analysis (parametric paired t-test, p