mechanisms of intestinal drug absorption from aprepitant

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In vivo mechanisms of intestinal drug absorption from aprepitant nanoformulations Carl Roos, David Dahlgren, Staffan Berg, Jan Westergren, Bertil Abrahamsson, Christer Tannergren, E. Sjögren, and H. Lennernas Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00294 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 30, 2017

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Molecular Pharmaceutics Roos, C Rat Perfusion Aprepitant 161231 CR

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Abstract

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Over recent decades there has been an increase in the proportion of BCS class II and IV drug

4

candidates in industrial drug development. To overcome the biopharmaceutical challenges associated

5

with the less favorable properties of solubility and/or intestinal permeation of these substances, the

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development of formulations containing nanosuspensions of the drugs has been suggested. The

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intestinal absorption of aprepitant from two nanosuspensions (20 µM and 200 µM total

8

concentrations) in phosphate buffer, one nanosuspension (200 µM) in fasted-state simulated intestinal

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fluid (FaSSIF), and one solution (20 µM) in FaSSIF was investigated in the rat single-pass intestinal

10

perfusion model. The disappearance flux from the lumen (Jdisapp) was faster for formulations

11

containing a total concentration of aprepitant of 200 µM than for those containing 20 µM, but was

12

unaffected by the presence of vesicles. The flux into the systemic circulation (Japp) and, subsequently,

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the effective diffusion constant (Deff) were calculated using the plasma concentrations. Japp was, like

14

Jdisapp, faster for the formulations containing higher total concentrations of aprepitant, but was also

15

faster for those containing vesicles (ratios of 2 and 1.5). This suggests that aprepitant is retained in the

16

lumen when presented as nanoparticles in the absence of vesicles. In conclusion, increased numbers of

17

nanoparticles and the presence of vesicles increased the rate of transport and availability of aprepitant

18

in plasma. This effect can be attributed to an increased rate of mass transport through the aqueous

19

boundary layer (ABL) adjacent to the gut wall.

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ACS Paragon Plus Environment


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Introduction

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Approximately 60% of the 50 most-sold drug products in the world are taken orally by patients (IMS

23

Health 2013). It is the most attractive administration route for both healthcare systems and patients

24

because of its acceptability to patients and associated dosage compliance [1, 2]. However, sufficient

25

intestinal absorption of the active pharmaceutical ingredient (API) and an acceptable intra- and

26

interindividual variability rating are necessary for an oral drug product to be therapeutically effective.

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Highly soluble APIs associated with good intestinal membrane permeability, i.e. Class I drugs

28

according to the Biopharmaceutics Classification System (BCS), can readily be developed into both

29

immediate and modified-release oral dosage forms [3]. However, during recent decades, increased

30

biopharmaceutical challenges have been encountered as a consequence of an increasing number of

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developmental APIs with less favorable biopharmaceutical properties, e.g., poor solubility and/or poor

32

intestinal permeation [4]. These compounds are expected to be associated with wider intra- and

33

interindividual variability in overall intestinal absorption because of extensive variability in

34

gastrointestinal (GI) transit times [5]. Highly lipophilic APIs (BCS class II) typically have good

35

permeation properties but are poorly soluble and slow to dissolve, which can lead to low, variable

36

bioavailability and plasma exposure, and pronounced food-drug effects, especially at high doses [4, 6].

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Several formulation strategies for improving the solubility or dissolution rates of these drugs have

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been developed, such as micronization of the API, formation of solvates and salts, solubilization in

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cyclodextrins, and the use of solid dispersions or co-solvents [7-10]. However, these pharmaceutical

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formulation methods may not always be sufficiently robust or successful to improve the rate/extent

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limited solubility, and alternative formulation approaches are often needed. One potentially viable oral

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delivery approach uses nano-sized API particles and/or drug carriers [11]. In addition, nano-based

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formulations can be used when high mass per volume loads are needed, due to the dense solid nature

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of the nanoparticles [11]. In these formulations, the API particles are typically in the size range of a

45

few hundred nanometers, which yields significantly larger surface areas than with microsuspensions,

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and thereby consequently increases the in vivo dissolution rates, according to the Noyes-Whitney

47

equation [7, 12-15]. However, concerns have been raised as to whether the increased intestinal


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absorption and subsequent increased bioavailability can be attributed solely to an increased luminal

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dissolution rate or whether other quantitative absorption processes are involved [16, 17]. One

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proposed hypothesis, which has not been mechanistically tested under in vivo conditions, is that

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nanoparticles contribute to the effective intraluminal diffusivity of the API, thus increasing the

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monomer concentrations of the API at the apical epithelial surface. This mechanism has also been

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suggested for API monomers solubilized into small colloidal structures, e.g. vesicles of approximately

54

50-100 nm diameter [16-19]. Other potential mechanisms for the increased absorption have also been

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postulated; for example: (1) intestinal absorption of intact nanoparticles into the enterocytes; (2)

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increased deposition and retention of particles due to mucoadhesion; (3) macrophagal phagocytosis;

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(4) penetration into the villous crypts where the permeability might be higher; and/or (5) lymphatic

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uptake [20, 21]. However, there is no compelling in vivo evidence to directly support any of these

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proposed mechanisms, and there is consequently a need for mechanistic studies in complex GI models.

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Aprepitant is a neurokinin NK-1 receptor antagonist that acts in the central nervous system; it is used

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to prevent acute and delayed chemotherapy-induced nausea and vomiting (CINV)[22]. It is a BCS

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class II drug, with poor solubility in phosphate buffer (0.37 µg/ml) and a high apparent permeability

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(Papp) value in Caco-2 cells (1.7 • 10-4 cm/s) [23]. It has a molecular mass of 534 Da, with a basic pKa

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of 2.4, and an acidic pKa of 9.2, meaning that it is primarily uncharged at a jejunal pH of 6.5 [23].

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Metabolism is primarily mediated by CYP3A4, with less contribution from CYP1A2 and CYP2C9

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[24]. In humans, aprepitant is a drug with low extraction across the liver with a total clearance of 60-

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85 ml/min and a bioavailability after oral administration of 60-70%, which indicates that the first-pass

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extraction is rather low [25]. The commercially available formulation of aprepitant (Emend®; Merck

70

& Co., Inc, NJ) contains nanoparticles of the drug with a diameter below 200 nm which are coated

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onto larger cellulose beads and encapsulated [26].

72 73

The main objective of this study was to increase understanding of the luminal and epithelial processes

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that determine the increase in overall intestinal absorption and bioavailability of nano-based

75

formulations of aprepitant. The intestinal absorption of different total amounts of API, in the presence



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or absence of bile acid and phospholipid vesicles, was investigated using the single-pass intestinal

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perfusion (SPIP) rat model with simultaneous plasma sampling. The vesicles were included to mimic

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the in vivo luminal conditions, where bile acids form macromolecular structures with digested food

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products and other bile components, such as cholesterol, phospholipids, monoglycerides, and fatty

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acids. The theory is that the API could be associated to these aqueous-soluble lipid colloidal structures

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which diffuse within the intestinal lumen and across the adjacent boundary layer to the intestinal

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epithelial membrane, at which point the API may be released to subsequently permeate the apical

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enterocyte membrane.

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Methods

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Drugs and chemicals

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Ketoprofen, phenol red, acetonitrile, trifluoroacetic acid, NaCl, and NaOH were bought from Sigma-

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Aldrich (St. Louis, MO). DMSO was manufactured by Fluka and formic acid by Scharlau (Barcelona,

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Spain). Emend® capsules containing aprepitant (80 mg) were produced by Merck Sharp & Dohme Ltd

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(Hertfordshire, UK) and were bought at the local pharmacy in Gothenburg, Sweden. The 80 mg

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aprepitant capsules also contained 16 mg hydroxypropyl cellulose, 80 mg sucrose, 39 mg

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microcrystalline cellulose, and 0.5 mg sodium lauryl sulphate (US patent number 8,258,132). Sodium

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taurocholate was obtained from Chemtronica (Sweden), lecithin was obtained from Lipoid (Germany)

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and NaH2PO4 was obtained from Merck KGaA (Darmstadt, Germany). Water was purified in a

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Millipore Milli-Q Advantage A10 system (Millipore Corporation, Billerica, MA).

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Experimental design

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The study was approved (no: 66-2014) by the local ethics committee for animal research in

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Gothenburg, Sweden. The animals arrived at the animal facility at least one week prior to the

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experiment. Male Wistar Han rats (Charles River, strain 273) 8-10 weeks old, weighing approximately

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280-320 g were kept with water and food ad libitum, with a 12-hour light and dark cycle, at 21°C, and

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at 50% relative humidity. The single-pass intestinal perfusion (SPIP) rat model in jejunum was used to

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investigate aprepitant absorption from four (I-IV) different perfusate formulations using four animals

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per formulation: (I) aprepitant nanosuspension (total concentration (Ctot) = 20 µM) in phosphate

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buffer, (II) aprepitant solution (Ctot =20 µM) in fasted-state simulated intestinal fluid (FaSSIF,

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containing phospholipid vesicles), (III) aprepitant nanosuspension (Ctot =200 µM) in phosphate buffer

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and (IV) aprepitant nanosuspension (Ctot =200 µM) in FaSSIF. In formulation II, aprepitant was fully

108

dissolved in the perfusate (FaSSIF), meaning that most of it was partitioned into the colloidal

109

structures present in the medium. The colloidal structures were characterized as vesicles of

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approximately 50-100 nm in diameter, which is consistent with those used previously [27]. This

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experimental design allowed us to determine the effect of increasing the total concentration of



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aprepitant (i.e. formulation I vs III, and II vs IV), as well as the effect of adding vesicles, which could

113

theoretically increase the absorption by binding the API before traversing the aqueous boundary layer

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(ABL) (i.e. formulation I vs II, and III vs IV). Ketoprofen and phenol red were added to all perfusion

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formulations as solutions at 139 µM and 25 µM to function as transcellular permeability and fluid

116

volume markers, respectively. In addition, aprepitant was administered intravenously to a fifth group

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(V, n=3) as a nanosuspension (1 ml, 1 µmol/ml) as a control. The formulations are summarized in

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Table 1.

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Preparation of the formulations containing nano-sized aprepitant for the single-pass perfusion

120

experiments

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Two buffers were prepared at pH 6.5. The first, phosphate buffer, contained 3.44 g NaH2PO4, 6.19 g

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NaCl, and 0.34 g NaOH per 1000 ml water. The second, FaSSIF, contained 3.44 g NaH2PO4, 6.19 g

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NaCl, 0.34 g NaOH, 1.613 g sodium taurocholate, and 0.157 g lecithin per 1000 ml water. The only

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differences between the phosphate buffer and FaSSIF were the addition of sodium taurocholate and

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lecithin to FaSSIF. The buffers were prepared each morning to ensure stability throughout the course

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of the experiment.

127 128

On the day of the study, an Emend® capsule containing 80 mg aprepitant was suspended in 2.0 mL 25

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°C MilliQ water while stirring for 45 minutes. The opaque nanosuspension was aspirated from the

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beaker using a syringe equipped with a 0.7 mm needle, leaving the cellulose beads in the beaker. The

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nanosuspension was sonicated in an ultrasonication bath for 1 minute. The concentration of aprepitant

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in this stock nanosuspension was 74.8 mM.

133 134

To produce the 20 µM aprepitant nanosuspension or solution, 80.2 µL of the stock nanosuspension

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was added to the FaSSIF or phosphate buffer, to a final volume of 300 mL. To produce the 200 µM

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formulations, 802 µL of the stock suspension was added to the FaSSIF or phosphate buffer, to a final

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volume of 300 mL. The final nanoformulations in the perfusate were stable and no sedimentation

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occurred over the course of the experiment. In addition, all formulations contained 25 µM phenol red

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and 139 µM ketoprofen in solution. These were added from DMSO stock solutions: 428 µL of a 17.5


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mM phenol red stock solution and 1.87 mL of a 22.3 mM stock solution of ketoprofen. The final

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concentration of DMSO in the formulations was 0.17 %, which has previously been validated

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regarding tissue integrity [28].

143 144

The particle size distribution of the formulations was measured using a Zetasizer Nano ZS instrument

145

(Malvern Instruments, Worcestershire, UK). The total aprepitant concentration in the final

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formulations was quantified using a Waters Acquity UPLC system (Milford, MA) equipped with a

147

photo diode array (PDA) detector. The column used was an Acquity BEH C18 (2.1x50 mm, 1.7 µm

148

particle size; Waters, Milford, MA) and the column temperature during analysis was 40 °C. The

149

mobile phases were water containing 0.03% trifluoroacetic acid (A) and acetonitrile containing 0.03 %

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trifluoroacetic acid (B). A gradient was run as follows: initially 20 % B, 20-99 % B for 1 min, 99 % B

151

for 0.15 min, and finally 20 % B for 1.85 min. The mobile phase flow rate was 1.0 mL/min and the

152

total run time was 3.0 minutes. Aprepitant was detected using UV detection at 264 nm. The injection

153

volume was 15 µL. The formulation samples (500 µL) were dissolved in an equal quantity of

154

acetonitrile (500 µL). Single standard concentrations were used to assess linearity, precision and

155

accuracy (method validated in-house at AstraZeneca). The concentrations of the standards were either

156

20 or 200 µM.

157 158

Single-pass intestinal perfusion (SPIP)

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The intestinal absorption model applied in this study was modified from that of Fagerholm et al., and

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has been described previously [29]. Briefly, the animals were anesthetized with inhalation Attane

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

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body temperature of 37°C. The abdomen was opened by a 4-6 cm longitudinal incision along the

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midline of the animal. A jejunal segment of 6-10 cm (measured in situ prior the start of the

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experiment; individual measurements were used in the corresponding calculations), located

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approximately 10 cm distal to the ligament of Treitz (preventing entrance of endogenous bile into the

166

perfused segment), was cannulated with a tube (polypropylene, O.D. 4 mm). Each segment was



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flushed with 20-30 ml 25°C saline solution for 1-2 minutes to remove luminal mucus and non-

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adherent debris to ensure an even flow. Approximately 10 cm of the ingoing tube was placed within

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the abdomen to ensure that the solution entering the segment was at body temperature. To prevent

170

heat loss and evaporation of fluid, the abdomen was sutured, leaving the ends of the tube exposed

171

outside the animal. The experiment started by filling the segment with 4 ml of drug suspension or

172

solution, and thereafter a perfusion of 0.2 ml/min was started with the same suspension or solution.

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This perfusion rate was kept constant throughout the remainder of the experiment (105 min). The time

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was set to 0 when the perfusion was initiated. The perfusate leaving the jejunal segment was

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quantitatively collected at 45, 60, 75, 90 and 105 minutes, and immediately stored at -20°C awaiting

176

analysis.

177 178

Blood samples of 200 µl were drawn from the vena jugularis at designated time points throughout the

179

experiment. The sampling times were 0, 15, 30, 45, 60, 75, 90, and 105 min. After each sampling the

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catheter was rinsed with saline solution to prevent clotting. The samples were collected in Li-heparin

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tubes (Sarstedt, Nümbrecht, Germany), and were centrifuged immediately at 4°C at 10000 rpm for 5

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minutes. The plasma was then transferred to Eppendorf tubes and stored at -20°C, awaiting analysis.

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Intravenous single-dose administration

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An aprepitant nanosuspension in saline was made by ultrasonication crystallization, with 3%

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dimethylaniline (DMA), 0.05 mM sodium dodecyl sulfate (SDS) and 0.04% polyvinylpyrrolidone

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(PVP) as stabilizers. The mean particle size was determined to be 271 nm using dynamic light

188

scattering. 1 mL (1µmol/mL) of the suspension was administered i.v. through a temporary catheter

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inserted into the tail vein (Table 1). The drug was infused over 1 min to 3 rats (284-313 g). The

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catheter was rinsed with 1 mL saline after administration. Blood samples of 200 µL were drawn from

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a vena cava catheter to a total volume of 2.6 mL. Blood was sampled immediately before drug

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administration and subsequently over 27 h (at 5, 10, 20, 30, 40, 50, 60 min, and 2, 4, 6, 21 and 27 h).

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The blood samples were put on ice and centrifuged (3000 × g, 10 min at 4 °C) within 5 min. 100 µL of


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the plasma was transferred to an Eppendorf tube. The plasma samples were frozen and stored at -20 °C

195

until analysis. The rats were conscious throughout the study period.

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Perfusate analysis

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The perfusate samples were thawed at room temperature and vortex-mixed for 1 minute. 200 µL of

199

sample and 200 µL acetonitrile were added to a 1.5 mL Eppendorf tube and vortex-mixed for 1 minute

200

to dissolve all the aprepitant in the sample. In order to remove the cellular debris in the samples, they

201

were centrifuged for 10 minutes at 20 ºC, at 11000 RCF, on a Hettich Rotina 46 R centrifuge

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(Tuttlingen, Germany) equipped with a 100 mm rotor. 150 µL of each sample was taken for

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concentration analysis.

204 205

The concentrations of aprepitant, ketoprofen and phenol red were quantified simultaneously on a

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Waters Aquity UPLC instrument equipped with a Waters BEH C18 column (2.1x50 mm, 1.7 µm

207

particle size) and a PDA detector (Waters Corporation, Milford, MA). The column temperature was 40

208

°C. The mobile phases were water containing 0.03% trifluoroacetic acid (A) and acetonitrile

209

containing 0.03 % trifluoroacetic acid (B). A gradient was run as follows: initially 20 % B, 20-99 % B

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for 1 min, 99 % B for 0.15 min, and finally 20 % B for 1.85 min. Total run time was 3.0 minutes.

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Aprepitant, ketoprofen and phenol red were detected using UV detection at 264 nm, 255 nm and 428

212

nm, respectively. The injection volume was 2 µL when analyzing the 200 µM aprepitant samples and

213

5 µL when analyzing the 20 µM aprepitant samples. Single standard concentrations were used to

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assess linearity, precision and accuracy (AstraZeneca, method validated in-house).

215

The concentrations of these standards were: 100 µM or 10 µM aprepitant, 50 µM ketoprofen and 12.5

216

µM phenol red. The limits of quantitation (LOQs) for aprepitant, ketoprofen and phenol red were not

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determined as the investigated samples were much higher in concentration.

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Plasma analysis

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The plasma samples were thawed at room temperature and vortex-mixed for 1 minute. 40 µL of

220

plasma sample and 80 µL of acetonitrile were added to a 1.5 mL Eppendorf tube and vortex-mixed for



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1 minute. The samples were centrifuged for 10 minutes at 20 ºC, at 11000 RCF, on a Hettich Rotina

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46 R centrifuge (Tuttlingen, Germany) equipped with a 100 mm rotor to remove plasma proteins and

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other cellular debris. The supernatant was transferred to liquid chromatography (LC) vials for

224

quantification. The concentrations of aprepitant and ketoprofen were quantified simultaneously on a

225

Waters Acquity UPLC instrument equipped with a Waters BEH C18 column (2.1 x 50 mm, 1.7 µm

226

particle size), a PDA detector and a single quadropole MS operating in electrospray ionization mode.

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The column temperature was 40 °C. The mobile phases were water containing 0.1 % formic acid (A)

228

and acetonitrile containing 0.1 % formic acid (B). A gradient was run as follows: initially 20 % B, 20-

229

99 % B for 1 min, 99 % B for 0.15 min, and finally 20 % B for 1.75 min. Total run time was 3.0

230

minutes. Aprepitant was detected by mass spectrometry at 535.2 m/z. Ketoprofen was detected using

231

UV detection at 255 nm. The injection volume was 5 µL for all samples. Standards were prepared in a

232

50:50 mixture of H2O and acetonitrile. The calibration curve was 5-500 nM for aprepitant and 1-50

233

µM for ketoprofen. The LOQ was 5nM for aprepitant. The LOQ for ketoprofen was not determined as

234

the investigated samples were much higher in concentration. The precision, expressed as relative

235

standard deviation of quality control samples, was 6-21 %.

236

Theoretical considerations

237

The traditional equation (see equation 1 below) for determining the effective intestinal permeability

238

(Peff) from a SPIP study relates the ratio of disappeared API to the surface area of the perfused

239

segment described as a smooth cylinder, and assumes the parallel tube hydrodynamics model [30]. For

240

compounds with a non-saturable mechanism of transport, Peff is considered to be concentration-

241

independent. When applying this traditional equation for Peff to formulations other than solutions, it is

242

obvious that the choice of the entering (Cin) and leaving (Cout) drug concentrations in the perfused

243

segment needs careful consideration in order to accurately interpret the absorption parameters.

244 245

For an API molecule to be transported from the intestinal lumen to the systemic circulation it must

246

diffuse through the adjacent ABL as well as pass across the epithelial cell layer. In addition to API

247

monomers, nanoparticles of API and small colloidal structures containing API are considered able to


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248

diffuse through the ABL, thereby increasing the total transport rate of API through the ABL, and

249

subsequently the concentration of free monomer at the apical cell membrane surface, yielding a higher

250

absorptive flux (Figures 1 and 2 a-b). Consequently, it is possible to directly correlate changes in

251

absorptive flux to the diffusion (drifting) of nano-sized particles and colloidal structures (e.g. micelles

252

and vesicles) through the ABL. The increased total transport can in turn be expressed as an increase in

253

the effective diffusion coefficient, and this can be compared to the diffusion coefficient of the API

254

fully dissolved in water. The effective diffusion coefficient is defined from = × × ∇

255

Equation 1

256

where J is the total flux of drug, f is the ratio of aprepitant solubility in phosphate buffer to that in

257

FaSSIF (0.016) [23], and C is concentration of dissolved drug, including drug partitioned into

258

colloidal structures. This means that f × C is the concentration of dissolved drug not partitioned to

259

micelles. On the basis of this theory, we developed two models for explaining the increase in

260

absorption associated with nano-sized material and colloidal partitioning diffusion in the ABL. The

261

first approach was to calculate the API disappearance flux from the perfusate, in analogy with the Peff

262

calculations, i.e. based on the change in total API concentration in the perfusate entering and leaving

263

the segment. The second approach for flux calculations was based on the appearance rate of aprepitant

264

in plasma, which is directly related to the flux from the intestinal lumen, across the intestinal wall, to

265

the systemic circulation. As the total clearance of aprepitant in both rat and man is low, variability in

266

the first-pass effect has only a minor influence on the flux calculations (see below) [25].

267

Permeability calculations

268

Peff was calculated from the total perfusate concentrations of aprepitant (i.e. both monomers and

269

particulates) by applying the parallel tube hydrodynamic model:

270

= ×

Ĉ ⁄Ĉ

Equation 2

271

where Qin is the perfusate flow rate, Ĉout is the total concentration of aprepitant, including particles, in

272

the perfusate leaving the segment, Ĉin is the total concentration of aprepitant entering the segment,

273

including particles, and A is the area of the perfused jejunal segment described as a smooth cylinder


Molecular Pharmaceutics

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Roos, C Rat Perfusion Aprepitant 161231 CR

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274

with a radius of 0.2 cm. Ĉout was calculated from Equation 3, to compensate for potential fluid

275

absorption from the perfused segment: Ĉ = Ĉ

276

!

"

× " ,$%&'( × CF ,$%&'(

Equation 3

277

where Ĉsample is the total aprepitant concentration measured in the perfusate leaving the segment,

278

Cin,PhRed and Cout,PhRed are the measured concentrations of phenol red in the perfusate entering and

279

leaving the segment, respectively, and CF is a correction factor (=1.15) to account for phenol red

280

absorption [31].

281

Pharmacokinetic calculations

282

Disposition pharmacokinetic (PK) parameters were estimated from the plasma concentration-time

283

profiles after i.v. administration using first-order compartmental kinetics (Phoenix WinNonlin v6.3,

284

Certara, L.P., St. Louis, MO, USA). The areas under the plasma concentration-time curves (AUCs)

285

for the intestinal perfusions were calculated using GraphPad Prism software (v7.00, GraphPad

286

Software, San Diego, CA) from time 0 (at the start of the SPIP) to 105 min using the linear trapezoidal

287

method, as no descending phase in the plasma concentration-time profiles was determined.

288 289

Small intestinal in vivo flux (Jdisapp) based on perfusate concentrations

290

To quantify the intestinal absorption of API from the lumen during the SPIP, a disappearance flux

291

(Jdisapp) based on the total perfusate concentrations (dissolved and particulate API), was calculated

292

according to equation 4:

293

+ !! =

Ĉ Ĉ ×,

Equation 4

294

where Q is the perfusion rate (0.2 ml/min), and A is the surface area of the perfused segment,

295

described as a smooth cylinder with a radius of 0.2 cm. To compensate for potential fluid flux in the

296

segment, Cout was corrected according to equation 3.

297


Page 13 of 36


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13 (28)

298

Small intestinal in vivo flux (Japp) based on deconvolution of plasma concentrations

299

A deconvolution method was used to calculate the intestinal absorption flux (Japp) for aprepitant, based

300

on its plasma concentrations, as previously described by Sjögren et al. [32]. This method has

301

previously been used and validated for the determination of regional intestinal in vivo Peff in humans

302

and dogs in two studies [33, 34]. Briefly, an input rate was calculated by deconvolution of the plasma-

303

concentration profiles from the SPIP, using the disposition PK parameters acquired from the

304

intravenous reference dose as a unit impulse response (Table 2). The absorption flux was then

305

calculated according to equation 5: !! =

306

! - ./-0

Equation 5

307

where r is the radius of the jejunum (0.2cm) and L is the length of the individual segment. Japp was

308

calculated from 0 minutes to 105 minutes and the median from each individual was used as

309

representative for further data analysis.

310 311

Effective diffusion coefficient calculations

312

The contribution of each of the aprepitant forms (dissolved molecules, nanoparticles and/or small

313

colloidal structures) being transported through the ABL can be described by the increase in diffusivity,

314

as described schematically in Figure 2 a-b. The flux through the ABL (JABL) can be expressed by

315

equation 6:

316

10 =

2'33 4

× × ( 6 − )

Equation 6

317

where Deff is the effective diffusion coefficient, δ is the length of the ABL (see below), Cb is the mean

318

bulk concentration in the segment assuming an exponential decrease according to the parallel tube

319

model (in cases where the concentration is higher than solubility throughout the entire segment, Cb =

320

solubility), f is the ratio of aprepitant solubility in phosphate buffer to that in FaSSIF (0.016) [23] , and

321

Cm is the concentration at the apical membrane. JABL is the total flux through the ABL since Deff

322

includes the transport due to dissolved molecules, molecules in colloidal structures and transport in

323

particles. Note that Cb and Cm is the concentration of dissolved molecules including colloidal


Molecular Pharmaceutics

Page 14 of 36

Roos, C Rat Perfusion Aprepitant 161231 CR

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14 (28)

324

structures, but excluding particles. The flux across the intestinal membrane (Jw) can in analogy be

325

expressed by equation 7: 9 =

326

× ( × − 6 )

Equation 7

327

where Pmem is the membrane permeability, f is the ratio of the solubility in phosphate buffer to that in

328

FaSSIF (0.016) [23], and Cm and Cbas are the concentrations of API at the apical surface and

329

basolateral side of the intestinal membrane, respectively. Under sink conditions, Cbas

mechanisms of intestinal drug absorption from aprepitant

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