Kolliphor Surfactants Affect Solubilization and Bioavailability of

Subscriber access provided by The Chinese University of Hong Kong

Article

Kolliphor™ Surfactants affect Solubilisation and Bioavailability of Fenofibrate – Studies of In Vitro Digestion and Absorption in Rats Ragna Berthelsen, Rene Holm, Jette Jacobsen, Jakob Kristensen, Bertil Abrahamsson, and Anette Mullertz Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp500545k • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1

Kolliphor™ Surfactants Affect Solubilisation and

2

Bioavailability of Fenofibrate – Studies of In Vitro

3

Digestion and Absorption in Rats

4

Ragna Berthelsen§, René Holm†§, Jette Jacobsen§, Jakob Kristensen€, Bertil Abrahamsson‡ and

5

Anette Müllertz§£*.

6

§

Department of Pharmacy, University of Copenhagen, Denmark.

7

†

Biologics and Pharmaceutical Science, H.Lundbeck A/S, Valby, Denmark.

8

€

Ferring Pharmaceuticals A/S, Copenhagen, Denmark.

9

‡

AstraZeneca Pharmaceutics, R&D, Mölndal, Sweden.

10

£

Bioneer:FARMA, Department of Pharmacy, University of Copenhagen, Denmark.

11 12 13 14 15 16 17 18 19

*Corresponding author: Anette Müllertz Department of Pharmacy Universitetsparken 2 2100 Copenhagen + 45 35 33 64 40 [email protected]

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Page 2 of 38

Abstract Graphic

3 4


2

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Abstract

3

Selection of excipients for drug formulations requires both intellectual and experimental

4

considerations as many of the used excipients are affected by physiological factors, e.g. they may

5

be digested by pancreatic enzymes in the gastrointestinal tract. In the present paper we have

6

looked systematically into the differences between Kolliphor™ ELP, EL and RH40 and how they

7

affect the bioavailability of fenofibrate, through pharmacokinetic studies in rats and in vitro

8

lipolysis studies. The study design was made as simple as possible to avoid confounding factors,

9

why the tested formulations only comprised an aqueous micellar solution of the model drug

10

(fenofibrate) in varying concentrations (2-25 % (w/v)) of the three tested surfactants.

11

Increased concentrations of Kolliphor™ ELP and EL led to increased fenofibrate AUC0-24h

12

values. For the Kolliphor™ RH40 formulations, an apparent fenofibrate absorption optimum was

13

seen at 15 % (w/v) surfactant, displaying both the highest AUC0-24h and Cmax. The reduced

14

absorption of fenofibrate from the formulation containing the highest level of surfactant (25 %

15

w/v), was thought to be caused by some degree of trapping within Kolliphor™ RH40 micelles.

16

In vitro, Kolliphor™ ELP and EL were found to be more prone to digestion than Kolliphor™

17

RH40, though not affecting the in vivo results. The highest fenofibrate bioavailability was

18

attained from formulations with high Kolliphor™ ELP/EL levels (25 % (w/v)), indicating that

19

these surfactants are the better choice for solubilising fenofibrate in order to increase the

20

absorption upon oral administration. Due to drug dependant effects of the different types of

21

Kolliphor™ more studies are recommended in order to understand which type of Kolliphor™ is

22

best suited for a given drug.

23


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 38

1 2

KEYWORDS:

3

Fenofibrate, Kolliphor™ EL, Kolliphor™ ELP, Kolliphor™ RH40, Cremophor, bioavailability,

4

rats, in vitro lipolysis, surfactant digestion, micellar trapping, IVIVC.

5


4

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Introduction

3

In the pharmaceutical industry many different excipients are utilized during drug formulation to

4

achieve the desired therapeutic profile for a given drug. These excipients cover a broad range

5

from inert materials used as fillers, binders or disintegrants in conventional dosage forms (tablets

6

and capsules), to excipients that may directly affect the overall drug absorption. For poorly water

7

soluble drugs, which at present are the majority of small molecules in development for oral

8

administration1 one way of improving drug absorption is to facilitate the solubilisation of the

9

drug in the gastrointestinal (GI) fluids. Increasing the concentration of solubilised drug in the GI

10

fluids will affect the concentration gradient across the intestinal membrane and thereby possibly

11

the rate and extent of absorption2. For poorly water soluble drugs, surfactants represent a very

12

commonly used type of excipients. In general, surfactants increase the drug absorption by

13

increasing the concentration of solubilised drug in the GI tract, by increasing the apparent drug

14

solubility and/or the dissolution rate. However, the surfactant induced effect on the drug

15

absorption can also be affected by e.g. the surfactant inhibiting efflux and influx transporters

16

involved in the drug absorption, surfactant induced membrane damage, modified or delayed

17

gastric emptying, micellar entrapment of the drug and degradation of the surfactant3-6.

18

Furthermore, it should be noted the use of surfactants also has the potential to decrease the

19

intestinal permeability of highly permeable drugs by decreasing the free fraction of the drug7. In

20

depth knowledge of how surfactants affect the drug absorption is, hence, key during formulation

21

development, as it will aid the pharmaceutical scientist choosing the best surfactant for a given

22

drug and formulation. Kolliphor™ ELP (ELP), Kolliphor™ EL (EL) and Kolliphor™ RH40

23

(RH40) (formerly known as Cremophor®) are all non-ionic surfactants commonly used in

24

pharmaceutical formulations (solid dosage forms and lipid based delivery systems) to improve


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 38

1

the bioavailability of poorly water soluble drugs8,9. EL is made by reacting castor oil with

2

ethylene oxide in a molar ratio of 1:35, while ELP is a purified grade of EL. RH40 is made by

3

reacting 1 mole of hydrogenated castor oil with 40 moles of ethylene oxide. The main constituent

4

of ELP, EL and RH40 is the tri-ricinoleate ester of ethoxylated glycerol. Other constituents

5

include polyethylene glycol ricinoleate and the corresponding free glycols

6

Kolliphor™ have been used in the formulation of poorly water soluble drugs, e.g EL has been

7

used as a vehicle for the solubilisation of a series of hydrophobic drugs, including cyclosporin A,

8

diazepam, propofol and paclitaxel9. Despite its common use, the intraluminal effects of the

9

Kolliphor™ surfactants are not fully understood. Furthermore, several in vitro studies have failed

10

to predict the amount of drug absorbed in vivo when administered in a Kolliphor™ containing

11

formulation. Sparreboom et al.11, Bardelmeijer et al.4 and Malingre et al.12 all reported that

12

despite a positive in vitro drug solubilising effect of EL, the surfactant limited the absorption of

13

paclitaxel from the gut in humans and mice. This lead to a general conclusion and understanding

14

that the most likely mechanism behind the inconsistent results was that EL formed micelles in

15

the aqueous environment in the GI tract, trapping paclitaxel in their hydrophobic core and

16

thereby limiting the drug absorption4,11,12. In a study by Cuiné et al.13 the bioavailability of

17

danazol in beagle dogs was reduced when the proportional content of EL, relative to lipid, in

18

self-emulsifying lipid based drug delivery systems (SEDDS), was increased. They hypothesized

19

that the poor in vivo solubilisation properties of EL may reflect its susceptibility to digestion by

20

pancreatic enzymes. In vitro lipolysis studies, performed by the same research group, revealed

21

that EL was indeed readily susceptible to digestion by pancreatic lipase. In comparison, RH40

22

was relatively poorly digested during in vitro lipolysis studies5. SEDDS comprising high

23

quantities of digestible surfactant (EL) appeared less effective in preventing danazol


10

. All three types of

6

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

precipitation in vitro, however in vivo no significant difference in bioavailability was seen for

2

formulations based on pure surfactants (100 % (w/w) EL and RH40).

3

With the purpose of gaining a better and broader understanding of the interplay between an

4

active pharmaceutical ingredient (API) and Kolliphor™ surfactants, the present study was

5

performed to investigate the in vivo-in vitro correlation between bioavailability of fenofibrate

6

solubilised in EL, ELP or RH40 and solubilisation during in vitro lipolysis. The study design was

7

made as simple as possible to avoid confounding factors, why the tested formulations only

8

comprised an aqueous solution of fenofibrate with varying concentrations of the three tested

9

surfactants. Fenofibrate was chosen as the model drug in the present work as it is a well-known

10

class II drug according to the biopharmaceutics classification system (BCS)14 displaying a very

11

low aqueous solubility (< 0.5 mg/l)15 and has a high logP (5.1)16. Upon oral administration,

12

fenofibrate is fully hydrolysed to fenofibric acid by tissue and plasma esterases17,18.

13 14

Experimental Section

15

Chemicals

16

Kolliphor™ EL, ELP and RH40 was kindly donated by BASF (Ludwigshafen, Germany).

17

Fenofibrate (min. 99 %), clofibric acid (97 %), pancreatin (porcine) (P1625), soybean oil,

18

polyethylene glycol (average MW 400) (PEG 400) and 4-bromobenzeneboronic acid (BBBA)

19

(95 %) was obtained from Sigma-Aldrich (St Louis, USA). Bile extract (bovine) (B3883) and

20

maleic acid were purchased from Fluka Chemie AG (Buchs, Switzerland), whereas calcium

21

chloride dehydrate, sodium hydroxide and sodium chloride from E.Merck (Darmstadt,

22

Germany). Fenofibric acid (98 % purity) was from AK Scientific Inc (Union City, Ca, USA),


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 38

1

and phosphatidylcholine (Lipoid S PC) (99 % purity) was purchased from Lipoid

2

(Ludwigshafen, Germany). TRIS ultrapure (TRIS-(hydroxymethyl)aminomethanol) was

3

purchased from ICN Biomedicals Inc. (California, US). Purified water was obtained from a

4

Milli-Q water purification system (Millipore, Bedford, Massachusetts). All other chemicals and

5

solvent were of analytical purity or high performance liquid chromatography (HPLC) grade.

6

Preparation of fenofibrate formulations

7

Fenofibrate was completely solubilised in different formulations prepared for oral administration

8

(Table 1). The formulations were tested in vivo applying a fixed dose at 2 mg/kg body weight in

9

order to correlate with the clinical dose of 145 mg for an adult person (70 kg). As the dose was

10

fixed across all formulations, the percentage of saturation varied, see Table 1. Four formulations

11

not including Kolliphor™ were included in the study comprising two different drug vehicles;

12

PEG 400 and soybean oil, in two different drug to excipient ratios (see Table 1).

13

All aqueous formulations were prepared iso-osmolar by adding sodium chloride to obtain

14

220±40 mOsm/kg.

15

Characterisation of the fenofibrate formulations

16

The saturation solubility of fenofibrate in all the formulations was determined. Fenofibrate was

17

added in excess to 10 mL of each formulation in a test tube and the tubes were placed on an end-

18

over-end rotator at 37°C. Samples (1 mL) were withdrawn after 1, 4, 24 and 48 h, and

19

centrifuged twice at 4,500 rpm and 15,000 rpm in an Eppendorf Centrifuge (Labnet Prism™

20

microcentrifuge, Labnet Internation, Inc., Edison, NJ, USA) for 10 minutes each. The

21

supernatant was diluted appropriately and analysed by high performance liquid chromatography


8

Page 9 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

(HPLC). Saturated solubility was reached when there was less than 5 % difference between two

2

consecutive measurements.

3

Particle size, size distribution by volume (polydispersity index, PdI) and zeta potential of the

4

aqueous Kolliphor™ formulations were measured by Zetasizer (Nano-ZS, Malvern instruments,

5

UK). Samples were measured undiluted in triplicates at ambient temperature.

6

In vivo pharmacokinetic studies

7

The protocol used for the in vivo studies in rats was approved by the institutional ethics

8

committee in accordance with Danish law regulating experiments on animals and in compliance

9

with EU directive 2010/63/EU, and the NIH guidelines on animal welfare. Male Spraque-

10

Dawley rats (296-343 g) were purchased from Charles River (Sulzfeld, Germany). The animals

11

were acclimatised and maintained on standard feed and carrots with free access to water for a

12

minimum of 5 days prior to the experiments.

13

Before entry into the experiments the animals were fasted for 16-20 h and randomly assigned to

14

receive one of the treatments. The animals were allowed access to drinking water 4 h after oral

15

dosing and carrots 10 h after dosing. Six animals were dosed by oral gavage with 2 mg/kg of

16

fenofibrate solubilised in each of the oral formulations (Table 1) with a fixed formulation volume

17

of 10 mL/kg. Formulations PEG-II and Soybean-II were dosed as a 4 mg/kg fenofibrate solution

18

in a volume of 5 mL/kg, ensuring equal dose of fenofibrate in all formulations. Blood samples of

19

0.2 mL were obtained from the tail vein and collected into 0.5 mL EDTA coated tubes at 30 min,

20

1, 2, 3, 4, 6, 8, 10 and 24 h after administration. Plasma was harvested immediately by 15 min of

21

centrifugation at 4 °C, 4000 rpm (Centrifuge Multifuge 1 S-R), and stored at -80 °C until

22

analysis. After the study the rats were euthanized.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 38

1

Bioanalytical procedure

2

Plasma samples were prepared for analysis based on a method modified from Borkar et al.

3

originally adapted from Hanafy et al.20. 50 µL of plasma sample and 20 µL of internal standard

4

(clofibric acid in methanol, 200 µg/mL) were mixed with 100 µL acetonitrile. The mixture was

5

briefly whirl mixed followed by sonification in an ultrasonic water bath for 10 min. The samples

6

were stored at -20 °C for 10 min, followed by centrifugation (14 min, 14,000 rpm) at 0 °C. The

7

clear supernatant was transferred into HPLC vials. The standard calibration curve was prepared

8

similar to the samples. The standard curves were run in the concentration range [0.06 µg/mL –

9

6.0 µg/mL] and were linear over the entire range. Recovery using this procedure was more than

10

90 % within the tested concentration range. The amount of fenofibric acid in the plasma samples

11

was determined by isocratic HPLC using the same column and apparatus as described below.

12

The mobile phase consisted of 68 % methanol and 32 % water, added 0.1 % formic acid. Flow

13

rate was 0.6 mL/min and injection volume 20 µL. Fenofibric and clofibric acid was measured at

14

UV detection wavelength 287 nm with approximate retention times of 9 min and 4.5 min,

15

respectively.

16

In vitro evaluation - digestion experiments

17

To study the digestion of lipids and surfactants in the formulations alongside the solubilisation of

18

fenofibrate in a simulated intestinal media, the dynamic in vitro lipolysis model developed by

19

Zangenberg et al. was used with some modifications21,22. The formulation to digestion media

20

ratio was set to 1:2.5, to correlate with the ratio between the volume of administrated formulation

21

and the amount of liquid in the rat intestine23. 10 mL formulation was added into a thermostat-

22

jacketed glass vessel and dispersed for 10 min in 25 mL digestion medium, comprising final


19

,

10

Page 11 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

concentrations of 2 mM tris-maleic acid, 2.95 mM bile salts, 0.26 mM phosphatidylcholine (PC)

2

and 50 mM sodium chloride, pH 6.5 (37 °C). Digestion was initiated by addition of 5 mL freshly

3

prepared pancreatic extract (550 USP units/mL). Sodium hydroxide solution (0.2 M) was

4

automatically added (controlled via the pH-stat controller) to the vessel to maintain a constant

5

pH (6.5) during digestion. Throughout the experiment calcium chloride was added continuously

6

(0.045 mmol/min) to remove fatty acids (FA) from the oil/surfactant-water interface by

7

precipitation of Ca2+-soaps (Ca(FA)2)21. Samples were taken at 0, 5, 15, 30, 45 and 60 min and

8

immediately treated with lipolysis inhibitor (1 M BBBA in methanol, 5µL per mL digestion

9

medium). All samples were centrifuged for 30 min at 37 °C and 13,500 rpm (17,135 g) in order

10

to separate the digests into two phases; a dispersed aqueous colloidal phase and a precipitated

11

pellet phase. The pellet phase was re-dissolved in methanol:water (4:1) and the amount of

12

fenofibrate in both phases was determined by HPLC after appropriate dilution with methanol.

13

Blank digestion experiments were carried out by replacing the fenofibrate formulations with 10

14

mL of purified water. The blank runs were carried out to account for the FA produced on

15

digestion of the lecithin present in the digestion medium. All digestion data obtained from the

16

different formulations were subsequently corrected for the background FA production by

17

subtraction of the amount of FA produced during blank digestion experiments.

18

Some FA released during digestion are partially ionized and therefore only partially titratable at

19

pH 6.524. The total amount FA released was, therefore, determined by “back” titrations; at the

20

end of each digestion experiment, the pH-stat was programmed to rapidly add sodium hydroxide

21

to the reaction vessel until pH 9.0, thereby ensuring ionisation of FA not ionised at pH 6.524,25.

22

The total volume of sodium hydroxide added during the experiment, corrected for the amount


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 38

1

needed to raise the pH to 9.0, in blank control experiments, was used to calculate the total

2

amount of FA (ionised and non-ionised at pH 6.5) produced during the digestion experiment.

3

Estimation of percent Kolliphor™ digested

4

The degree of digestion of the different types of Kolliphor™ during the in vitro digestion

5

experiments was estimated following the approach previously described by Cuiné et al.5. The

6

total amount of titratable FA present per gram of surfactant was estimated utilising the

7

assumption that 1 g of surfactant contained the same number of titratable FA molecules as would

8

be present in a physical mixture of the reactants (e.g. 1 mol of RH40 consists of 1 mol

9

hydrogenated castor oil and 40 mol ethylene oxide and will produce 3 mol of FA on digestion).

10

This assumption was necessary since ELP, EL and RH40 all are reaction products of glyceride

11

lipids (castor oil and hydrogenated castor oil) and polyethylene oxide, and typically comprise a

12

mixture of several components, why it is very difficult to obtain a precise number of FA present

13

in each of the surfactants.

14

HPLC analysis

15

Samples from the digestion experiments were analysed using an isocratic HPLC method

16

retaining fenofibrate on a Phenomenex® Kinetex® 5µ XB-C18 100 A (100 x 4.6 mm) HPLC

17

column protected by a Phenomenex® guard-column (Phenomenex, Torrance, USA). The mobile

18

phase consisted of 85 % (v/v) methanol and 15 % (v/v) purified water, the flow rate was 1.5

19

mL/min and the injection volume 20 µL. The detection wavelength was 280 nm and the drug

20

retention time was approximately 1.4 min. All samples were analysed at room temperature using

21

a Dionex ASI-100 Automated Sample injector, P680 HPLC Pump and PDA-100 Photodiode

22

Array Detector (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The chromatograms


12

Page 13 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

were evaluated using Thermo Scientific Dionex Chromeleon 7 Chromatography Data System

2

Software (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

3

Data and statistical analysis

4

Pharmacokinetic parameters were calculated using WinNonlin Professional software 4.1

5

(Pharsight Corporation, Mountain View, CA, USA). Based on a non-compartmental analysis the

6

area under the curve (AUC) of the plasma concentration-time profiles was determined by the

7

linear log trapezoidal method from t = 0 to t = 24 h (last plasma concentration measured after

8

dosing). Statistical comparison of the pharmacokinetic data, the recovery of fenofibrate in the

9

different phases after in vitro lipolysis experiments and the amount of fatty acids titrated during

10

lipolysis studies was conducted by use of a 2-way ANOVA test, followed by pair wise

11

comparison of means using the Tukey method (α = 0.05). The statistical tests were performed

12

using GraphPad Prism, 6.0 (GraphPad Software, San Diego, CA, USA).

13 14

Results

15

Sixteen different fenofibrate formulations were prepared and tested in vivo and in vitro. Prior to

16

the in vivo study, the formulations were characterised with respect to fenofibrate saturation

17

solubility and particle size distribution.

18

In vitro formulation characterization

19

The saturation solubility of fenofibrate in all tested formulations is presented in Table 1. All

20

three types of Kolliphor™ produced a linear relation between surfactant concentration and

21

fenofibrate solubility in the formulation (SI). This was well expected, as all the chosen surfactant


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 38

1

concentrations were above literature values of the critical micellar concentration (CMC) (0.01-

2

0.3 % (w/v) for ELP, EL and RH404,26). Of the three types of Kolliphor™, EL showed the highest

3

solubilising capacity with an increase in fenofibrate solubility of 0.27 mg/mL per % (w/v) EL,

4

followed by ELP (0.24 mg/mL fenofibrate / % (w/v) ELP) and RH40 (0.18 mg/mL fenofibrate /

5

% RH40).

6

The overall Z-average of the Kolliphor™ formulations was 11.8 ± 1.1 nm in diameter (PdI = 0.2)

7

for ELP, 11.3 ± 0.7 nm in diameter (PdI = 0.1) and 15.7 ± 1.7 nm in diameter (PdI = 0.2) for

8

RH40, without correcting for viscosity differences (which were apparent at the highest surfactant

9

concentrations). For all three types of Kolliphor™, at all concentrations below 20 % (w/v), only

10

one particle population was seen. For ELP, EL and RH40, levels of 20 and 25 % (w/v) surfactant

11

resulted in multiple particle populations probably as a result of aggregation.

12

In vivo data

13

Plasma concentration-time profiles of fenofibric acid following oral administration of 13

14

different fenofibrate formulations are shown in Figure 1 and 2. The pharmacokinetic parameters,

15

area under the curve (AUC0-24h), peak plasma concentrations (Cmax) and time to reach Cmax

16

(tmax)), following non-compartmental analysis are summarized in Table 2. The observed

17

bioavailability (estimated as the AUC0-24h) of fenofibrate after administration of the tested

18

formulations is in general agreement with data from previous studies20,27,28.


14

Page 15 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 1. Mean (± SEM) fenofibric acid plasma concentrations in rats (n = 6) after oral

3

administration of fenofibrate solutions (2 mg/kg) with varying concentrations of

4

Kolliphor™ ELP (A), EL (B) and RH40 (C).

5


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 38

1 2

Figure 2. Mean (± SEM) fenofibric acid plasma concentrations in rats (n = 6) after oral

3

administration of an equivalent dose of fenofibrate solutions (2 mg/kg) in PEG 400 (A) and

4

soybean oil (B), with varying dosing volumes.

5 6

Kolliphor™ formulations

7

Increasing levels of ELP (25% (w/v) vs 2% (w/v)) lead to an increased bioavailability of

8

fenofibrate (AUC0-24h, p ≤ 0.05) (Figure 1 and Table 2). The same tendency was apparent for the

9

EL formulations, but with no statistical significant difference between the AUC0-24h –values. For

10

the RH40 formulations, an apparent fenofibrate absorption optimum was seen at 15 % (w/v)


16

Page 17 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

surfactant, displaying both the highest AUC0-24h and Cmax. Comparisons of the formulations based

2

on the different types of Kolliphor™ showed that at high levels of surfactants (25 % (w/v)),

3

RH40 gave a significantly (p ≤ 0.05) lower bioavailability of fenofibrate (AUC0-24h and Cmax)

4

compared to ELP-25 and EL-25.

5

For the EL and ELP formulations, tmax showed a clear increasing tendency with increasing

6

surfactant concentrations, whereas absorption of fenofibrate from RH40 formulations showed no

7

difference in tmax with varying surfactant concentration.

8

PEG 400 and soybean oil formulations

9

PEG 400 and soybean oil were included in the study as control vehicles for relative comparison.

10

All control formulations displayed significant differences compared to the Kolliphor™

11

formulations with lower Cmax and longer tmax values (p ≤ 0.05). At both dosing levels, PEG 400

12

produced a plasma concentration-time profile for fenofibric acid displaying two peaks, one at 30

13

min (first sample after administration) and another 6-7 h after administration. When lowering the

14

dose volume and thereby the volume of the excipient, the AUC0-24h was slightly decreased along

15

with the two peak plasma concentrations, however; the overall shape of the profile remained

16

unchanged. PEG 400 dosed at 10 mL/kg (PEG-I) gave a significantly higher AUC0-24h compared

17

to RH40-2, but not compared to any of the other Kolliphor™ formulations.

18

For soybean oil, tmax was reduced from 8.83 ± 2.86 h to 6.7 ± 1.6 h by doubling the drug to

19

vehicle ratio and thereby reducing the dose volume from 10 to 5 mL/kg. Except for ELP-25,

20

there was no significant difference between AUC0-24h of soybean oil and that of all Kolliphor™

21

formulations. Compared to ELP-25, dosing soybean oil (both with 5 mL/kg and 10 mL/kg) led to

22

a significantly (p ≤ 0.05) lower AUC0-24h.


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 38

1

Dynamic in vitro lipolysis studies

2

The apparent FA titration profiles, measured at pH 6.5 during 60 min of in vitro lipolysis of the

3

three types of Kolliphor™ tested at three different concentration levels are depicted in Figure 3.

4

In general, all three types of Kolliphor™ were relatively poorly digested with less than

5

approximate 100 µmol titratable FA released during the 60 min digestion study. Compared to a

6

previous study by Cuiné et al. in which 1 g EL was found to produce 358 µmol FA5, the amount

7

of FA released from EL in this study was relatively low (1.5 g EL produced 106 µmol FA).

8

However, the lipolysis setup differed between the two studies with e.g. variations in digestion

9

medium composition and pH, as well as pancreatic lipase activity, which might account for the

10

observed differences.

11

After 60 min of lipolysis, the pH of the digestion medium was quickly raised to 9.0 to determine

12

the total amount of FA released, as described in the experimental section. The amount of FA

13

released after 60 min at pH 6.5 and the total amount of FA released at pH 9.0 (the FA released

14

pH 6.5 with the addition amount FA released at pH 9) are shown in Figure 4. With the increased

15

pH more FA were titrated for all the tested formulations showing that in all situations FA were

16

formed upon digestion of the surfactants, which were only partially ionised at pH 6.5.

17

Particularly for EL and ELP the back titration to pH 9 added to the total amount of FA generated,

18

demonstrating a structural differences between the formed FA from ELP/EL and RH40,

19

correlating well with the fact that these surfactants are made by reacting different molecules

20

(castor and hydrogenated castor oil) with ethylene oxide in different ratios. No significant

21

difference (p > 0.05) was found when comparing the total amount of FA released from the

22

different types of Kolliphor™ at the same concentration level. However, within each type of

23

surfactant a concentration-dependence was evident, i.e. higher concentrations of Kolliphor™ led


18

Page 19 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

to titration of more FA, demonstrating active digestion of the surfactants. The observed increase

2

in FA released upon back titration generating a higher total amount of FA released, led to a better

3

correlation to the amount of FA released in the study performed by Cuiné et al.5.

4

Table 3 shows the calculated percent of EL, ELP and RH40 digested after 60 min of in vitro

5

lipolysis. The highest relative level of surfactant digestion during lipolysis was seen for the 2 %

6

(w/v) formulations. The observed decrease in the level of digestion seen as a result of increased

7

amounts of surfactants was presumably caused by insufficient enzyme activity. The percent of

8

surfactant digested based on the total amount of FA showed a significant difference between

9

ELP-2/EL-2 and RH40-2, with ELP/EL being the more digestible surfactants.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 38

1 2

Figure 3. Profiles of FA titrated during lipase mediated digestion of fenofibrate solutions

3

with varying concentrations of Kolliphor™ ELP (A), EL (B) and RH40 (C). Digestion was

4

initiated at t = 0 min by addition of pancreatin extract and pH was maintained constant at


20

Page 21 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

pH 6.5. Values are expressed as mean ± SD (n = 3) and have been corrected for the level of

2

fatty acids released in background digestion tests.

3

4 5

Figure 4: Apparent titration of FA released after 60 min in vitro digestion of fenofibrate

6

solutions with varying concentrations of ELP, EL and RH40. Digestion was initiated at t =

7

0 min on addition of pancreatin, and pH was maintained at pH 6.5 until 60 min. At 60 min

8

the pH was shifted to pH 9.0 by addition of NaOH. Values are expressed as mean ± SD (n =

9

3) and have been corrected for the level of FA released in background digestion tests. Black

10

bars show FA released at pH 6.5 and grey bars show additional FA released at pH 9.0, the

11

bars are placed on top of each other representing the total amount of FA released. Stars

12

indicate statistical difference between the total amount FA released with p ≤ 0.01 (**), p ≤

13

0.005 (***).

14 15 16


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 38

1

Solubilisation of fenofibrate in the aqueous phase upon lipolysis

2

Figure 5 shows the percentage of the dose solubilised in the aqueous phase during 60 min of in

3

vitro lipolysis. For the majority of the fenofibrate formulations the entire dose (2 mg) was kept in

4

solution throughout the experiment. However, from the formulations with the lowest content of

5

EL and ELP (2 % (w/v)), fenofibrate precipitated during the digestion process resulting in

6

approximately 50 % of the fenofibrate dose being solubilised in the aqueous phase after 60 min

7

of lipolysis (37.8 % and 48.6 % for ELP-2 and EL-2, respectively).

8


22

Page 23 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

Figure 5. Dose solubilized (%) in the aqueous phase during 60 min in vitro lipase mediated

3

digestion of fenofibrate solutions with varying concentrations of Kolliphor™ ELP (A), EL

4

(B) and RH40 (C). Digestion was initiated at t = 0 min upon addition of pancreatin extract

5

and pH was maintained constant at pH 6.5. Values are expressed as mean ± SD (n = 3).


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 38

1

Discussion

2

Selection of excipients for drug formulations requires both intellectual and experimental

3

considerations as many of the used excipients are affected by physiological factors, e.g. they may

4

be digested by pancreatic enzymes in the GI tract thereby affecting their drug solubilising

5

capacity. In the present paper we have looked systematically into the differences between

6

Kolliphor™ ELP, EL and RH40, through investigations in rats and in vitro lipolysis studies.

7

In vivo data

8

The tmax was prolonged with increasing concentrations of all excipients except PEG 400. In the

9

case of soybean oil, the increased in tmax was seemingly caused by a delayed gastric emptying

10

due to intake of higher amounts of oil (calories), as several studies have shown that an increased

11

intake of calories, e.g. from soybean oil will delay the stomach emptying in both rats and

12

humans29-31. Based on this the prolonged tmax seen with higher concentrations of EL and ELP

13

might also suggest that these surfactants was more digestible compared to RH40. However, in

14

that case, it may also be explained by intestinal retention of fenofibrate by entrapment in

15

Kolliphor™ micelles.

16

The dual plasma peaks obtained after administration of fenofibrate solubilised in PEG 400 could

17

be explained by fast absorption of the initially dissolved fenofibrate with a high concentration

18

gradient across the intestinal barrier, followed by fenofibrate precipitation as the formulation was

19

diluted in the GI fluids and a very slow uptake, presumably rate-limited by the apparent

20

fenofibrate solubility.

21

The relatively long tmax and low Cmax values observed for both soybean oil formulations were

22

presumably the result of a delayed stomach emptying affected by the large dose of soybean oil


24

Page 25 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

administrated combined with a slow drug release leading to an incomplete absorption of the

2

administrated dose of fenofibrate. The tendency that the AUC0-24h increased with a decrease in

3

the amount of soybean oil administrated further supported the notion that the drug release

4

process was slow. The slow drug release from soybean oil was in this case expected to be a

5

consequence of fenofibrates high logP (5.1) and preference towards the soybean oil, which

6

should most probably be digested in order to release fenofibrate. In an older study by Palin

7

(1981) it was showed that the poorly water soluble compound DDT was absorbed to a lesser

8

degree when dissolved and administrated in 1 mL of indigestible oil (liquid paraffin) compared

9

to 1 mL of digestible oil (arachis oil and miglyol 812) or from an aqueous suspension32,

10

demonstrating the importance of digestion for drug release from lipid based formulations.

11

In vitro data

12

The levels of digestion presented in relation to the available amount of titratable FA (2 % (w/v)

13

formulations, Table 3) are comparable with previous studies, e.g. Cuiné et al. found that 30 % of

14

EL and 7.5 % of RH40 was digested during 60 min of in vitro lipolysis5. In another study by

15

Bardelmeijer et al. up to 30 %4 of EL administrated to mice as an oral solution with ethanol and

16

paclitaxel, was degraded in the GI tract to form free ricinoleic acid, which is also consistent with

17

the results presented here.

18

Christiansen et al. found that 14 % of EL and 6 % of RH40 was digested within 90 min of

19

lipolysis10, further supporting the range of digestion for the individual surfactants, but also the

20

relative digestion in all cases showing that EL was more susceptible to digestion compared to

21

RH40.

In an in vitro study utilising a rather different lipolysis setup,


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 38

1

The present data showed that the amount of dose solubilised during in vitro lipolysis correlated

2

with the in vivo data for ELP showing a statistical significant (p < 0.05) increase in fenofibrate

3

bioavailability (measured as the AUC0-24h) when increasing the surfactant concentration from 2

4

% (w/v) to 20/25 % (w/v), and suggested that the lower AUC0-24h at the lowest surfactant level

5

may be due to drug precipitation in the GI tract. In vivo there was no significant difference in

6

AUC0-24h or Cmax for ELP at all concentrations and increasing concentrations of EL above 5 %

7

(w/v), however, there was a tendency that both parameters increased with increasing amounts of

8

surfactant throughout the tested concentration range. This tendency was not reflected in vitro,

9

where both 15 % (w/v) and 25 % (w/v) ELP/EL solubilised the entire dose of fenofibrate

10

throughout the duration of the digestion experiment. RH40 kept the dose solubilised throughout

11

the lipolysis experiment at all concentration levels. The lack of variation within the solubilising

12

capacity of the RH40 formulations did not correlate with the corresponding plasma

13

concentration-time profiles after administration of the same formulations displaying an apparent

14

maximum in AUC0-24h at 15 % (w/v) RH40, (Figure 1C). The higher solubilising capacity of

15

RH40 compared to EL and ELP, relates well to the digested percentage of the surfactants, i.e.

16

ELP was digested to a higher degree compared to RH40 and therefore displays a lower

17

solubilising capacity upon digestion.

18

For all tested types of Kolliphor™ the amount of drug solubilised during in vitro lipolysis studies

19

did not sufficiently predict the in vivo performance of the different fenofibrate formulations.

20

These model shortcomings might reflect that the model was simply not predictive for the rat or

21

that digestion of the surfactants and solubilisation of the drug was not the only mechanisms

22

important when predicting the absorption of fenofibrate from surfactant based formulations. As

23

described in the introduction the use of surfactants may affect drug absorption through several


26

Page 27 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

mechanisms e.g. surfactants may inhibiting efflux and influx transporters involved in the drug

2

absorption, modify or delay gastric emptying and micellarly entrap the drug thereby decreasing

3

the free fraction of the drug and reducing the concentration gradient across the intestinal

4

membrane3-7. In the case of RH40, which was digested to a lesser degree compared to ELP and

5

EL, the in vivo absorption might be limited by micellar entrapment, which is not accounted for in

6

the lipolysis model. Furthermore, the in vivo data showed a tendency of delayed tmax values with

7

increasing surfactant concentrations (ELP and EL), which might reflect a prolonged and/or

8

delayed gastric emptying possibly affecting fenofibrates residence time in the small intestine and

9

thereby the absorption. This factor was not accounted for in the in vitro lipolysis model, either.

10

Thomas et al. also saw a lack of correlation between the amounts of fenofibrate solubilised

11

during in vitro lipolysis studies and the AUC0-24h of plasma concentration-time profiles from

12

lipid based drug delivery systems administrated orally to minipigs. In that study, the in vitro

13

studies predicted a difference between a series of formulations, which was not seen in vivo33.

14

This shows that one need to carefully design and optimize in vitro models to predict in vivo

15

performance.

16

In vivo-In vitro Correlations

17

Different hypotheses for the effect of EL, ELP and RH40 on the absorption of fenofibrate in rats

18

were tested in the present study. Based on results from a series of studies performed by

19

Sparreboom et al. and Malingre et al. showing that EL limited the absorption of paclitaxel from

20

the gut, it was hypothesised that Kolliphor™ would entrap fenofibrate in micelles within the GI

21

tract and thereby inhibiting the absorption. The interaction between paclitaxel and EL was

22

studied in vivo in humans and mice and in a series of binding and dialysis studies performed in

23

vitro. In vivo oral solutions with paclitaxel and different concentrations of EL were tested, with


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 38

1

analysis of paclitaxel, EL and their main metabolites in plasma and faeces. In vitro it was shown

2

that the affinity of paclitaxel was much greater towards EL (in concentrations above CMC)

3

compared to pure water, plasma and human serum albumin. Based on the results from both in

4

vivo and in vitro studies, it was concluded that EL decreased the absorption of paclitaxel by

5

entrapment in micelles4,11,12. A similar effect would be evident in the present study, if increasing

6

surfactant concentrations above CMC decreased the uptake of fenofibrate. The present data

7

showed no indication of ELP and EL micelles entrapping fenofibrate, as increasing levels of ELP

8

and EL (in concentrations above CMC) increased the absorption of fenofibrate. However, in the

9

case of RH40, the highest bioavailability of fenofibrate was obtained from RH40 formulations

10

with surfactant content of 15% (w/v). This apparent maximum was not seen in vitro where the

11

surfactants solubilising capacity remained unchanged throughout the digestion experiments.

12

However, this discrepancy between in vitro and in vivo results corresponds well with the micellar

13

trapping hypothesis, suggesting that the reduced absorption of fenofibrate from the formulation

14

containing the highest level of surfactant (25 % w/v), might be caused by some degree of

15

trapping within RH40 micelles. This idea was supported by the fact that RH40 was less

16

digestible compared to ELP and EL, i.e. the RH40 micelles were less degraded and therefore

17

more adept to keep fenofibrate trapped. According to the micellar trapping theory, which is

18

based on the same concepts as the solubility-permeability interplay described by Dahan and co-

19

workers7,34, increasing surfactant concentrations above CMC will increase micellar solubilisation

20

and decrease the free fraction of the drug thereby decreasing the amount of drug available for

21

intestinal permeation. However, the overall effect of the surfactants on the drug absorption will

22

depend on several possible effects aside from micellar solubilisation e.g. inhibition of efflux


28

Page 29 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

transporters and tight junction opening7,34. However, as the model drug used in the present study,

2

fenofibrate, is not a substrate for the Pgp-efflux35 this effect was ignored.

3

A second hypothesis proposed that ELP and EL are digested in the GI tract thereby lowering the

4

solubilising capacity and consequently decreasing the absorption of fenofibrate. Additionally,

5

RH40 should be digested to a lesser degree compared to ELP and EL in the GI tract producing a

6

higher absorption of fenofibrate compared to ELP and EL. This hypothesis was primarily based

7

on the studies performed by Cuiné et al. which showed that the bioavailability of danazol in

8

beagle dogs was reduced when the proportional content of EL relative to lipid (soybean oil) in

9

self-emulsifying drug delivery systems (SEDDS), was increased. In vitro, they found that EL

10

contributed to the formation of very fine dispersions and that increasing concentration (up to 50

11

% w/v) increased the drug load. However, compared to long-chain lipids, EL did not appear to

12

enhance the solubilising capacity in an intestinal environment as significantly. Cuiné and co-

13

workers suggested that the poor solubilisation properties of EL may reflect its susceptibility to

14

digestion by pancreatic enzymes. Formulations comprising high quantities of digestible

15

surfactant appeared less effective in preventing danazol precipitation in vitro. In vivo, no

16

significant difference in bioavailability was seen between formulations based on pure surfactants

17

(100 % w/w) EL and RH40. SEDDS comprised of 70 % EL or RH40, 20% soybean

18

oil:Maisine™ 35-1(1:1 w/w) and 10 % ethanol gave different results as the bioavailability of

19

RH40-SEDDS was approximately two-fold higher than the EL-SEDDS formulation5,13. In the

20

present study RH40 was found to be less digestible as compared to ELP and EL during in vitro

21

lipolysis studies, in agreement with the studies performed by Cuiné et al.5 and Christiansen et

22

al.10. However, in vivo, RH40 lowered the AUC0-24h of fenofibrate in rats after oral

23

administration of 25 % (w/v) aqueous Kolliphor™ solutions compared to ELP and EL,


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 38

1

contrasting the results from the danazol study5. This difference in results might be explained by

2

differences in the study designs, e.g. different formulations, different animal species and

3

different model drugs with different physico-chemical characteristics, e.g. fenofibrate is

4

approximately 10 times more soluble in soybean oil than danazol16, but may also reflect a more

5

complex difference where the excipients act differently in a drug specific manner.

6 7

In conclusion, the present study showed that Kolliphor™ ELP and EL, in the tested concentration

8

range, led to no indication of micellar entrapment of fenofibrate in vivo. Increased concentrations

9

of both surfactants (ELP and EL) increased fenofibrate AUC0-24h values. For the RH40

10

formulations, an apparent fenofibrate absorption optimum was seen at 15 % (w/v) surfactant,

11

displaying both the highest AUC0-24h and Cmax. The reduced absorption of fenofibrate from the

12

formulation containing the highest level of surfactant (25 % w/v), is thought to be caused by

13

some degree of trapping within RH40 micelles.

14

In vitro, ELP and EL were found to be more prone to digestion compared to RH40, though not

15

affecting the in vivo results. The highest fenofibrate bioavailability was attained from

16

formulations with high ELP/EL (25 % (w/v)), indicating that these surfactants are the better

17

choice for solubilising fenofibrate in order to increase the absorption upon oral administration.

18

Due to drug dependant effects of the different types of Kolliphor™ more drug-surfactant studies

19

are needed in order to understand which surfactants are best suited for a given drug.

20 21

Acknowledgement


30

Page 31 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

The personnel in the animal facilities at H.Lundbeck A/S are thanked for their skilful help during

2

the conduction of this study. Special thanks to Yanghwan Yun for carrying out a substantial

3

amount of the in vitro lipolysis experiments.

4

This work has been supported by the Predicting Drug Absorption Innovation Consortium (PDA).


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 38

1 2

Reference List

3 4 5 6

1.

Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. H. Strategies to Address Low Drug Solubility in Discovery and Development. Pharmacol. Rev. 2013, 65 (1), 315499.

7 8 9 10

2.

Norinder, U.; Haeberlein, M. Calculated Molecular Properties and Multivariate Statisical Analysis in Absorption Prediction. In Drug Bioavailability. Estimation of Solubility, Permeability, Absorption and Bioavailability., van de Waterbeemd, H., Lennernäs, H., Artursson, P., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim: 2003; pp 358-405.

11

3.

Gibaldi, M.; Feldman, S. Mechanisms of surfactant effects on drug absorption. J. Pharm. Sci. 1970, 59 (5), 579-589.

12 13 14

4.

Bardelmeijer, H. A.; Ouwehand, M.; Malingre, M. M.; Schellens, J. H.; Beijnen, J. H.; van, T. O. Entrapment by Cremophor EL decreases the absorption of paclitaxel from the gut. Cancer Chemother. Pharmacol. 2002, 49 (2), 119-125.

15 16 17

5.

Cuine, J. F.; Mcevoy, C. L.; Charman, W. N.; Pouton, C. W.; Edwards, G. A.; Benameur, H.; Porter, C. J. H. Evaluation of the impact of surfactant digestion on the bioavailability of danazol after oral administration of lipidic selfemulsifying formulations to dogs. J. Pharm. Sci. 2008, 97 (2), 995-1012.

18 19 20

6.

Martin-Facklam, M.; Burhenne, J.; Ding, R.; Fricker, R.; Mikus, G.; Walter-Sack, I.; Haefeli, W. E. Dose-dependent increase of saquinavir bioavailability by the pharmaceutic aid cremophor EL. Br. J. Clin. Pharmacol. 2002, 53 (6), 576-581.

21 22

7.

Dahan, A.; Miller, J. M. The solubility-permeability interplay and its implications in formulation design and development for poorly soluble drugs. AAPS J. 2012, 14 (2), 244-251.

23 24 25

8.

Ali, S.; Kolter, K. Challenges and Opportunies in Oral Formulation Development. American Pharmaceutical Review . 12-10-2012.

26 27

9.

Gelderblom, H.; Verweij, J.; Nooter, K.; Sparreboom, A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur. J. Cancer 2001, 37 (13), 1590-1598.

28 29

10.

Christiansen, A.; Backensfeld, T.; Weitschies, W. Effects of non-ionic surfactants on in vitro triglyceride digestion and their susceptibility to digestion by pancreatic enzymes. Eur. J. Pharm. Sci. 2010, 41 (2), 376-382.

30 31 32

11.

Sparreboom, A.; Van Zuylen, L.; Brouwer, E.; Loos, W. J.; De Bruijn, P.; Gelderblom, B.; Pillay, M.; Nooter, K.; Stoter, G.; Verweij, J. Cremophor EL-mediated alteration of paclitaxel distribution in human blood: Clinical pharmacokinetic implications. Cancer Res. 1999, 59 (7), 1454-1457.

33 34 35

12.

Malingre, M. M.; Schellens, J. H.; van, T. O.; Ouwehand, M.; Bardelmeijer, H. A.; Rosing, H.; Koopman, F. J.; Schot, M. E.; Ten Bokkel Huinink, W. W.; Beijnen, J. H. The co-solvent Cremophor EL limits absorption of orally administered paclitaxel in cancer patients. Br. J. Cancer 2001, 85 (10), 1472-1477.

36 37 38

13.

Cuine, J. F.; Charman, W. N.; Pouton, C. W.; Edwards, G. A.; Porter, C. J. H. Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs. Pharm. Res. 2007, 24 (4), 748-757.

39 40

14.

Amidon, G. L.; Lennernas, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: the correlation of in vitro drug product dissolution and in vivo bioavailability. Pharm. Res. 1995, 12 (3), 413-420.


32

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1 2

15.

Granero, G. E.; Ramachandran, C.; Amidon, G. L. Dissolution and solubility behavior of fenofibrate in sodium lauryl sulfate solutions. Drug Dev. Ind. Pharm. 2005, 31 (9), 917-922.

3 4

16.

Persson, L. C.; Porter, C. J.; Charman, W. N.; Bergstrom, C. A. Computational prediction of drug solubility in lipid based formulation excipients. Pharm. Res. 2013, 30 (12), 3225-3237.

5 6

17.

Balfour, J. A.; McTavish, D.; Heel, R. C. Fenofibrate. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic use in dyslipidaemia. Drugs 1990, 40 (2), 260-290.

7 8

18.

Zhu, T.; Ansquer, J. C.; Kelly, M. T.; Sleep, D. J.; Pradhan, R. S. Comparison of the gastrointestinal absorption and bioavailability of fenofibrate and fenofibric acid in humans. J. Clin. Pharmacol. 2010, 50 (8), 914-921.

9 10 11

19.

Borkar, N.; Xia, D.; Holm, R.; Gan, Y.; Mullertz, A.; Yang, M.; Mu, H. Investigating the correlation between in vivo absorption and in vitro release of fenofibrate from lipid matrix particles in biorelevant medium. Eur. J. Pharm. Sci. 2014, 51, 204-210.

12 13 14

20.

Hanafy, A.; Spahn-Langguth, H.; Vergnault, G.; Grenier, P.; Grozdanis, M. T.; Lenhardt, T.; Langguth, P. Pharmacokinetic evaluation of oral fenofibrate nanosuspensions and SLN in comparison to conventional suspensions of micronized drug. Advanced Drug Delivery Reviews 2007, 59 (6), 419-426.

15 16

21.

Zangenberg, N. H.; Mullertz, A.; Kristensen, H. G.; Hovgaard, L. A dynamic in vitro lipolysis model. I. Controlling the rate of lipolysis by continuous addition of calcium. Eur. J. Pharm. Sci. 2001, 14 (2), 115-122.

17 18

22.

Zangenberg, N. H.; Mullertz, A.; Kristensen, H. G.; Hovgaard, L. A dynamic in vitro lipolysis model. II: Evaluation of the model. Eur. J. Pharm. Sci. 2001, 14 (3), 237-244.

19 20

23.

Cizek, L. J. Total water content of laboratory animals with special reference to volume of fluid within the lumen of the gastrointestinal tract. Am. J. Physiol 1954, 179 (1), 104-110.

21 22

24.

Fernandez, S.; Rodier, J. D.; Ritter, N.; Mahler, B.; Demarne, F.; Carriere, F.; Jannin, V. Lipolysis of the semi-solid selfemulsifying excipient Gelucire 44/14 by digestive lipases. Biochim. Biophys. Acta 2008, 1781 (8), 367-375.

23 24 25 26 27

25.

Williams, H. D.; Sassene, P.; Kleberg, K.; Bakala-N'Goma, J. C.; Calderone, M.; Jannin, V.; Igonin, A.; Partheil, A.; Marchaud, D.; Jule, E.; Vertommen, J.; Maio, M.; Blundell, R.; Benameur, H.; Carriere, F.; Mullertz, A.; Porter, C. J.; Pouton, C. W. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations. J. Pharm. Sci. 2012, 101 (9), 3360-3380.

28 29 30

26.

Matsaridou, I.; Barmpalexis, P.; Salis, A.; Nikolakakis, I. The influence of surfactant HLB and oil/surfactant ratio on the formation and properties of self-emulsifying pellets and microemulsion reconstitution. AAPS PharmSciTech. 2012, 13 (4), 1319-1330.

31 32

27.

Buch, P.; Langguth, P.; Kataoka, M.; Yamashita, S. IVIVC in oral absorption for fenofibrate immediate release tablets using a dissolution/permeation system. J. Pharm. Sci. 2009, 98 (6), 2001-2009.

33 34

28.

Li, X.; Gu, L.; Xu, Y.; Wang, Y. Preparation of fenofibrate nanosuspension and study of its pharmacokinetic behavior in rats. Drug Dev. Ind. Pharm. 2009, 35 (7), 827-833.

35 36

29.

Sunesen, V. H.; Vedelsdal, R.; Kristensen, H. G.; Christrup, L.; Mullertz, A. Effect of liquid volume and food intake on the absolute bioavailability of danazol, a poorly soluble drug. Eur. J. Pharm. Sci. 2005, 24 (4), 297-303.

37 38 39

30.

Hirata, T.; Keto, Y.; Yamano, M.; Yokoyama, T.; Sengoku, T.; Seki, N. Inhibitory effect of ramosetron on corticotropin releasing factor- and soybean oil-induced delays in gastric emptying in rats. J. Gastroenterol. Hepatol. 2012, 27 (9), 1505-1511.

40 41

31.

Hunt, J. N.; Stubbs, D. F. The volume and energy content of meals as determinants of gastric emptying. J. Physiol 1975, 245 (1), 209-225.


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 38

1 2

32.

K.J.Palin Effect of Oils on Drug Absorption. Department of Pharmacy and Physiology and Pharmacology, University of Nottingham, 1981.

3 4 5

33.

Thomas, N.; Richter, K.; Pedersen, T. B.; Holm, R.; Mullertz, A.; Rades, T. In vitro lipolysis data does not adequately predict the in vivo performance of lipid-based drug delivery systems containing fenofibrate. AAPS J. 2014, 16 (3), 539-549.

6 7 8

34.

Miller, J. M.; Beig, A.; Krieg, B. J.; Carr, R. A.; Borchardt, T. B.; Amidon, G. E.; Amidon, G. L.; Dahan, A. The solubility-permeability interplay: mechanistic modeling and predictive application of the impact of micellar solubilization on intestinal permeation. Mol. Pharm. 2011, 8 (5), 1848-1856.

9 10 11 12 13

35.

Ehrhardt, M.; Lindenmaier, H.; Burhenne, J.; Haefeli, W. E.; Weiss, J. Influence of lipid lowering fibrates on Pglycoprotein activity in vitro. Biochem. Pharmacol. 2004, 67 (2), 285-292.


34

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

AUTHOR INFORMATION

2

Corresponding Author

3

*Anette Müllertz

4

Department of Pharmacy

5

Universitetsparken 2

6

2100 Copenhagen

7

+ 45 35 33 64 40

8

[email protected]

9 10

Author Contributions

11

The manuscript was written through contributions of all authors. All authors have given approval

12

to the final version of the manuscript.


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 36 of 38

Table 1. Composition and fenofibrate solubility of formulations utilised for in vivo pharmacokinetic studies and in vitro lipolysis studies. Formulation

Fenofibrate concentration [mg/mL]

Volume administrated [ml/kg]

0.2 10 PEG-Ia a 0.4 5 PEG-II a 0.2 10 Soybean-I 0.4 5 Soybean-IIa 0.2 10 ELP-2 0.2 10 ELP-5a a 0.2 10 ELP-10 0.2 10 ELP-15 0.2 10 ELP-20a 0.2 10 ELP-25 0.2 10 EL-2 0.2 10 EL-15 0.2 10 EL-25 0.2 10 RH40-2 0.2 10 RH40-15 0.2 10 RH40-25 a Not included in the in the in vitro lipolysis studies

Excipient [% w/v] ELP EL RH40 PEG Soybean 400 oil

2 5 10 15 20 25 -

2 15 25 -

2 15 25

100 100 -


100 100 -

Solubility of fenofibrate in formulation [mg/mL] Mean ± SD > 20 > 20 >5 >5 0.50 ± 0.005 1.18 ± 0.02 2.47 ± 0.06 3.62 ± 0.05 4.73 ± 0.19 5.86 ± 0.18 0.46 ± 0.01 4.06 ± 0.17 6.72 ± 0.10 0.31 ± 0.01 2.91 ± 0.11 4.53 ± 0.19

Dose as percentage of equilibrium solubility Mean

Kolliphor Surfactants Affect Solubilization and Bioavailability of

Recommend Documents