ARTICLE
Auteur(s) : Vincent Jannin
Pharmaceutical R&D, Gattefossé S.A.S., Saint-Priest, 69804,
France
Introduction
Most of the new chemical entities developed by the pharmaceutical
industry are practically insoluble in water and consequently
possess a low oral bioavailability [1]. These poorly-water soluble
molecules are classified in the class 2 and 4 of the
Biopharmaceutics Classification System [2]. In order to efficiently
formulate these active substances for the oral route, formulators
should either increase the dissolution of the drug in the
gastro-intestinal tract or pre-dissolve the drug into the
formulation and avoid its precipitation when in contact with the
biological fluids. Many formulation techniques can increase the
dissolution of poorly-water soluble drugs such as micronization,
inclusion in cyclodextrins, addition of surfactants or lipid-based
excipients.
The use of lipids and lipid-based excipients in self-emulsifying
systems is more and more described in the literature as well as
used in marketed products. Recently a classification of these
lipid-based systems was introduced and characterized [3] and in
addition, many formulation techniques have been developed to
produce solid or semi-solid systems [4].
Among these self-emulsifying systems
Gelucire® 44/14, a PEG-32 lauroyl
polyoxylglycerides (Gattefossé SAS, Saint-Priest, France), is
obtained by polyglycolysis of hydrogenated coconut oil (medium and
long chain triacylglycerols) and PEG-32. It is composed of a
defined admixture of C8-C18 mono-, di- and triacylglycerols
(20% w/w), PEG-32 mono- and diesters and free PEG-32 (80%
w/w). The main fatty acid present is lauric acid which accounts for
45% on average of the total fatty acids [5-7].
Gelucire® 44/14 has been widely used and
characterized during the last five years in order to increase the
solubility and bioavailability of many drugs: carbamazepine [8],
glibenclamide [9], antiviral agent PG301026 [10], piroxicam [11,
12], propranolol [13, 14], flurbiprofen [15], aceclofenac [16],
carvedilol [17], griseofulvin [18], spironolactone [19], and
cinnarizine [11].
The aim of this paper is to present the physical and
biopharmaceutical characterizations needed to develop a successful
formulation with Gelucire® 44/14.
Physical characterization
As Gelucire® 44/14 is a semi-solid crystalline
excipient, formulators should characterize the structure of the
mixture containing this lipid-based vehicle and the drug substance
to ascertain that their formulation is in its most stable form and
retains its self-emulsifying properties. In this chapter, the main
physical characterization tests needed are presented firstly on the
raw material and secondly with two model drug substances.
Thermal analysis
Figure 1 shows
the thermogram of the first melting of a Gelucire®
44/14 sample recorded with a Differential Scanning Calorimeter
(Pyris Diamond, Perkin-Elmer, USA) calibrated with benzoic acid
(Tm = 122.4 °C) and indium (Tm =
156.6 °C, ΔHm = 26.6 J/g). The thermal
analysis was carried out between − 20 and 120 °C at a
heating rate of 3 °C/min on a 10 mg sample.
Gelucire® 44/14 presents a broad endotherm ranging
from 10 to 45 °C with an onset melting temperature of
38.2 °C and a peak melting temperature of 43.2 °C. This
thermal behaviour can be explained by the composition of the
excipient, a mixture of acylglycerols and PEG esters. The
Differential Scanning Calorimetry (DSC) analyses of these two
fractions separately (figure 1) show that the
acylglycerol fraction melts first (representing the first two
endothermic events), and then the PEG ester and free PEG fraction
melts last (representing the last and main endothermic event).
Crystalline structure
The combined use of X-Ray Diffraction (XRD) and DSC allows the
detection of all polymorphs formed after various thermal treatments
ranging from quenching into liquid nitrogen to slow
crystallization. XRD allows the study of the structure and the
polymorphism of lipid-based compound. Wide-Angle X-ray Scattering
(WAXS) region corresponds to short reticular distances between
hydrocarbon chains while Small-Angle X-ray Scattering (SAXS) domain
corresponds to long spacing. DSC, by temperature and enthalpy of
phase-transition measurement, shows energy transfers that occur
during the heating or cooling of the sample. By combining these two
techniques, one can link structural changes to phase transitions.
Gelucire® 44/14 is crystallized in lamellar
phases with the PEG chains under a helical conformation. This
crystalline structure has already been reported for another
polyoxylglycerides containing the same ethylene oxide unit:
Gelucire® 50/13 [20]. SAXS analysis of the
untreated sample shows a lamellar phase of 120 Å (figure 2). No signal of
the acylglycerol fraction was detected. Various polymorphs with
shorter lamellar phases such as 90, 94, 99 or 105 Å were
detected when the product is melted and crystallized with different
crystallization rates. Shorter structures were due to the more or
less important tilt of the PEG chains. However during the heating
of these freshly crystallized samples, we observed a progressive
phase transition from the tilted lamellar phases to the most stable
phase of 120 Å. Figure 3 shows that the
sample evolves by itself to its most stable form after storage of
21 hours at 25 °C. This phenomenon was confirmed with
WAXS analysis of samples crystallized slowly at 0.1 °C/min or
by quenching into liquid nitrogen (figure 4). WAXS
measurement also allowed detecting acylglycerols under a hexagonal
lattice.
This study demonstrates that Gelucire®
44/14 evolves to the most stable form (lamellar phase of
120 Å) whatever the crystallization rate applied during the
formulation process if left 21 hours at 25 °C.
Hydration and emulsification performance
Naproxen and sodium naproxen were chosen as model drugs to evaluate
the impact of drug polarity on the wettability and emulsification
performance of Gelucire® 44/14. Model drugs were added
at 10% w/w to the molten excipient under stirring. The mixture
obtained with Gelucire® 44/14 was either a
solid solution in the case of naproxen or a solid dispersion with
sodium naproxen as detected by DSC and XRD [21].
Gelucire® 44/14 and these two mixtures were used to
form films with an Automatic Film Applicator (Sheen-1137, height =
2 mm, spreading rate = 0.05 ms−1).
Gelucire® 44/14 film shows an irregular surface,
with slopes and localized folds [21]. The inclusion of naproxen
results in a smoother surface. The inclusion of sodium naproxen
gives a surface which is more broken; numerous edges and deep
cavities of 50 μm of diameter can be observed. This film
crystallizes quicker than the other ones, giving a more porous
structure. In this case the drug acts as a nucleation enhancer
where newly-formed crystals diffuse toward existing drug crystals
creating cavities in the film. The surface of the film containing
sodium naproxen is also affected by the morphology of the drug
crystals as this substance is not soluble in Gelucire®
44/14 [22].
Wettability of these films by water was characterized by
goniometry (G1 Krüss goniometer, Krüss GmbH, Germany). Initial
contact angles are similar whatever the drug used and are identical
to those obtained with Gelucire® (53.1 ± 4.9°, 57.9 ±
5.6°, and 64.8 ± 7.8° for Gelucire® 44/14, the mixture
containing naproxen, and the mixture with sodium naproxen,
respectively). This shows that
Gelucire® 44/14 hydrophilicity dominates the
polarity differences of the two drug models as all initial contact
angles were below 65°, implying a hydrophilic surface which is
required for rapid emulsification in a lipid-based self emulsifying
system. However the contact angle at the equilibrium was
statistically higher for the mixture containing naproxen due to the
hydrophobicity of the drug. On the other hand, the rate of
spreading of the water drop (i.e. wettability) is similar for
Gelucire® 44/14 alone and the mixture with naproxen
(– 1.68 ± 0.5°/s, and – 1.86 ± 0.5°/s, respectively), but
dramatically decreased for the mixture with sodium naproxen due to
the broken surface of the sample (– 2.89 ± 0.5°/s) [21].
Figure 5
presents the variation of viscosity and refraction index of
Gelucire® 44/14 during the discrete addition
of water at 45 °C under stirring (100 rpm). The addition
of up to 13% w/w of water into the molten Gelucire®
44/14 leads to a liquid solution with the same viscosity as
the excipient itself (below 100 mPa.s). This amount of water
is needed to completely hydrate the ethylene oxide units in the PEG
ester and free PEG fraction [23]. The system slowly becomes a
transparent gel with the addition of 13 to 43% w/w of water,
the viscosity steadily increasing from 0.1 to 1 Pa.s.
Then a high viscosity gel is formed between 43 and 61% w/w of
water. The increase of viscosity measured up to 7 Pa.s is due
to the formation of cubic mesophases. This gel slowly erodes and
emulsifies with the addition of increasing amounts of water to
become a turbid gel with low viscosity (61 to 75% w/w of
water), then a turbid liquid system (75 to 90% w/w of water)
and finally a translucent liquid system for high aqueous dilutions.
This last system is characterized as a microemulsion with a
particle size distribution of 80 nm (measured with a photon
correlation spectrophotometer, PSS Nicomp, USA).
These studies demonstrate that Gelucire® 44/14
hydrophilicity dominates the polarity of the model drugs and favour
the hydration of the lipid-based systems leading to a cubic phase
system that erodes/emulsifies with the hydrodynamic of the aqueous
environment.
Biopharmaceutical characterization
In the previous chapter the ability of
Gelucire® 44/14 to evolve into a stable
crystalline form and to self-emulsify in contact with water in
vitro was presented. However, this excipient is a lipid-based
system, containing both acylglycerols and PEG esters that can be
hydrolyzed by lipases. In this chapter enzymes able to lipolyze
Gelucire® 44/14 and the effects of this
hydrolysis on drug performance are presented. In addition, the
influence of Gelucire® 44/14 on
enterocyte-based proteins and the absorption of drugs into the
enterocyte are briefly discussed.
Lipolysis
The lipolysis of Gelucire® 44/14 and its
fractions was evaluated by measuring the release of free fatty
acids (FFAs) with a pH-stat apparatus (718 STAT Titrino,
Metrohm, Switzerland) adjusted to a constant end-point value [24].
An emulsion of 500 mg Gelucire® 44/14 in
15.0 mL of an assay solution (NaCl 150 mM; NaTDC
4 mM; CaCl2 1.4 mM; Tris-HCl 1 mM) was
mechanically stirred (450 rpm) in a temperature-controlled
reaction vessel at 37 °C. Before adding enzymatic solution, we
waited for 5 min until the gel phase disappeared and a
translucent medium is obtained. The pH was kept constant using an
automated burette to titrate FFAs with a 0.1 M NaOH solution.
Activities were expressed in international units: 1 U
corresponds to 1 μmol of FFAs released per minute. Specific
activities were expressed as U per mg of pure enzyme.
Table 1 presents the specific
activities of four lipases on
Gelucire® 44/14 and its components:
acylglycerol fraction and PEG fraction [7]. Human Pancreatic Lipase
(HPL), the main lipase involved in the digestion of dietary
triacylglycerols, does not show any significant activity on
Gelucire® 44/14 (2 ± 2 U/mg) nor on either of
its fractions. Other pancreatic lipases such as Human Pancreatic
Lipase-Related Protein 2 (HPLRP2) show low activity on
Gelucire® 44/14 (12 ± 1 U/mg) although the
highest activity of HPLRP2 is that observed on the
acylglycerol fraction (333 ± 0 U/mg). In addition, this enzyme
shows low activity on the PEG ester fraction. Carboxyl Ester
Hydrolase (CEH) shows high activity on
Gelucire® 44/14 (96 ± 2 U/mg), and the
highest activity of CEH is that recorded on the PEG ester fraction
(50 ± 12 U/mg). The highest activity of all the enzymes tested
is that of Dog Gastric Lipase (DGL) on
Gelucire® 44/14 (108 ± 10 U/mg), although
DGL shows low activity on the PEG ester fraction.
Gastric lipase probably plays an essential role in the in vivo
digestion of Gelucire® 44/14, although it is less
abundant than pancreatic lipase in the human digestive system. In
addition, the main pancreatic enzyme involved in the intestinal
digestion step of Gelucire® 44/14 is probably
CEH.
Table 1 Specific activities of Human Pancreatic Lipase
(HPL), Human Pancreatic Lipase Related Protein 2 (HPLRP2), Carboxyl
Ester Hydrolase (CEH), and Dog Gastric Lipase (DGL) on
Gelucire® 44/14 and its two fractions: acylglycerols and
PEG esters. Specific activities are expressed in U/mg as mean ±
standard deviation (n = 2).
|
Enzymes
|
Specific activity (U/mg)
|
|
Gelucire® 44/14
|
Acylglycerols fraction
|
PEG esters fraction
|
|
rHPL
|
2 ± 2
|
20 ± 0
|
6 ± 0
|
|
rHPLRP2
|
12 ± 1
|
333 ± 0
|
5 ± 2
|
|
CEH
|
96 ± 2
|
163 ± 7
|
50 ± 12
|
|
DGL
|
108 ± 10
|
106 ± 3
|
21 ± 6
|
Impact of lipolysis on the solubilising performance of
Gelucire® 44/14
The in vitro gastrointestinal lipolysis of
Gelucire® 44/14 was then investigated to
understand which compounds are, after digestion, responsible for
keeping poorly water-soluble drugs in solution [11]. Experimental
conditions were adapted from in vivo data recorded at 50% gastric
emptying of test meals, both in the stomach and in the duodenum,
and enzymatic solutions were prepared according to in vivo
secretions of lipases during a meal [25]. Experiments were
performed over a period of 90 minutes to simulate the
gastrointestinal digestion of lipids. An emulsion of
Gelucire® 44/14 with either piroxicam or
cinnarizine in the assay solution was mechanically stirred in a
temperature-controlled reaction vessel at 37 °C. Then a
freshly prepared gastric enzymatic solution (rDGL) was added to the
reaction vessel and the pH was kept constant at 5.5 during
30 min (gastric step of lipolysis), via an automated titration
of FFAs with 0.1 M NaOH using a pH-stat device. After the
gastric step, a freshly prepared pancreatic enzymatic solution was
added to the mixture (dilution by 1.7-fold) and the pH was shifted
to 6.25 and kept constant for 60 min. At different time
points, samples were taken to assay each component of
Gelucire® 44/14 and also the percentage of
drug dissolved in the aqueous phase.
During the gastrointestinal lipolysis of
Gelucire® 44/14, monoacylglycerols and PEG esters
are the largest compounds present at the end of gastric phase, and
PEG mono and diesters are the largest compounds after the duodenal
phase.
Solutions of Gelucire® 44/14 with either
piroxicam or cinnarizine were formulated to evaluate the
precipitation of these active substances during the
gastrointestinal lipolysis of the excipient [11]. The precipitation
of piroxicam is mainly due to the gastric lipolysis of
Gelucire® 44/14 nevertheless the aqueous solubility
of this drug is increased 4-fold due to the metabolites of the
lipid-based excipient. With respect to the formulation of
cinnarizine with Gelucire® 44/14, drug
precipitation is only associated with the dilution of the gastric
medium by the pancreatic juice until it reaches the composition of
the duodenal medium. However, at the end of simulation of the
gastrointestinal lipolysis, the aqueous solubility of cinnarizine
formulated with Gelucire® 44/14 is increased
132-fold when compared with its aqueous solubility without
excipient. This study highlights the importance of gastrointestinal
lipolysis and the associated phenomena such as the dilution of
chyme by biliary and pancreatic secretions in vivo, on the
solubilisation of poorly water-soluble drugs formulated with
Gelucire® 44/14 [11].
Interaction with enterocyte-based proteins
The absorption and bioavailability of active substances can be
limited by enterocyte-based proteins such as P-glycoprotein (P-gp),
an efflux protein that transports the drug out of the cell, or by
cytochrome P450 enzyme that transform active substances into
metabolites. In both cases the access of the drug to systemic
circulation is limited and its bioavailability reduced.
Gelucire® 44/14 demonstrated its inhibitory
effect on efflux proteins such as P-gp both in vitro diffusion
chambers [26] and with Caco-2 cells monolayer [27, 28]. The
inhibition of the efflux of Rhodamine 123 in diffusion
chambers is observed for concentrations of Gelucire®
44/14 ranging from 1.0 to 10.0% v/v. These concentrations
are higher than the critical micellar concentration of the
excipient (0.01% v/v) suggesting that the drug should be
included into micelles in order to increase its absorption [26].
Recently it has been proposed that Gelucire®
44/14 specifically inhibits P-gp and not Breast Cancer
Resistance Protein (BCRP) another efflux protein [29].
Gelucire® 44/14 has also shown its ability to
inhibit the metabolism of active substances by cytochrome
P450 on human liver microsomes [28, 30].
Acknowledgements
We gratefully thank all our academic partners who help us
characterizing Gelucire® 44/14 over the past five years:
Prof. Odile Chambin (Pharmaceutical Technology Group, EMMA Team, EA
581, Faculté de Pharmacie, Dijon, France), Dr. Frédéric Carrière
and Dr. Sylvie Fernandez (Laboratoire d’Enzymologie Interfaciale et
de Physiologie de la Lipolyse, CNRS UPR 9025, Marseille, France),
Dr. Jean-Blaise Brubach and late Dr. Michel Ollivon (Laboratoire de
Physicochimie des Systèmes Polyphasés, UMR 8612, Faculté de
Pharmacie, Châtenay Malabry, France).
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