ARTICLE
Auteur(s) : Sylvie Fernandez1,2, Frédéric
Carrière2, Vincent Jannin1
1Gattefossé SAS, 36, chemin de Genas, 69804
Saint-Priest, France
2Laboratoire d'enzymologie interfaciale et de
physiologie de la lipolyse, CNRS UPR 9025, 31, chemin
Joseph-Aiguier, 13402 Marseille cedex 20, France
Oral administration of drugs exhibiting poor solubility in
gastrointestinal fluids is one of the main challenges of the
pharmaceutical industry. To improve oral bioavailability of poorly
water-soluble drugs, innovative formulations such as
self-emulsifying drug delivery systems (SEDDS), and
self-microemulsifying drug delivery systems (SMEDDS), are developed
(Charman et al., 1997; Holm et al., 2003). SEDDS and
SMEDDS are isotropic mixtures of oil, surfactant, cosurfactant, and
drug that form, under gentle stirring, oil-in-water emulsions or
microemulsions, respectively (Charman et al., 1992). Among the
different self-emulsifying excipients available,
Labrasol® and Gelucire® 44/14 are
macrogolglycerides able to form microemulsions in contact
with gastrointestinal fluids. Labrasol® and
Gelucire® 44/14 are obtained by polyglycolysis of
medium-chain triacylglycerols or medium- and long-chain
triacylglycerols and PEG-8 or PEG-32, respectively. These
excipients are a mixture of mono-, di-, and triacylglycerols and
mono- and diesters of PEG, and free PEG. Labrasol® and
Gelucire® 44/14 significantly increase the
bioavailability of various poorly water-soluble drugs (Hauss
et al., 1998; Hu et al., 2001; Eaimtrakarn et al.,
2002; Barker et al., 2003; Rama Prasad et al., 2003; Rama
Prasad et al., 2004), but the mechanisms underlining these
effects still remain to be elucidated.
In humans, the digestion of dietary triacylglycerols starts in
the stomach with the gastric lipase (Carriere et al., 1993).
The free fatty acids (FFAs) released in the stomach are essential
to stimulate biliary and pancreatic secretions, as well as for the
subsequent action of pancreatic lipase (Gargouri et al., 1986;
Bernback et al., 1989). Thanks to the gastric emptying, the
lipolysis continues in the duodenum where the chyme is mixed with
bile and pancreatic juice which contains several lipolytic enzymes
able to hydrolyze various natural substrates such as
triacylglycerols, phospholipids, galactolipids, and vitamin esters
(De Caro et al., 2004; Eydoux et al., 2007). We have
already shown that in vitro, Labrasol® and
Gelucire® 44/14 were hydrolyzed by several digestive
lipases (Fernandez et al., 2007; Fernandez et al., 2008).
These findings suppose that in vivo, these excipients are
hydrolyzed by digestive lipases and their lipolytic products might
play an important role in the transport of the drug from the
formulation to the mixed micelles and/or the unstirred water layer
next to the enterocytes.
To mimic the gastrointestinal lipolysis, we developed an in
vitro method that considers both gastric and duodenal steps of
digestion, the first step being not taken into consideration in
most of the studies on bioavailability and/or solubility of poorly
water-soluble drugs (Sek et al., 2001; Sek et al., 2002).
Using this model, we studied the changes in the composition of both
Labrasol® and Gelucire® 44/14 during
their in vitro lipolysis. The aim of this study was to perform
similar experiments in presence of a poorly water-soluble drug,
cinnarizine, a piperazine derivative with antihistaminic activity
and mainly used for the control of vomiting due to motion sickness,
and to follow the concentration of cinnarizine dissolved in the
aqueous phase during the lipolysis of Labrasol® or
Gelucire® 44/14.
Experimental procedure
Labrasol® and Gelucire® 44/14 are lipid-based
excipients composed of acylglycerols (mono-, di-, and
triacylglycerols, 10% and 20% w/w, respectively) and of PEG esters
(mono- and diesters) and free PEG corresponding to 90% w/w and 80%
w/w of Labrasol® and Gelucire® 44/14,
respectively.
Experimental conditions were based on in vivo data recorded at
50% gastric emptying of test meals, both in the stomach and in the
duodenum. Enzyme solutions were prepared based on the lipase levels
recorded in vivo during a meal (Carriere et al., 2000). The
gastric enzyme solution was prepared using recombinant
dog gastric lipase (rDGL) and the pancreatic enzyme solution
was prepared using porcine pancreatic extracts (PPE).
Experiments were performed during 90 min to simulate
gastrointestinal digestion of lipids (Fernandez et al., 2009).
An emulsion of either Labrasol® or
Gelucire® 44/14 in the assay solution was
mechanically stirred in a temperature-controlled reaction vessel at
37 °C. At t = 0 min, the gastric lipase solution was added to
the reaction vessel and, using a pH-stat device, the pH was kept
constant at 5.5 for 30 min (gastric step of lipolysis), via an
automated titration of FFAs with 0.1 M NaOH. At t =
30 min, pancreatic enzyme solution was added to the mixture
and the pH was shifted to 6.25 and kept constant for 60 min.
At several time points, samples were taken and treated to be
analyzed. Acylglycerols were assayed by GPC-FID method, whereas the
quantification of PEG derivatives (PEG esters and free PEG) was
done by HPLC (Fernandez et al., 2009).
The same experiments as described above were performed using 25
mg cinnarizine formulated in 1.5 g of either
Labrasol® or Gelucire® 44/14. The amount
of cinnarizine dissolved in aqueous solution was determined by
HPLC. A control experiment was also performed without enzymes
to determine the influence of experimental conditions such as
dilution and pH on cinnarizine solubilization.
In vitro gastrointestinal lipolysis
of Labrasol®
and Gelucire® 44/14
To identify which compounds and/or lipolysis products of
Labrasol® and Gelucire® 44/14 were
responsible for maintaining poorly water-soluble drugs in aqueous
solution, we studied the changes in the composition of both
excipients during their gastrointestinal lipolysis. The
experiments, performed here, were based on in vivo data recorded
during test meal digestion. The gastric lipolysis step was
simulated using rDGL which is known to be a good model of human
gastric lipase (Bodmer et al., 1987; Carriere et al.,
1991; Vaganay et al., 1998), at pH 5.5, the mean pH value
found in the stomach at 50% of gastric emptying. A solution of
PPE was then added to the reaction mixture in order to perform the
duodenal step of lipolysis. PPE were used as a model of human
pancreatic juice. Indeed, we previously showed that human
pancreatic juice and porcine pancreatic extracts presented similar
lipolytic activities on both Labrasol® and
Gelucire® 44/14 (Fernandez et al., 2007; Fernandez
et al., 2008), although their enzyme compositions are slightly
different. Moreover, the volume of pancreatic enzyme solution added
was adapted to obtain a 1.7-fold dilution of the gastric phase
which corresponds to the dilution of the chyme by biliary and
pancreatic secretions that occurs in vivo (Carriere et al.,
2000). It is important to mention that dilution can also affect
drug solubility. The pH value was increased to 6.25 during the
duodenal step of lipolysis which corresponds to the mean pH value
found in the duodenum during digestion of a test meal at 50% of
gastric emptying.
Figure 1
presents the variations in the composition of Labrasol®
and Gelucire® 44/14 during their in vitro
gastrointestinal lipolysis. For each family of compounds, the
results are expressed in percent of the initial composition and as
a function of time. Concerning free PEG, the results shown here
correspond to the free PEG released, expressed in percent of the
amounts of free PEG initially present in excipients.
During the gastric lipolysis step of Labrasol®,
C8-C10 di- and triacylglycerols and PEG-8 diesters were rapidly
hydrolyzed, whereas C8-C10 monoacylglycerols and PEG-8 monoesters
were poor substrates of gastric lipase (figure 1A).
Consequently, at the end of gastric lipolysis phase, C8-C10
monoacylglycerols and PEG-8 monoesters were the main hydrolyzable
compounds of Labrasol® remaining before duodenal
lipolysis.
The addition of pancreatic enzymes led to major changes. Indeed,
C8-C10 monoacylglycerols were rapidly and totally hydrolyzed within
5 min after the addition of pancreatic enzymes contrary to the
residual C8-C10 di- and triacylglycerols and PEG-8 diesters, which
were slightly hydrolyzed during the duodenal step of lipolysis.
PEG-8 monoesters were also hydrolyzed as shown by the increase in
free PEG-8. At the end of the gastrointestinal simulation, the main
compounds of Labrasol® remaining were PEG-8 monoesters
and free PEG-8 (Fernandez et al., 2009).
During the gastric lipolysis step of
Gelucire® 44/14, results were quite different from
those found with Labrasol® (figure 1B). During
the gastric phase of lipolysis, C8-C18 di- and triacylglycerols
were rapidly hydrolyzed and an accumulation of C8-C18
monoacylglycerols was observed. PEG-32 mono- and diesters were not
hydrolyzed. Consequently, at the end of gastric lipolysis phase,
C8-C18 monoacylglycerols and PEG-32 mono- and diesters were the
main hydrolysable compounds of Gelucire® 44/14 remaining
before duodenal lipolysis.
After addition of pancreatic lipases, C8-C18 monoacylglycerols
and PEG-32 mono- and diesters rapidly decreased and free PEG-32 was
rapidly released. Residual C8-C18 diacylglycerols were also
hydrolyzed. At the end of the gastrointestinal simulation, the
main compounds of Gelucire® 44/14 remaining were
PEG-32 monoesters and free PEG-32 (Fernandez et al.,
2009).
Labrasol® and Gelucire® 44/14
correspond to a mixture of acylglycerols, responsible for the
solubilization of lipophilic drugs (Pouton and Porter, 2008), PEG
esters, amphiphilic molecules responsible for the surfactant
properties, and free PEG which plays a role of cosolvent.
In vivo, the hydrolysis of acylglycerols and PEG esters
contained in both excipients might have a major influence on the
fate of a poorly water-soluble drug in the gut. The results found
here suggest that in vivo, monoacylglycerols and PEG esters, and
particularly PEG monoesters, will have a major role to maintain
poorly water-soluble drugs in aqueous solution during the gastric
digestion.
Concerning the duodenal lipolysis phase, the results suggest
that PEG monoesters probably play a crucial role in vivo to
maintain the drug in solution in the small intestine. Concerning
free PEG, it seems difficult that this cosolvent has a major role
in the fate of the drug during the in vivo lipolysis. Indeed,
water-soluble cosolvents such as PEG-8 are used to increase the
solvent capacity of the formulation, but they loose rapidly their
solvent capacity after dispersion of the formulation in an aqueous
phase (Pouton and Porter, 2008).
Solubilization of cinnarizine during in vitro
gastrointestinal lipolysis of Labrasol®
and Gelucire® 44/14
We studied the concentration of cinnarizine dissolved in the
aqueous phase, during in vitro gastrointestinal lipolysis of either
Labrasol® or Gelucire® 44/14. For each
formulation, a blank experiment was performed without any enzyme,
to evaluate the precipitation of cinnarizine due to experimental
parameters such as assay solution, dilution, and pH variation.
Figure 2 shows
the variation with time of cinnarizine dissolved in the aqueous
phase during in vitro gastrointestinal lipolysis of the two
excipients. In both cases, lipid excipients led to a complete
solubilization of cinnarizine in the gastric milieu after
dispersion (t = 0 min).
When cinnarizine was formulated with Labrasol®, the
results obtained during the Labrasol® lipolysis were
significantly different from those obtained without enzymes (figure 2A). In
the absence of enzymes and therefore lipolysis, the dilution of the
formulation induced a dramatic precipitation of the drug which
increased with time until the end of the experiment. Under these
conditions, the percentage of cinnarizine remaining in solution was
63-times higher than its solubility in the assay solution at pH
6.25. Hence, Labrasol® helps keeping the drug in
supersaturation (63-fold) even after dilution and change of pH.
When Labrasol® was hydrolyzed by digestive lipases, the
overall cinnarizine precipitation was however much lower and the
concentration of cinnarizine dissolved in the aqueous phase was
equivalent to a 142-fold supersaturation. It is worth noticing
that, in this case, cinnarizine precipitation was initiated during
the gastric lipolysis step but was not significantly affected by
the dilution of the gastric medium by the pancreatic enzyme
solution.
During the gastrointestinal lipolysis of Labrasol®,
PEG-8 monoesters were the only compounds which remained at a high
level (figure 1A). One can
therefore assume that PEG-8 monoesters, the main surface active
components initially present in Labrasol®, were involved
in maintaining the drug dissolved during gastrointestinal lipolysis
of this excipient.
When cinnarizine was formulated with
Gelucire® 44/14, no differences were observed in
absence or in presence of enzymes (figure 2B).
Cinnarizine precipitation only occurred when the gastric medium was
diluted whatever the fate of Gelucire® 44/14
(hydrolyzed or not).
Since the percentage of PEG-32 monoesters remained high during
the gastrointestinal lipolysis (figure 1B), PEG-32
monoesters might play a key role to maintain cinnarizine in aqueous
solution, as in the case of Labrasol®. Cinnarizine
precipitation due to the dilution of the gastric medium might be
attributable to a greater solubilization of PEG-32 monoesters.
Whatever the experimental conditions, when cinnarizine was
formulated with Gelucire® 44/14, the solubility of
cinnarizine was increased as compared to solubility of cinnarizine
in the assay solution, leading to a 144-fold and 133-fold
supersaturation of cinnarizine with and without enzymes,
respectively.
Conclusion
We studied in parallel the evolution of the composition of
lipid-based excipients during their in vitro gastrointestinal
lipolysis and the resulting precipitation of a poorly water-soluble
drug. We showed that gastrointestinal lipolysis of both
Labrasol® and Gelucire® 44/14 had a
significant impact on the composition of both excipients. Our
results highlight the importance of gastric digestion step which
was not taken into account in the in vitro bioavailability studies
performed so far.
The cinnarizine precipitation strongly depended on the excipient
used. When cinnarizine was formulated with Labrasol®,
the lipolysis of Labrasol® significantly affected the
fate of cinnarizine. Precipitation of cinnarizine due to the
dilution of the gastric medium did not occur when
Labrasol® was hydrolyzed. This finding supposes that in
vivo Labrasol® lipolysis is a prerequisite to prevent
cinnarizine form precipitation and keep the drug in supersaturation
in the gastrointestinal milieu. No effect of the lipolysis was
observed on cinnarizine precipitation when using
Gelucire® 44/14 as lipid-based vehicle.
Thèse soutenue le 31 octobre 2008 à Marseille devant le
jury suivant :
Pr Frédéric Fotiadu : École centrale Marseille
(examinateur)
Dr Frédéric Carrière : CNRS (examinateur)
Dr Vincent Jannin (examinateur) : Gattefossé
Pr Anette Müllertz (rapporteur) : University of
Copenhagen, Denmark
Pr Thierry Vandamme (rapporteur) : Université Louis Pasteur
Acknowledgments
Sylvie Fernandez's PhD research was supported by a CIFRE contract
from Association Nationale de la Recherche Technique (ANRT,
France).
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