Biochimie 125 (2016) 297e305

Contents lists available at ScienceDirect

Biochimie
journal homepage: www.elsevier.com/locate/biochi

Review

Impact of gastrointestinal lipolysis on oral lipid-based formulations

and bioavailability of lipophilic drugs
de
ric Carrie
re
Fre
CNRS, Aix Marseille Universit
e, UMR7282 Enzymologie Interfaciale et Physiologie de la Lipolyse, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 6 September 2015
Accepted 15 November 2015
Available online 29 November 2015

Oil-in-water emulsions are common vehicles for lipids as nutrients and for the delivery of poorly watersoluble drugs. Enhancing oral bioavailability of these drugs using lipid-based formulations (LBF) or selfemulsifying drug delivery systems is one of the current challenges in pharmaceutical industry. Many of
the compounds found in LBF (acylglycerols, surfactants with esteried fatty acids, ) are however potential substrates for digestive enzymes. Their digestion (or lipolysis) in the gastrointestinal (GI) tract is
critical for drug dissolution and absorption: it can be benecial (drug solubilization/dispersion) or
deleterous (drug precipitation) depending on the drug-LBF association. A better understanding of the
fate of LBF in the GI tract is therefore required to engineer efcient lipid-based drug delivery systems.
In vitro models for testing simultaneously LBF digestion and drug dispersion are in development to
predict drug solubilization and bioavailability, select the best drug-LBF association and obtain better
in vitro-in vivo correlations. So far, research in this area has focused on LBF lipolysis under intestinal
conditions because the small intestine is the main target for drug delivery and absorption, as well as the
main site of digestion by pancreatic enzymes. Lipolysis however starts within the stomach through the
action of gastric lipase, the rst enzyme involved in fat digestion in humans. In vitro digestion experiments show that most LBFs are submitted to gastric lipolysis, and therefore, both intragastric and intestinal digestions are critical for the fate of LBF and drug solubility.
 te
 Franaise de Biochimie et Biologie Mole
culaire (SFBBM). All rights
2015 Elsevier B.V. and Socie
reserved.

Keywords:
Drug delivery
In vitro digestion
Lipase
Lipids
Lipolysis
o/w emulsions

Contents
1.
2.
3.
4.
5.
6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Lipid-based formulations: a novel way forward in drug development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Improving the solubility/dispersion and the bioavailability of poorly soluble drugs using LBFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Changing pharmacokinetics and optimizing drug bioavailability using LBFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
Gastrointestinal lipolysis of LBFs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304

1. Introduction
Abbreviations: CEH, pancreatic carboxyl ester hydrolase; GI, gastrointestinal; GL,
gastric lipase; LBF, lipid-based formulation; LC, long chain acylglycerols; MC, medium chain acylglycerols; PL, classical pancreatic lipase; PLRP2, pancreatic lipaserelated protein 2; rDGL, recombinant dog gastric lipase; SEDDS, self-emulsifying
drug delivery system; SMEDDS, self-microemulsifying drug delivery system; sPLA2IB, pancreatic phospholipase A2.
E-mail address: carriere@imm.cnrs.fr.

Oral delivery is the preferred route for drug administration

thanks to several advantages over other routes (non invasive, less
expensive, less potential for side effects such as injection site reactions, etc.) and is the easiest and most convenient method of
drug delivery for treatment of chronic pathologies. The importance of the oral drug administration route is illustrated by the

http://dx.doi.org/10.1016/j.biochi.2015.11.016
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 Franaise de Biochimie et Biologie Mole
culaire (SFBBM). All rights reserved.
0300-9084/ 2015 Elsevier B.V. and Socie

F. Carriere / Biochimie 125 (2016) 297e305

298

fact that >60% new molecular entities excluding biologics

received FDA approval as oral formulations in 2014 [1]. The majority of new chemical entities (NCEs) identied as drug candidates via high throughput screening procedures are however
insoluble or poorly soluble in water, and around 70% of these NCEs
belong to the Biopharmaceutics Classication System (BCS [2])
class II of drug substances presenting low solubility and high
permeability. BCS class II drugs represent 30% of the top 200 drugs
marketed in the USA and this is equivalent to the proportion of
marketed BCS class I drugs presenting both high solubility and
permeability [3]. Formulating NCEs for oral delivery is currently
one of the greatest challenges faced by scientists in the pharmaceutical industry. Although it is still believed that combinatorial
chemistry can be used for improving the water solubility of
pharmacophores, one has to keep in mind that the lipophilicity of
drugs is often tightly associated with their target-binding characteristics and therefore, their efcacy. The large diversity of NCEs
remains today under-exploited.
Lipid-based formulations (LBFs) or lipid-based drug delivery
systems (LBDDS) of lipophilic drugs are becoming more and more
attractive in that context [4] but their development and association
with drugs remains largely empirical. In vitro-in vivo correlations
are usually very poor, mainly because current standard methods for
testing drug dissolution and predict bioavailability are not adapted.
Indeed, these simple dissolution methods do not include the
digestive enzymes found in the gastrointestinal (GI) tract while
most components of LBFs are potential substrates for lipolytic enzymes [5]. LBFs can therefore be digested along the GI tract before
reaching the site of drug absorption and this digestion process can
affect drug dispersion and solubilization. The digestibility of LBFs is
today well recognized and novel methods for testing LBFs under
more physiologically relevant conditions are currently under
development, notably in the framework of academia-industry
partnerships like the Lipid Formulation Classication System
(LFCS; see http://www.lfcsconsortium.org/) [6e11]. In addition to
some highlights on the use of LBFs for oral drug delivery, this review is dedicated to LBF lipolysis by the various digestive lipases
found in the GI tract and its impact on drug solubilization. While
research in this area has focused on LBF digestion by pancreatic
enzymes under intestinal conditions, the role of gastric lipase in LBF
lipolysis is emerging and new ndings in this area are reported
here.
2. Lipid-based formulations: a novel way forward in drug
development
LBF can enhance oral bioavailability of a poorly water soluble
drug primarily by improving its solubility and/or dispersion in the
aqueous environment of the GI tract. They can also favorably

impact intestinal permeability, stabilize the drug and reduce in

some particular cases its potential degradation by the digestive
enzymes present in the GI tract. Overall, LBF can improve drug
delivery to the patient's systemic circulation. It is also envisioned
that LBF could be used for the controlled release of active agents
embedded in a digestible lipid matrix.
Additionally, LBFs offer several advantages in the formulation of
the drug itself. Nonpolar drugs with low aqueous solubility (i.e.
<20 mg/mL) are more easily solubilized in a lipid matrix. Formulating BCS class II drug is often challenging due to difculties in
achieving content uniformity and LBFs are well adapted for the
obtention of isotropic mixtures. Substances with low melting point
(i.e. <100
C), including those that are liquid at room temperature,
are often hard to formulate into dry powders and this process
usually requires large quantities of excipients. Sensitivity to moisture can also affect the chemical stability of drugs and hence their
in vivo performance. This can be addressed by formulating the drug
in a lipophilic matrix.
LBFs are usually based on various mixtures of oils, surfactants,
and cosolvents. They have been classied according to their
composition (Table 1) [12,13]. Depending on this composition, LBFs
can present various physical characteristics in the presence of water
(non-dispersed oil phase, emulsions of different sizes, micellar and
lamellar structures) [14,15], each characteristic presenting various
kinds of advantages for monitoring drug solubilization, dispersion
and absorption (Table 1). For instance, lipid formulations of type III
including an oil phase and surfactants are the basis for the so-called
Self-Emulsifying Drug delivery Systems (SEDDS) that spontaneously generates a dispersed oil phase in the presence of water. The
self-emulsication process is illustrated in Fig. 1 with optical microscope pictures taken from a drop of Labral M2125CS, a
mixture of corn oil glycerides (oil phase), PEG-6 (co-solvent) and
PEG-6 esters (surfactants), added to water. The generation of small
oil droplets from an initial large drop of oil-surfactant mixture is
due to the presence of surfactants that lower surface tension at the
oil-water interface. Various mechanisms can be involved in
dispersion of small lipid droplets by self-emulsication, such as
diffusion and stranding, osmotic pressures imbalances, phase
transformations and changes in pH conditions. Diffusion and
stranding depends on the initial presence of a solute (surfactant)
in one phase (e.g., oil phase made of glyceride mixture), that is also
soluble in the second phase (water) and couples these phases
within a limited composition range [16]. It is generally assumed
that drug bioavailability enhancement by SEDDS results from the
nely dispersed state of the drug-containing lipid droplets and
increased surface area of interaction with the GI uids that favors
the partitioning of the drug from the lipid phase to the colloidal
aqueous phase containing micelles and lamellar aggregates of
biliary lipids and lipolysis products. Nevertheless, the droplet size

Table 1
The lipid formulation classication system (LFCS).a
Compounds in formulation

Oils: triglycerides or mixture of tri-, di- and

monoglycerides
Lipophilic surfactants (HLB<12)
Hydrophilic surfactants (HLB>12)
Cosolvents (e.g. PEG, propylene glycol, Transcutol)
Appearance on dispersion in water
a

Content of formulation (%, w/w)

Type I

Type II

Type IIIb

Type IV

Oils

SEDDS

SMEDDS

Lipid-free

100

35e80

40e80

e
e
e
No or limited dispersion

20e65
e
e
Coarse/opaque o/w
emulsion; >100 nm

e
20e40
0e40
Fine emulsion to transparent
dispersion; 20e100 nm

e
50
50
Micellar solution; z10 nm

Inspired by Professor Colin Pouton's Lipid Formulation Classication System [12,13].

Type III LBFs can be further divided into Type IIIA and Type IIIB LBFs in order to identify more hydrophilic systems (Type IIIB) where the content of hydrophilic surfactants
and cosolvents increases and the lipid content reduces [44].
b

F. Carriere / Biochimie 125 (2016) 297e305

299

Fig. 1. Self-microemulsifying drug delivery system. The pictures sequence shows the self-microemulsication of a drop of Labral M2125CS in water by a mechanism of diffusion
and stranding. These pictures were taken at 5, 10, 15, 20 and 25 s after the Labral M2125CS drop was added to water. They were extracted with permission from a movie made by
 S.A.S, St Priest, France; copyright 2007). Labral M2125CS is a mixture of tri-, di and monoglycerides (oil phase), PEG-6 (co-solvent) and PEG-6
C. Bruley C. and V. Jannin (Gattefosse
esters (surfactants) obtained from the reaction of PEG-6 with corn oil. Other names of Labral are Linoleoyl macrogol-6 glycerides (EP), Linoleoyl polyoxyl-6 glycerides (NF), Corn oil
PEG-6 esters (FDA IIG).

distribution might not be preserved upon GI lipolysis because of

dynamic variations in LBF composition.
The size of the lipid particles dispersed in the aqueous phase can
be engineered using various lipid/surfactant/cosolvent mixtures to
obtain microemulsions (SMEDDS) and nanoemulsions (SNEDDS).
3. Improving the solubility/dispersion and the bioavailability
of poorly soluble drugs using LBFs
Drug solubility and absorption are key factors in a drug's efcacy. A drug can only be effective once it has dissolved and
permeated through the intestinal barrier where the absorption of
nutrients and most drugs naturally occurs. The fate of a drug in the
GI tract depends on various physical changes that occur during its
dispersion in the contents of the GI tract, the dilution of these
contents at various stages of the GI tract by the endogeneous secretions (saliva, gastric juice, pancreatic juice, bile), and the digestive process that might in some cases affect the drug chemical
stability (Fig. 2). Poorly water soluble drugs would precipitate to a

large extent and would be inactive if specic vehicles were not used
to solubilize/disperse these drugs in the gastric and intestinal
contents after oral administration. The main advantage of LBF is
that a lipophilic drug can remain dispersed in solution throughout
its transit in the GI tract. The absorption of the drug in the intestine
is then achieved by either passive diffusion of the drug from the
lipid vehicle to the membranes of enterocytes (interfacial transfer)
or release of the drug upon degradation of the lipid matrix by
gastrointestinal lipolysis (Fig. 2). These processes are certainly not
exclusive and the lipid components of LBFs, their lipolysis products
and the lipophilic drug are probably re-organized in complex
structures with bile lipids (bile acids, phospholipids and cholesterol) before reaching the brush border of the small intestine where
absorption occurs (Fig. 2).
Conventional excipients are not well adapted for the formulation of lipophilic drugs. LBFs were mainly developed after it was
observed that the oral bioavailability of poorly water soluble drugs
could be increased when they were administered with high fat
meals [17,18]. Several lipid-based vehicles for these drugs were

Fig. 2. Oral drug delivery with LBF in the GI tract. Unlike traditional drug formulations, the chemical composition and physical structure of LBFs is changed after oral administration
in a process analogous to the digestion of dietary lipids [4,5,42]. Lipid digestion is mediated rst by gastric lipase in the stomach and then by pancreatic enzymes in the small
intestine. The lipid digestion products generated are subsequently solubilized by bile salts and mixed with other biliary lipids (phospholipids, cholesterol) to form a range of
colloidal structures in the small intestine uids, mainly mixed micelles. These micelles play the role of shuttle to transfer lipolysis products (fatty acids, monoglycerides, lysophopsholipids) from the intestinal lumen to the intestinal wall for absorption by enterocytes. LBFs exploit these processes naturally present in the GI tract to deliver poorly watersoluble drugs in the intestinal lumen, solubilize them in colloidal structures resulting from lipolysis and mixture with bile, and promote their absorption by enterocytes. Like dietary
lipids, lipophilic drugs can be further delivered to lymph and systemic circulation using the chylomicrons secreted from enterocytes as vehicles.

300

F. Carriere / Biochimie 125 (2016) 297e305

used in clinical trials over the last 20 years and LBFs have enabled
many new drugs to be evaluated. The use of LBFs also augments the
uptake of highly lipophilic drugs (log P > 5, lipid solubility > 50 mg/
g) into the lymph system, the favorite route for the lipoproteins
(chylomicrons) assembled in the enterocytes and further excreted
into the lymph. This pathway may reduce rst pass metabolism of
the drug in the liver, thus increasing the drug's oral bioavailability
[18e20]. Direct drug access to intestinal lymphatic system may
alter systemic distribution patterns. This could result in enhanced
exposure to the drug and improved efcacy, especially if the agent
has a direct effect on the immune system, e.g. antiretroviral and
anticancer drugs. It was shown with Halofantrine that the
lymphatic drug transport is low in absence of lipid (fasting conditions), but the co-administration of the drug with microemulsions
of long chain lipids increased signicantly the cumulative recovery
of the drug in the lymph of dogs [19] and rats [20].
4. Changing pharmacokinetics and optimizing drug
bioavailability using LBFs
Reformulating existing drugs with poor aqueous solubility using
LBFs can improve their pharmacokinetics and bioavailability, thus
achieving a better therapeutic effect. As an illustration, results obtained with Probucol, an anti-hyperlipidemic drug for the treatment of coronary artery disease, are shown in Fig. 3A and Table 2.

Table 2
Pharmacokinetics of Probucol absorption in minipigs following oral administration
in either self-nano-emulsifying drug delivery system (SNEDDS), oil solution or
classical powder formulation. Cmax, maximum plasma concentration values; Tmax,
time for measuring the maximum plasma concentration of the drug; AUC, areaunder-the-curve deduced from the variation of Probucol plasma concentration as
a function of time (see Fig. 3A). Adapted with from Ref. [24].
Probulcol formulation

Cmax (mg.mL1)

Tmax (h)

AUC0e48h (h.mg.mL1)

SNEDDS
Oil solution
Powder formulation

1.84 0.53
0.98 0.23
0.24 0.05

5.0 [5.0e6.0]
7.0 [5.0e8.0]
24.0 [5.0e48]

26.2 8.8
19.9 4.1
7.5 2.9

The rate of Probucol absorption estimated from plasma levels (areaunder-the-curve, AUC) and maximum plasma concentration values
(Cmax) was found to be increased with lipid formulations, while the
kinetics of absorption (time for measuring the maximum plasma
concentration of the drug, Tmax) was accelerated. The bioavailability
of several drugs was thus enhanced when they were formulated in
LBFs e.g. coenzyme Q10, a-tocopherol, cyclosporine A, Danazol [21],
Paclitaxel [22,23], Probucol [24], Erlotinib [25].
In addition to an increased oral bioavailability, drugs formulated
in LBFs have more predictable release kinetics. For instance, the
dose linearity of cyclosporine pharmacokinetics from a microemulsion formulation (Neoral) is improved when compared to the

Fig. 3. Various effects of LBFs on the pharmacokinetics of lipophilic drug absorption. Panel A: Fasted state study of Probucol oral bioavailability in minipigs. Mean plasma concentration versus time proles (n 6) for Probucol following oral administration (10 mg/kg) to minipigs fasted overnight and fed 4 h after drug administration. Probucol was
administered in gelatine capsules containing either self-nano-emulsifying drug delivery system (SNEDDS), oil solution or classical powder formulation. Adapted from Ref. [24];
Panel B: Comparison of Sandimmune and Neoral (LBF) formulations of cyclosporine, based on the relationship between cyclosporine dose and AUC deduced from the variation of
cyclosporine plasma concentration as a function of time. Adapted from Ref. [26]; Panel CeD: Cyclosporine plasma concentration-time proles following single oral administration of
the 300-mg reference formulation (C) and 180-mg LBF (Neoral) formulation (D) under fasting conditions or with a fat-rich meal (mean values from 24 healthy male volunteers).
Adapted from Ref. [18]. Panel E: mean plasma concentration (n 8) of vitamin E as a function of time following oral administration of 400 IU in the form of a self-emulsifying
preparation and soft gelatin capsule containing pure soybean oil. Adapted from Ref. [27].

F. Carriere / Biochimie 125 (2016) 297e305

pharmacokinetics obtained with the oil-and-alcohol Sandimmune

formulation (Fig. 3B) [26].
Moreover, it has been shown that the absorption of the LBF
Neoral formulation of cyclosporine was less affected by food than
the standard formulation. Whereas the rate of cyclosporine absorption is slowed by a fat-reach meal using standard formulation
(Tmax 4.8 1.8 h vs. 2.5 0.9 h in fasting conditions; Fig. 3C), the
rate of absorption of the LBF Neoral formulated cyclosporine is
faster and is not signicantly changed by food intake
(Tmax 1.5 0.4 h in fasting conditions vs. 1.8 0.7 h in fed conditions; Fig. 3D) [18]. In addition, the bioavailability of 180-mg
cyclosporine LBF formulation (Cmax 1011 192 ng/mL) is 1.6fold the bioavailability of 300-mg cyclosporine reference formulation (Cmax 645 248 ng/mL) in fasting conditions.
As shown in the case of cyclosporine, LBFs have already been
used to re-formulate several drugs on the market. Danazol,
launched in 1970 for the treatment of endometriosis, was reformulated in 2008 using a LBF containing a digestible surfactant
[21]. Vitamin E (Natopherol) marketed as an OTC drug in the US by
Abbott since 1972 was re-formulated in 2000 with LBF to improve
its absorption [27]. The self-emulsifying preparation achieved a
faster rate and higher extent of absorption than the Natopherol
formulation available as soft gelatin capsules, under fasting condition. The AUC values of the self-emulsifying preparation are much
higher (between 2- and 4-fold) than those of the soft gelatin
capsule product (Fig. 3E). Ibuprofen, mainly available in the form of
tablets for oral administration, was re-formulated in a liquid form
using lipid nanocapsules and positive effects on drug absorption
were observed with an 18% increase in the AUC and a 27% higher
mean residence time when compared to tablets.
Drug re-formulation using LBF can be used not only for
improving pharmacokinetics and bioavailability, but also to extend
product lines (lipid formulations, tablets, etc.) or provide new opportunities to extend intellectual property on new applications of
existing drug substances. An oil-in-water emulsion was used for
instance for encapsulating the poorly soluble anti-cancer drug
Paclitaxel [23]. The appropriate selection of lipid excipients and
formulation strategies at the earliest stages of drug development
can lead to considerable savings in both the cost and time to show
drug efcacy and bring it to the market. Potent new oral drugs
would not have reached the clinic in the absence of these improved
formulations. Some LBFs facilitate controlled release of the drug,
resulting in more patient friendly dosing schedules e which may
lead to better tolerability and, as a result of improved compliance,
enhanced efcacy. The pharmacokinetics and bioavailability of
LBFs-formulated drugs are less affected by the presence of food
than those of drugs formulated with non-lipid excipients (Fig. 3C
and D). As a result, patients may not have to co-ordinate their dietary and drug taking schedules, and this exibility could improve
compliance. LBFs also allows presenting adapted formulation to
patients, such as oral suspension instead of tablets for children e.g.
Griseofulvin (Grifulvin) oral suspension.

301

5. Gastrointestinal lipolysis of LBFs

There is a new interest in lipid digestion in the context of LBF
development because the lipolysis of LBF compounds, along with
that of dietary fats, affects the solubility, dispersion and bioavailability of poorly water-soluble drugs. LBF digestion (or lipolysis) in
the GI tract is critical for drug dissolution and absorption: it can be
benecial (drug solubilization/dispersion by lipolysis products such
as fatty acids) or deleterous (drug precipitation after digestion of
the oil phase or changes in colloidal structures) depending on the
drug-LBF association. Indeed, many of the compounds present in
LBFs, such as acylglycerols, phospholipids, polyethyleneglycol
mono- and di-esters, polysorbates (Tweens), contain ester functions and can be hydrolyzed by the various esterases present in the
GI tract. This includes gastric lipase (GL), the rst enzyme involved
in fat digestion in humans [28], and several pancreatic enzymes,
among which classical colipase-dependent pancreatic lipase (PL),
pancreatic carboxyl ester hydrolase (CEH), pancreatic lipase-related
protein 2 (PLRP2) and pancreatic phospholipase A2 (sPLA2-IB). The
respective roles and targets of these enzymes in LBF digestion was
recently reviewed [5]. GL and LP are true lipases that preferentially
hydrolyze substrates forming oil-in-water emulsions like triacylglycerols (TAG) and diacylglycerols (DAG) while CEH, PLRP2
and sPLA2-IB have a preference for micellar substrates. These three
latter enzymes are all active on phospholipids found in mixed micelles and display various levels of activity and specicity (phospholipase A1 activity for CEH and PLRP2; phospholipase A2 activity
for sPLA2-IB). While sPLA2-IB is strictly specic of phospholipids,
CEH and PLRP2 have broad substrate specicity and they also hydrolyze monoacylglycerols (MAG) at high rates. Among natural
substrates, they are also active on galactolipids (PLRP2 >> CEH),
vitamin esters (CEH, PLRP2) and cholesterol esters (CEH only).
Some of these enzymes are also active on non-natural esters and
surfactants present in LBFs, like PEG esters [29,30], polysorbates
(Tweens) and glycerol-polyethylene glycol ricinoleate (polyethoxylated castor oil) [11]. Although the various lipolytic enzymes
present in the GI tract are well characterized, it is still assumed in
most publications dealing with LBF digestion that pancreatic lipase
is the main enzyme involved in the GI lipolysis of LBFs. Moreover,
since the small intestine is the main site of digestion by pancreatic
enzymes and the main target for drug delivery and absorption,
research in this area has focused on LBF lipolysis under intestinal
conditions and most models of in vitro GI lipolysis of LBFs use
porcine pancreatic extracts (pancreatin) as the sole source of
digestive enzymes [31]. These models are not optimum since they
do not take into account potential lipolysis by gastric lipase already
occurring in gastric contents and conditions. Nevertheless, they
have shed light on the relationship between LBF lipolysis and drug
solubilization depending on the type of LBF [7] and they have
allowed better in vitro-in vivo correlations [32]. The work of the
LFCS consortium has shown for instance that model LBFs spanning
the range of the four LFCS classes (type I to IV; Table 3) were all

Table 3
Compositions of representative LBFs spanning the LFCS classes. LC, long chain acylglycerols; MC, medium chain acylglycerols. A more detailed description of
acylglycerol mixtures can be found in Ref. [7].
LBF type

Composition (% w/w)

I-LC
II-LC
IIIA-LC
I-MC
II-MC
IIIA-MC
IIIB-MC
IV

Corn oil: mixed glycerides of predominantly linoleic acid (50/50)

Corn oil: mixed glycerides of predominantly linoleic acid: Tween 85 (32.5/32.5/35)
Corn oil: mixed glycerides of predominantly linoleic acid: polyethoxylated castor oil (32.5/32.5/35)
Tricaprate/tricaprylate triglycerides: mixed glycerides of capric/caprylic acid (50/50)
Tricaprate/tricaprylate triglycerides: mixed glycerides of capric/caprylic acid: Tween 85 (32.5/32.5/35)
Tricaprate/tricaprylate triglycerides: mixed glycerides of capric/caprylic acid: polyethoxylated castor oil (32.5/32.5/35)
Mixed glycerides of capric/caprylic acid: polyethoxylated castor oil: di-ethylene glycol monoethyl ether (25/50/25)
Polyethoxylated castor oil: di-ethylene glycol monoethyl ether (50/50)

302

F. Carriere / Biochimie 125 (2016) 297e305

subjected to lipolysis at various rates and extent depending on their

composition (long chain (LC) versus medium chain (MC) acylglycerols; amounts and type of surfactants; presence of a cosolvent)
[7]. In the same study, the solubilization of a model drug, Danazol,
was tested using the panel of eight representative LBFs (Table 3)
and a standard procedure involving a 30-min step of GI lipolysis
monitored with a pHstat device, followed by phase separation (oily
phase, aqueous colloid phase and insoluble pellet) by

ultracentrifugation and estimation of Danazol partitioning between

these phases. The oily phase contained a mixture of incompletely
digested lipid and a fraction of the lipophilic drug initially incorporated in LBF, the aqueous colloid phase contained the solubilized
drug and the pellet phase contained the precipitated drug. Danazol
solubilization was highly dependent on LBF composition, but
poorly correlated with simple performance indicators such as
dispersion efciency. No attempt was made for correlating the

Fig. 4. Danazol solubilization in the course of in vitro lipolysis of various LBFs. The solubilization of a model drug, Danazol, was tested using eight representative LBFs ranging from
type I to IV LFCS classes (see Table 1 for denition and Table 3 for composition; LC and MC abbreviations correspond to long chain and medium chain acylglycerols, respectively) and
a standard procedure involving a 30-min step of GI lipolysis monitored with a pHstat device, followed by phase separation by centrifugation and estimation of Danazol partitioning
between the phases. The plots presented here were built from the data reported in Ref. [7] (Figure 8 and Table 3). Panels A, C and E show Danazol distribution in the aqueous phase,
oil phase and insoluble pellet, respectively, as a function of the lipolysis level (% of total fatty acids release upon lipolysis). Panels B, D and F show Danazol distribution in the same
phases as a function of the amount of fatty acids that are released upon lipolysis (mmoles).

F. Carriere / Biochimie 125 (2016) 297e305

303

extent of LBF lipolysis or free fatty acid levels with drug solubilization in this work. In order to investigate new correlations, the
data on LBF lipolysis and Danazol solubilization reported separately
in Ref. [7] were used here to draw Fig. 4. As shown in Fig. 4A, Danazol solubilization in the aqueous phase appeared to be higher
(40e60%) with LBFs showing the highest lipolysis levels, most of
them consisting of MC acylglycerols (types I-MC, II-MV, IIIA-MC).
MC acylglycerols are usually hydrolyzed at high rates by digestive
lipases because of their dispersion properties and the fact that they
are less dependent on the presence of surfactants to form ne
dispersions upon stirring. A formulation with LC acylglycerol from
corn oil (IIIA-LC) was also hydrolyzed at high levels and was efcient for solubilizing Danazol, probably because the presence of
hydrophilic surfactants (Table 3; polyethoxylated castor oil; Cremophor EL from BASF) at high levels allowed the spontaneous
formation of a corn oil microemulsion (SMEDDS; particle size of
around 60 nm). The activity of digestive lipases is usually increased
upon increasing the specic area (surface unit per volume unit) of
the oil-water interface [33], unless lipase adsorption at this interface is impaired by competition with surfactants [34,35]. LC formulations with no (type I-LC) or low amounts of surfactants (type
II-LC) were much less subjected to lipolysis and as a consequence
most Danazol (>80%) remained in the oil phase (Fig. 4B). These
latter LBFs are not efcient for a fast solubilization of the drug in the
aqueous phase under these experimental conditions but, nevertheless, they avoid drug precipitation (Fig. 4E) and they might be
good vehicles for delivering the drug at a slower rate in the small
intestine.
Some positive correlation between Danazol solubilization in the
aqueous phase and the lipolysis level was therefore observed, with
the exception of type IIIB-MC that showed low solubilization capacity in water (<20%) although it was hydrolyzed at a high level
(>70% lipolysis). This MC LBF contains however high levels of surfactants and co-solvent compared to the oil phase (Table 3) and the
amounts of fatty acid released upon lipolysis are much lower than
those released from other MC LBFs. Another representation of Danazol solubilization as a function of fatty acid levels gives a better
correlation (Fig. 4B), suggesting that free fatty acids are important
for solubilizing the drug in the aqueous phase. Formulation IIIA-LC
deviates from these ndings, probably because long chain fatty
acids have a higher capacity to form colloidal structures with

lipophilic drugs.
Paradoxically, the formulations leading to the highest precipitation of Danazol (>85%; Fig. 4EeF) and weak solubilization in
water (Fig. 4AeB) were those in which Danazol shows the highest
initial solubility: 48.7 mg/g and 65.5 mg/g for types IIIB-MC and
type IV formulations, respectively (Fig. 5). There is in fact no correlation between the initial Danazol solubility in LBF and the solubilization of Danazol in aqueous phase (Fig. 5). Both type IIIB-MC
and type IV formulations contain large amounts of hydrophilic
surfactants (polyethoxylated castor oil; Cremophor EL from BASF)
and co-solvent (Table 3) that are efcient for dissolving Danazol in
the LBF. But upon mixing with water, bile salts and phospholipids
present in simulated intestinal uid, these compounds probably
reorganized in colloidal structures that do not allow Danazol solubilization and lead to its precipitation. These in vitro ndings
correlate well with the reduction of Danazol oral bioavailability in
beagle dogs when the proportional content of Cremophor EL relative to lipid is increased in SEDDS formulations [36].
The dominating idea that pancreatic lipase (PL) was the main
enzyme involved in LBF digestion was challenged when the lipolysis of two SMEDDS, Labrasol and Gelucire 44/14 was tested
in vitro with several puried lipases [29,30]. It was established that
gastric lipase (GL), pancreatic carboxyl ester hydrolase (CEH) and
pancreatic lipase-related protein 2 (PLRP2) had a much higher
lipolytic activity than PL on these LBFs containing a mixture of
acylglycerols and PEG esters. These ndings have shown that the
lipolysis of LBFs may actually start in the stomach and involve
several lipolytic enzymes, like the digestion of dietary lipids. For
testing LBF digestion by GL, in vitro experiments were carried using
a pHstat device, eight representative LBFs previously tested with
pancreatin and pH values covering both fasted and fed conditions
[11]. All representative LBFs were found to be hydrolyzed by GL
with an optimum activity in the pH range 4e5.5. Fig. 6 shows the
specic activities of GL on the various LBFs at pH 4. The highest
activities were recorded on MC LBFs (1268 62 U/mg on type IIIAMC LBF) and were similar to the maximum activity of GL recorded
on TAG substrate [37e40].
A few in vitro studies have already taken into account the fact
that LBF lipolysis starts into the stomach and may affect LBF
structure and drug solubilization before the drug is delivered into
the small intestine. The solubilization of two model drugs,

Fig. 5. Relationship between Danazol initial solubility in LBF and Danazol solubilization in the aqueous phase in the course of in vitro lipolysis experiments. Same
experimental conditions and LBFs as in Fig. 4. The plot presented here was built from
the data reported in Ref. [7] (Figures 1 and 8).

Fig. 6. Specic activity of gastric lipase on various LBFs. The lipolytic activity of recombinant dog gastric lipase (rDGL) on eight representative LBFs ranging from type I to
IV LFCS classes (see Table 1 for denition and Table 3 for composition) was measured at
pH 4 with a pHstat device, Specic activity is expressed in U per mg of enzyme, with 1
U 1 mmole of fatty acid released per min. Adapted from data in Ref. [11].

304

F. Carriere / Biochimie 125 (2016) 297e305

Cinnarizine and Piroxicam, formulated with either Labrasol or

Gelucire 44/14, was thus tested using a two-step digestion model
involving a gastric phase with GL followed by a duodenal phase
with pancreatic enzymes (porcine pancreatin) and addition of bile
[41]. Fig. 7 shows the results obtained with Cinnarizine, an antihistaminic drug belonging to BCS class 2, formulated in the
SMEDDS Labrasol. Several compounds entering in the composition of Labrasol were already hydrolyzed to a large extent during
the gastric phase, like TAG, DAG and PEG-8 diesters (Fig. 7A) but
Cinnarizine solubilization in the aqueous phase remained high
(>90%; Fig. 7B). Lipolysis was extended during the intestinal phase
with the complete digestion of MAG and additional lipolysis of
DAG, PEG-8 mono- and diesters (Fig. 7A). Cinnarizine solubilization

remained however >85% under these conditions (Fig. 7B). Hence,

LBF lipolysis and changes in lipolysis products composition in both
gastric and duodenal phases had a limited effect on Cinnarizine
solubilization. Similar experiments performed with same pH
values, dilution factor and addition of bile but without addition of
enzymes revealed however a drastic drop in Cinnarizine solubilization after 30 min when the reaction mixture was diluted and the
pH changed to simulate intestinal conditions (Fig. 7B). Enzymatic
lipolysis of LBF was therefore required to keep the drug solubilized
during the duodenal phase, thus revealing that LBF lipolysis products play an important role in the overall process of lipophilic drug
delivery by LBF.
6. Conclusion
Although their mechanism of action still remains to be better
explored at the molecular level and under gastrointestinal conditions, LBFs have already enabled several new drugs to be developed
to treat a range of diseases. LBFs can also change the pharmacokinetics of existing lipophilic drugs and improve their bioavailability
and efcacy. Further development and improvement of LBFs requires however physiologically relevant in vitro digestion models to
investigate the fate of LBFs in the GI tract, solubilization and
bioavailability of oral lipophilic drugs. Since LBFs are submitted to
lipolysis by gastric lipase and pancreatic enzymes, in vitro digestion
models should take into account both intragastric and intestinal
digestion steps.
References

Fig. 7. Study on the solubility of Cinnarizine formulated with Labrasol using a twostep in vitro digestion model simulating the gastric and duodenal phases of digestion. Panel A: variations in the composition of Labrasol constituents and lipolysis
products as a function of time. These variations are expressed in percent of mono(MAG), di- (DAG), and triacylglycerols (TAG), PEG-8 mono- and diesters initially present in Labrasol. For free PEG-8, only the amounts generated upon lipolysis are shown
and expressed in percent of the amounts of PEG-8 present in Labrasol. The gastric
(0e30 min) and duodenal (30e90 min) phases of lipolysis were performed using rDGL
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Control experiments were performed with same pH values, dilution factor and addition of bile but without addition of enzymes. Adapted from Ref. [41].

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