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Introduction
The lack of oral options for peptide/protein drugs limits their commercial use as well as their clinical application. To obtain
an optimistic bioavailability of peptide/protein drugs by the oral route, various obstacles must be overcome, of which harsh
conditions of the gastrointestinal tract and low membrane permeability are the main barriers. Enzyme inhibitors, permeation
enhancers, multifunctional polymers and encapsulation technology such as microspheres and nano-particles have been set
forth to boost oral insulin
bioavailability[1_5]. Recently, oil-based self-emulsifying formulations have attracted much attention
as a promising oral delivery form for improving drug solubilization and dissolution and/or intestinal absorption enhancement.
Both the self-emulsifying drug delivery systems (SEDDS) for lipophilic drugs and water-in-oil (W/O) microemulsion for
hydrophilic molecules are within the context of self-emulsifying formulation. SEDDS are isotropic mixtures of oil, surfactant,
co-surfactant and lipophilic drugs. As an outstanding
successful oral formulation based on SEDDS, cyclosporine A (Sandimmun
Neoral®, Basel, Basel-Stadt, Switzerland) has been
developed and marketed. Generally, W/O microemul-sion developed to overcome metabolic and physical barriers
impermeable to water-soluble molecules, such as peptides and proteins, contains mixtures of surfactants and oil with
the aqueous phase being maintained to below 20%
(w/w)[6_9].
We successfully developed a novel oral insulin formulation which can form fine microemulsion with a droplet size of
about 260 nm when introduced into water under mild agitation.
In this study, insulin-loaded formulation in a narrow size range was well prepared. The formulation was evaluated by
various physicochemical parameters such as droplet size, insulin encapsulation efficiency and stability. The influence of this
formulation on Caco-2 monolayer cells was assessed in terms of
Papp and TEER. In addition, a 2-cross-over trial was
conducted in diabetic beagles as an in vivo model to evaluate the potential application of this preparation in blood glucose
control.
Materials and methods
Materials and animals Porcine zinc insulin (28.1 IU/mg) was purchased from Wanbang Pharmaceutical Company (Xuzhou,
China). Polyethylene glycol-8-glycol octanoate/decanoate and polyglycerol-3-oleate were supplied by Cerdex
(Gennee-villiers, Cerdex, France). Fetal bovine serum and trypsin-EDTA were supplied by Hyclone (Hyclone, UT, USA). Dulbecco's
modified Eagle's media (DMEM), L-glutamine, penicillin and streptomycin were obtained from Gibco Laboratory (Invitrogen
CO, Grand Island, NY, USA). Ranitidine and propranonol were purchased from the National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, Beijing, China). All other chemical reagents were of high purity or HPLC
grade.
Male Beagle dogs (weight: 10 kg) were supplied by the large animal house of the China Academy of Military Medical
Sciences (Beijing, Beijing, China). All animal experiments were compliant with local legislation "Principles of Laboratory
Animal Care".
Formulation preparation Formulation with or without insulin was prepared according to the
patent[10]. Briefly, the weighed amount of insulin was first dissolved in a few drops of 0.1 mol/L HCl in order to facilitate dissolution; NaCl solution
(0.9%, w/v) was then added and the pH was adjusted to 3.0, resulting in solution A with a final insulin concentration of 60
mg/mL. Solution A was mixed with 5-fold liquid non-ionic surfactant polyethylene glycol-8-glycol octanoate/decanoate, then
magnetically stirred for a few minutes at 20 °C until an optically clear formulation was observed, resulted in solution B.
Solution B was then added to another liquid non-ionic surfactant polyglycerol-3-oleate, wherein the volume ratio of solution
B to that of the polyglycerol-3-oleate was 1:2. The solution was mixed quickly to form a homogeneous formulation, which was
stored at 4 °C. The insulin-free formulation was prepared exactly the same as the insulin-loaded formulation.
Evaluation of the formulation
Droplet size: Microemul-sions were formed by 1:800 dilution of the formulation with
purified water. The droplet sizes of the microemulsions were determined by the photon correlation spectroscopy method
using Zeta/sizer 3000HS (Malvern, Worcestershire, UK).
Encapsulation efficiency: A dialysis bag
(mw cutoff:
12 000_14 000) containing 2 mL insulin-loaded formulation and 8 mL phosphate-buffered saline buffer (PBS, pH=7.4) was
stirred in 200 mL PBS buffer up to 24 h. Samples 1 mL were extracted from the PBS buffer at the time points of 0, 1, 2, 5, 10 and
24 h, and the same volume of fresh PBS was supplied to keep constant volume. The amount of insulin in the water phase was
measured with Radio Immunoassay (RIA) by the method described in RIA kit instruction.
The encapsulation of insulin in the formulation was
calculated according to Equation 1:
En=(Tinsulin_Winsulin
)/Tinsulin×100% (1)
Where En indicates encapsulation efficiency,
Tinsulin (in IU/mL) is the amount of insulin added in the formulation and
Winsulin (in IU/mL) is the amount of insulin in the water phase.
Physical stability: The physical stability of the formulation stored at 4 °C was evaluated by periodic visual inspection of
the presence of macroscopic phase separation, as shown by cloudiness or the formation of 2 distinct layers. The
pharmaceutical activity of insulin encapsulated in the formulation was measured by RP-HPLC. Before analyzing, the insulin-loaded
microemulsion samples were pretreated with 0.02% Triton X-100 solution.
Proteolytic studies Native insulin (400 µg) or equimolar amounts of insulin-loaded formulation was dissolved in 920 µL
(0.1 mol/L) Tris-HCl (pH 8.0); 80 µL of a 200 µg/mL trypsin solution was added into the same buffer and the mixtures were then
incubated at 37 °C. At scheduled points (0, 5, 20, 35, 60, 90, 120, 150 and 180 min), 50 µL samples were withdrawn and diluted
with the same volume of 0.1% trifluoro-acetic acid to terminate the enzymatic reaction. Samples were stored at 4 °C before
analysis.
Cell culture Caco-2 cell line was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were
cultured as described by the supplier with DMEM in tissue culture flasks (75
cm2) in a humidified (95% relative humidity), 37
°C, 5% CO2 incubator. DMEM contained glucose (4.5 g/L) supplemented with 10% FBS, 1% NEAA, 1%
L-glutamine, 100 IU/mL penicillin and 100 mg/mL strepto-mycin. For the transport study, cell passages between 40_45 were used. After reaching
80% confluence in a week, cells were harvested with trypsin-EDTA and seeded in the apical sides of 12-well trans-well plates
(pore size 3.0 µm, diameter 24 mm; Costar, NL, USA) at a density of
1.0×105 cells/mL. The integrity of Caco-2 monolayers was
monitored by measuring the resistance across the cell layer (TEER) with an epithelial voltohmmeter (World Precision Instrument,
Sarasota, Florida, USA).
Transport study After 21 d growth, the TEER across the Caco-2 monolayer cells reached an equilibrium value, indicating
that the monolayer's tight junctions had well formed and could be used for transport
study[4,11]. The test drugs, insulin (50
IU/mL), ranitidine (1.5 mg/mL) and propranonol (1.5 mg/mL), were respectively dissolved in Hanks' Balanced Salt Solution
(HBSS) (insulin was first dissolved in 0.01 mol/L HCl solution) and added to the different apical sides (each 0.5mL) of the
chambers, Fresh HBSS was added into the basolateral side of these chambers (each 1.5 mL). In the test group, 2%
(v/v) formulation was added into apical sides, while no formulation was added to the control group. Samples 200 µL were taken at
given time intervals (0, 30, 60, 90, 120 and 150 min) from the basolateral sides of the chambers, and the same volume of fresh
HBSS was supplied to keep constant volume in the basolateral sides. TEER values were monitored during the experiment.
The chambers were washed twice with DMEM as soon as the transport study finished. After 0.5 mL and 1.5 mL DMEM
containing 10% FBS were respectively added to the apical and basolateral sides, the chambers were placed in an incubator.
TEER values were measured 3 h later.
Papp (in cm/s) of drugs were calculated according to Equation 2:
Papp=(DQ/Dt)/(C
0×A) (2)
Where DQ/Dt (in mmol/s) indicates linear apparent rate of mass in basolateral side,
C0 (in mmol/cm3) is the initial
concentration of drugs in the apical side; and A (in
cm2) is the surface area of the well.
Hypoglycemic effects in diabetic beagles
A 60 mg/kg dose of alloxan was intravenously injected to beagles to render
diabetes. Aβout 1 week later, the blood glucose levels were measured in 3 consecutive days in order to verify the diabetic
state of the dogs. Only the animal with a glycemia level around 250 mg/dL in all 3 occasions could be considered the
successful diabetic dog. Eventually, 6 diabetic dogs with similar blood glucose concentrations were used for the study. The
serum glucose levels of these 6 diabetic dogs were measured (blank control) during 24 h. A week later, a 2-cross test was
employed to minimize individual varieties, in which 6 diabetic dogs were randomly divided into 2 groups (each consisting of
3 dogs):, (A) oral administration of insulin-loaded formulation (2.5 IU/kg) and (B) subcutaneous administration of native
insulin (0.5 IU/kg). In another week, group A were treated by the subcutaneous injection of native insulin (0.5 IU/kg), and
group B were orally administered with insulin-loaded formulation (2.5 IU/kg). Blood samples were collected from the jugular
vein at the given time intervals and determined using genuine test strips (Life Scan, Milpitas, CA, USA). Before the
experiment, the animals were made to fast overnight, but had access to water.
The oral bioavailability (BA) was calculated by utilizing Equation 3:
BA=[AUC0_24
oral×(weight/dose)oral]/
[AUC0_24
sc×(weight/dose)sc]×100% (3)
The AUC0_24 is the area under the reduction of serum glucose levels from 0 to 24 h; weight (in kg) is the body weight of the
beagles and dose (in IU) is the amount of insulin administered to the animals.
HPLC analysis The chromatography was equipped with a Waters HPLC system (Waters, Milford, Mass, USA). A 5 µL
sample was injected. A VYDAC-218TP54 C18 reversed-phase column (Vydac, Hesperia, CA, USA) was used in the study.
For insulin analysis, the mobile phase consisted of 50 mmol/L
NaH2PO4 (pH 3.00; A) and acetonitrile (B). The procedure
was as follows: a linear gradient from 80% channel A to 20% in 20 min, channel B correspondingly changed from 20% to 80%,
then returned to 80% A and 20% B within 5 min and stayed that way for 5 min before the next injection. The flow rate was
maintained at 1.00 mL/min. Detection was performed at 214 nm.
Both ranitidine and propranolol were analyzed by VYDAC-218TP54 C18 column with isocratic elution at a flow rate at
1.0 mL/min. Ranitidine was analyzed at 320 nm, and the mobile phase used for analysis consisted of acetonitrile -30 mmol/L
ammonium acetate (10:90, v/v, pH 3.0). Propranolol was detected at 294
nm[12] and the mobile phase was composed of
acetonitrile -50 mmol/L phosphate buffer (40:60,
v/v, pH 3.0).
Statistics Each set of experiments described was
repeated at least 3 times. All values were expressed as
mean±SD. Statistical differences were assumed to be reproducible with
P<0.05.
Results
Characterization of the
formulation Self-emulsifying properties of the formulations were demonstrated when the
formulations were introduced into water under mild agitation. The mean droplet diameters of insulin-loaded formulation and
insulin-free formulation were 264.7 nm and 257.2 nm, respectively, with 800-fold dilution.
Most of the insulin was still encapsulated by the microemulsion, even it had been dialyzed in bulk buffer over 24 h (Table
1).
Stability study Based on our research, a single and optically clear phase was still maintained and no visible sign of
change was found in both formulations during the 6-month period of storage at 4 °C. As seen in Figure 1, the HPLC
chromatogram of native insulin (Figure 1A) was identical to that of the encapsulated insulin in the formulation stored for 6
months at 4 °C (Figure 1C); no other peaks appeared (regardless of surfactants; Figure 1B) compared with
native insulin. Therefore, neither degradation nor conglomeration of insulin occurred in the insulin-loaded formulation.
Degradation
characterization The degradation profiles of native insulin and encapsulated insulin in the formulations are
shown in Figure 2. More than 80% native insulin was rapidly degraded within 1 h. In contrast, the encapsulated insulin
displayed a significantly higher resistance to proteolysis.
Transport study Transport studies were carried out to verify the influence of the formulation on drug transportation
through the intestinal mucosa. As shown in Figure 3, the Papp of Ranitidine, a paracellular marker, increased from
0.317×10_6 to
6.26×10_6, about 20-fold higher than the control when 2% formulation was added. Insulin also showed similar behavior:
its Papp increased from 0.103×10-6 to
1.23×10-6, about 12-fold higher than the control. However, the permeability of Propranolol,
a transcellular marker, seemed unchanged
(2.273×10_5 vs
2.533×10_5, P=0.438).
TEER studies No significant changes of TEER with time were observed in the control group. In the presence of the
formulation, TEER values decreased significantly (within 150 min), but were constantly above 250
W/cm2, suggesting that the monolayers maintained their integrity. The effect of the formulation on Caco-2 cells appeared to be reversible as TEER values
recovered (>400 W/cm2) after 3 h following the transportation study (Figure 4).
Oral efficacy of the
formulation It is noteworthy that low glucose levels were maintained throughout the 24-h period.
Compared with the hypoglycemic percent of native insulin (0.5 IU/kg) administered subcutaneously, the bioavailability
(AUC) of this oral formulation (2.5 IU/kg) reached 15.21%. Additionally, a sustainable release was found in the diabetic dogs,
which had an orally administed insulin-loaded formulation (Figure 5).
Discussion
A stable self-emulsifying formulation was developed to encapsulate insulin, which can form fine emulsions with a droplet
diameter about 260 nm when introduced into water under mild agitation.
It was supposed that the ability of the formulation to encapsulate and protect insulin from proteolytic degradation may be
attributed to the location of insulin, which was dissolved in the inner aqueous layer upon the effect of surfactants. Because
the oil/water partition coefficient of insulin is almost 0, the diffusion of insulin across the surfactant membrane after dilution
would be negligible[13]. As a result, insulin is encapsulated and protected from degrada-tion. This structure feature makes the
appropriate pretreatment necessary for a satisfactory recovery (>95%) of insulin in the formulation as determined by
RP-HPLC. Without the pretreatment, the recovery of insulin in the formulation was around
30%[14].
Proteins have been reported in some studies to be absorbed at the oil/water interface and involved in the stabilization of
emulsion[15,16]. The stability of our formulation was measured by visual inspection and RP-HPLC analysis, and the results
showed neither physical change nor the new peak appearing in the insulin-loaded formulation even after 6 months of storage
at 4 °C.
Caco-2 monolayer cells were chosen as an
in vitro model to measure the ability of the formulation to enhance absorption
of insulin and to explore mechanisms involved in the systemic uptake of the insulin through the gastrointestinal epithelium.
Although Caco-2 cells come from the human colon carcinoma, they acquire many features of absorptive intestinal cells in
culture, such as microvillous structures, hydrolysis enzymes, carrier transport systems for sugars, amino acids and drugs and
well-formed tight junctions between the adjacent cells. Several reports have already shown the possibility of predicting the
oral absorption of drugs in human from studies of their permeability to Caco-2
monolayers[11,17_20].
Based on the different behaviors of the 2 marker drugs,
ranitidine[21] and
propranolol[22], and the changes of TEER in the
transport study, a safe conclusion was drawn that the tight junctions (TJ) between adjacent cells were loosened upon the
influence of this formulation. So the increase of insulin transportation through Caco-2 monolayers
(Papp increased from
0.103×10_6 to
1.23×10_6 with 2% formulation) may be mainly due to the changed TJ structures which allowed insulin, a
hydrophilic macromolecule with 51 amino acids, to transport through epithelium membrane barriers via the transcellular route. The
mechanism of the TJ "opening" was related to the C10 fatty acid, one of the main components in the formulation, which has
been accepted nowadays as a substance to trigger a series of reactions leading to the widening of the TJ, thus increasing
membrane permeability for hydrophilic
macromolecules[23_26].
During the transport study, Caco-2 monolayers maintained integrity (TEER constantly above 250
W/cm2) and the TEER values recovered to 400
W/cm2 and above after 3 h of incubation following the transport study. Therefore, the influence of
this preparation on Caco-2 cells was uninjurious and reversible with 2% formulation.
In this assay, hypoglycemia was induced by a comparative small dose of insulin (2.5 IU/kg) and the bioavailability of this
oral formulation was up to 15.21% compared with sc native insulin. This posted a great cost advantage and was much more
effective than the other reported studies on oral insulin
delivery[1,2,27_30]. Furthermore, at a dose of 300 times more than that
administered to man, no adverse effect of this preparation on the intestine, spleen and liver in mice was found, suggesting the
possible use for human beings. This formulation has now been approved by the China State Food and Drug Administration
to enter clinical trials.
Conclusion
A novel formulation with the capacity of encapsulating insulin was prepared. The high efficacy of blood glucose control
of this formulation was attributed to such possible positive factors as degradation resistance, permeation increase and so on.
Stability, efficacy and safety of the preparation make it a promising oral formulation to achieve optimistic bioavailability.
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