Extract
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Introduction
Hirudin, a 65-66 amino acid polypeptide (7 kDa), is one
of the most potent inhibitors of thrombin and has proven to
have outstanding anticoagulant and antithrombotic
activities[1]. Recombinant hirudin (rHV), which can now be
produced through DNA techniques, has a similar
anticoagulative effect to natural
hirudin[2]. Compared with other anticoagulants, including heparin, rHV possesses many
advantages with respect to safety, antigenicity, and
toxicity[3]. Currently, rHV has been used for the prophylaxis and
treatment of heparin-induced thrombocytopenia (HIT), venous
and arterial thrombosis, and shunt thrombosis, and the
treatment of disseminated intravascular coagulation
(DIC)[4,5].
However, because of its susceptibility to protease
degradation and low mucosal permeability, only parenteral
injection (iv or sc) is available for the delivery of rHV.
Frequent injections, especially when rHV is indicated by chronic
symptoms (and during prophylactic usage) would cause
considerable discomfort to patients. Therefore, considerable
effort has been directed towards developing alternative
administration routes other than injection. Although
recombinant hirudin-1 (rHV1) and rHV2 can be absorbed in the
gastrointestinal tracts of rats after duodenal and oral
administration, the absorption was limited or it varied
depending on the analytical methods used, which indicated
that the results were unreliable[6,7]. As a convenient method
of administration, nasal delivery has many benefits relative
to oral administration, including the avoidance of the liver
first-pass effect and a higher bioavailability. Additionally,
for polypeptides, nasal delivery is one of a few non-parenteral
administrative routes that have gained regulatory approval
so far. Nasal delivery formats for polypeptide drugs such as
calcitonin, insulin, desmopressin and growth hormone are
already commercially available or in clinical trials. However,
there is limited information about intranasal delivery of rHV.
The main barriers to the nasal administration of
hydrophilic peptides are mucosal penetration and mucociliary
clearance. Chitosan, a positively charged bioadhesive
polysaccharide, has been found to be able to improve the
nasal absorption of peptides and reduce the clearance of
liquid formulations from the nasal cavity through its
bioadhesive characteristics, while causing negligible
damage to the nasal mucosal
membrane[8,9].
The present study was therefore intended to investigate
the nasal administration of rHV using chitosan. To further
improve the enhancing activity of chitosan, we studied the
effects of chitosan with some enhancers on the permeation
of rHV across excised rabbit nasal epithelium in
vitro and
the absorption of rHV by nasal delivery in rats. Furthermore,
the mucosal ciliotoxicity of different formulations was also
evaluated by using an in situ toad palate
model[10]. In the present study, we chose rHV2 as the model drug. For the
assays, the rHV2 was labeled with fluorescein isothiocyanate
(FITC), which formed a stable covalent conjugate
(FITC-rHV2) that could be assayed by fluorometry.
Materials and methods
Materials Recombinant hirudin-2 (rHV2, rHV-Lys47) was
obtained from the College of Life Science (Peking University,
Beijing, China), and chitosan (Mr
250 kDa, degree of deacetylation >85%) was from Yuhuan Ocean Biochemical
Co (Zhejiang, China). FITC, Brij35, ethylenediamine
tetraacetic acid (EDTA), lecithin and Sephadex G-25 were
purchased from Sigma (St Louis, MO, USA), and sodium
dodecylsulfate (SDS), hydroxyl-propyl-beta-cyclodextrin
(HP-b-CD), menthol, l-dodecylazacycloheptan-2-one (Azone)
and Tween 80 were from Beijing Chemical Co (Beijing, China).
Glycyrrhizic acid monoammonium salt (GAM) was the
product of Xinjiang Tianshan Pharmaceutical Industry
Co (Wulumuqi, Xinjiang, China). All other chemicals were in
analytical grade.
Animals Male rats (Sprague-Dawley, weighing
280-300 g) and male rabbits (Japanese White, weighing
2.5-3.0 kg) were obtained from the Experimental Animal Center of
Weitonglihua (Beijing, China); toads (weighing 30-40 g) were
from the Experimental Animal Center of the Health Science
Center of Peking University (Beijing, China).
The care and handling of animals was performed with the
approval of the Institutional Authority for Laboratory
Animal Care.
Preparation and purification of FITC-rHV2
The
synthesis of FITC-labeled rHV2 was based on the reaction
between the isothiocyanate group of FITC and the tyrosine
in rHV2[11]. FITC in 0.5 mol/L carbonate buffer (pH 9.5) was
added into a 1/10 volume of rHV2 solution (20 g/L in 0.01
mol/L phosphate buffered saline [PBS]; pH 7.1); the molar
rate of the two compounds was 3:1. After 4 h of reaction with
magnetic mixture in the dark at 0-9 °C, FITC-labeled rHV2
was separated from unreacted FITC in a Sephadex G-25
column (2.0 cm ID×30 cm L) pre-washed with PBS (pH 7.4).
A sample of approximately 1 mL was put on the top of the
column and then washed with PBS as an elution solvent.
The first yellow band was collected. After the collections
were mixed, FITC-rHV2 was obtained by freeze-drying. All
experiments were carried out under light exclusion conditions.
The molecular weight of FITC-rHV2 was determined to be
7388.99 by mass spectrometry (data not shown). This result
indicated that one rHV2 molecule had combined with one
molecule of FITC. FITC-rHV2 and rHV2 were found to have
similar activities when measured using the chromogenic
thrombin substrate assay[12].
Preparation of FITC-rHV2 formulations For the
in vitro studies, FITC-rHV2 was dissolved in 0.5% chitosan
(pH 5.0, w/v) at a concentration of 400 mg/L for transport. In
the in vivo studies, FITC-rHV2 was dissolved in 0.5%
chitosan solution at a concentration of 36 g/L for intranasal
administra-tion. When required, the enhancers were added
into these formulations (menthol, Azone and lecithin were
initially dissolved in propylene glycol). The concentration
of FITC-rHV2 for transport control was 400
mg/L in Ringer’s solution and for subcutaneous administration was 0.5 g/L in
0.9% NaCl. All the formulations were prepared on the day of
the experiments.
FITC-rHV2 assay The concentration of FITC-rHV2 was
determined in a fluorescence spectrofluorometer
(650-60; Hitachi, Japan) at an excitation wavelength of 495 nm and an
emission wavelength of 515 nm. A standard curve was
prepared using FITC-rHV2 at concentrations between 4
µg/L and 200 µg/L. The FITC-rHV2 concentrations of the test
samples were estimated using the standard curve.
Nasal epithelium preparation Rabbit nasal epithelium
was prepared as described by previous
reports[13]. After each rabbit was killed, its nasal septum was surgically
removed with a scalpel immediately, and then the
epithelium was carefully excised from the septum and stored in ice-cold
Ringer’s solution (pH 7.4; 125 mmol/L NaCl, 5 mmol/L KCl,
10 mmol/L NaHCO3, 1.2 mmol/L
NaH2PO4, 1.4 mmol/L
CaCl2 and 11 mmol/L D-glucose). The epithelium was used within
0.5 h of removal.
In vitro permeability experiments
Prior to the experiment, Ringer’s solution was added to both sides of the
horizontal diffusion chamber with a 4 mL volume in each
side, and the excised rabbit nasal epithelium was mounted in
the diffusion chamber at 37 °C. After an equilibration period
of approximately 0.5 h, the buffer was replaced with the
FITC-rHV2 solution on the donor side (mucosal), and fresh buffer
on the receiver side (serosal). A 200 µL aliquot of sample
was taken from the receiver side at particular times (0, 0.5, 1,
1.5, 2, 3, 4 and 6 h), and at the same time an equal volume of
Ringer’s solution was added to the receiver side. The
FITC-rHV2 concentration in the receiver side
(Cr) was determined by FITC-rHV2 assay. All experiments were carried out under
light exclusion conditions.
Calculation of the permeability coefficient
The Cr values were plotted as a function of time
(t) from 0 h to 6 h. The
permeability coefficient (P) was calculated according to the
following equation:
P = (dq/dt) /
(C0 A)
where dq/dt (µg/s) represents the permeability rate,
C0 (µg/mL) was the initial concentration in the donor chamber,
and A (cm2) is the effective cross-sectional area available for
diffusion (0.126 cm2). The transport enhancement ratios (ER)
were calculated using the following equation:
ER = Penh / Pctrl
where Penh and
Pctrl refer to the P values with added and
no added enhancers, respectively. Student’s
t-test was used to determine statistical significance.
In vivo studies The in
vivo studies were performed as previously
described[14]. The rats were fasted overnight
before the study. Anesthesia was induced by intraperitoneal
injection of 40 mg/kg sodium pentobarbital and maintained
by additional 15 mg/kg doses as required. The rats were
fixed on their backs on boards and were surgically prepared
by cannulation of the trachea to enable breathing,
cannulation of the carotid artery to facilitate blood sample collection,
and ligation of the esophagus to prevent samples being
swallowed. A 50 µL dose of the formulations was
administered into the left nares via a flexible polyethylene tube
attached to a microsyringe. In addition, 0.9% NaCl was
administered as a control to ensure that there was no interference
with FITC-rHV2. For the calculation of Fr, FITC-rHV2
solution (0.5 mg/kg) was subcutaneously administered by bolus
injection. Blood samples (400 µL) were withdrawn from
the carotid artery into the plastic microfuge tubes at particular
times (0, 0.5, 1, 1.5, 2, 3, 4 and 6 h). The blood samples were
anticoagulated with 3.8% (w/v) trisodium citrate solution at
a ratio of 8.25:1.75 (v/v), and plasma was separated after
centrifugation at 1 300×g for 5 min .
The fluorescence intensity in the plasma before and after
trichloroacetic acid (TCA) precipitation was determined, and
the intensity of the insoluble portion was used to calculate
the intact FITC-rHV2 concentration[15].
Data analysis The area under the FITC-rHV2
concentration time curves (AUC) was calculated by using the
trapezoidal method. The relative bioavailability (Fr) was
calculated by comparing the area under the
curve obtained after intranasal administration with that obtained after
subcutaneous injection. Statistical analysis was performed using
Student’s t-test. Differences were considered to be
significant for values of P<0.05.
Ciliotoxicity evaluation An in
situ toad palate model[10] was used. SDS (1%), Brij35 (5%),
HP-b-CD (5%), Tween 80 (5%), EDTA (0.1%), and GAM (1%) were directly dissolved
in a chitosan solution, and menthol (1.5%), Azone (4%), and
lecithin (5%) were initially dissolved in propylene glycol and
then in chitosan solution. The toads were fixed on their
backs and their mouths were opened with pincers. The test
formulations (0.5 mL) were applied to the upper palate of the
toads for 30 min, and then the palates were rinsed twice with
0.9% NaCl. The palates were dissected out, and the mucocilia
were examined with an optical microscope (Olympus, Japan).
The duration of ciliary movement in different formulations
was recorded and then the relative durations compared with
0.9% NaCl were calculated. Each group was duplicated 3
times and Student’s t-test was used to determine statistical
significance.
Results
The time versus the amount of FITC-rHV2 that had
permeated across the excised rabbit nasal epithelium after
application of the chitosan solution with or without enhancers to
the mucosal side is shown in Figure 1. The corresponding
permeability coefficients and the transport enhancement
ratios (ER) are listed in Table 1. When chitosan or chitosan
with various enhancers was added into the FITC-rHV2
formulation, the amount of FITC-rHV2 moving across the
epithelium increased, and there were significant increases in
the permeability coefficient. We found that the permeability
coefficient of FITC-rHV2 in various chitosan formulations
was 1.7-fold to 33-fold higher than that without chitosan. In
addition, there was a marked increase in the permeability
coefficient of FITC-rHV2 in chitosan formulations with 1%
SDS, 5% Brij35, 5% Tween 80, 1.5% menthol, 1% GAM or 4%
Azone (P<0.05) compared with chitosan alone. No
significant difference (P>0.05) in the permeability coefficient was
observed between the chitosan formulations containing 5%
HP-b-CD or 0.1% EDTA and chitosan alone, although there
was a significant decrease in the permeability coefficient in
the formulation containing 5% lecithin.
The mean plasma concentration of FITC-rHV2 versus
time after nasal administration to rats using various
formulations is shown in Figure 2, and the Fr values are given in
Table 2. When administered intranasally, in a formulation
containing neither chitosan nor enhancer (as a control),
FITC-rHV2 was only poorly absorbed, with an Fr value of 1.86%
compared with subcutaneous injection (Figure 2, Table 2).
The addition of chitosan at a concentration of 0.5% resulted
in a significant improvement, with an Fr value of 8.26%,
which is 4-fold that of the control. After enhancers were
added to the 0.5% chitosan solution, the Fr values of
FITC-rHV2 changed markedly in some formulations. Compared
with chitosan alone, the addition of 1% SDS, 5% Tween 80,
4% Azone, 1.5% menthol, 1% GAM or 5% Brij35 significantly
increased the Fr of FITC-rHV2 (P<0.05), whereas the
addition of 5% HP-b-CD, 5% lecithin or 0.1% EDTA did not
increase Fr to any significant extent.
Regarding the permeability or absorption of FITC-rHV2
in chitosan formulations with or without enhancers, the
results of the in vitro experiment (ER values) were in
agreement with the in vivo results (Fr values), as shown in
Figure 3 (R2=0.9059).
The effects of chitosan with or without enhancers on the
ciliary movement duration in toad palate are shown in Figure 4.
Ciliary movements were significantly inhibited by
co-administration of chitosan with 1% SDS, 5% Brij35, 4% Azone, 5%
lecithin, 0.1% EDTA or 1.5% menthol compared with the
0.9% NaCl control, but not significantly inhibited by
0.5% chitosan alone or 0.5% chitosan with 5% HP-b-CD, 5%
tween80 or 1% GAM. Based on these data, the rank order of
ciliotoxicity for different chitosan formulations based on the
relative ciliary movement durations was as follows: no
enhancer<0.5% chitosan<+5% HP-b-CD<+1% GAM<+5%
Tween80<+5% lecithin<+0.1% EDTA<+5% Brij35<+4%
Azone<+1.5% menthol<+1% SDS.
Discussion
For a nasal solution formulation, 0.5%-1.0% chitosan and
a molecular weight greater than 100 kDa are
preferred[16,17]. In the present study, we used 0.5% chitosan and found that
at this concentration, the chitosan solution could
significantly enhance the efflux as well as the nasal absorption of
FITC-rHV2 both in vitro and in vivo. This result can be
attributed to a combination of bioadhesion and a transient
opening of the tight junctions in the cell membrane to allow
hydrophilic macromolecules to pass
through[8,9].
SDS, Brij35, Tween 80 and Azone are all percutaneous
enhancers[18-20]. In our experiments, the addition of any of
these agents into the chitosan solution significantly
increased nasal absorption of FITC-rHV2 compared with
chitosan alone. This result may be due to the fact that these
compounds can all change the arrangement of the epithelial
cell membrane phospholipids and increase the fluidity of the
membrane lipid bilayers or interact with the membrane protein,
therefore resulting in a transcellular pathway transport of
FITC-rHV2. Chitosan was able to affect the paracellular
pathway transport of FITC-rHV2 by its mucoadhesive properties
and opening of the tight junction
effect[8,9]. The combined effect of the two agents consequently increased the
absorption of FITC-rHV2 compared with chitosan alone.
When EDTA was added to the FITC-rHV2 solution con
taining chitosan, EDTA activated protein kinase C by
depletion of extracellular calcium via chelation, resulting in an
expansion of the paracellular route[21], and chitosan interacted
with the membrane protein, also causing the tight junctions
to open. The combination of these two effects could
potentially result in a marked enhancement of FITC-rHV2
absorption. However, the negatively charged carboxyl
groups of EDTA interact with the positively charged amino
groups of chitosan, which may inhibit the enhancing action
of both agents. Therefore, in the present study we did not
observe any absorption enhancement of FITC-rHV2 with
EDTA added to chitosan compared with chitosan alone.
There exists a controversial explanation for the
enhancing mechanism of cyclodextrins; that is, that the enhancing
effects include the disaggregation of protein aggregates
(insulin), an interaction with lipids and divalent cations on
the membrane surface, and a direct effect on the paracellular
pathway by a transient effect on tight
junctions[22,23]. However, in most cases, cyclodextrin systems are used as a
means of enhancing drug
solubilisation[24]. In our experiment,
when HP-b-CD was co-administered with chitosan, we did
not observe the reported synergistic effect for FITC-rHV2
absorption[25]. The most likely reason is that FITC-rHV2 is
not like insulin, which usually aggregates in hexamers in
solution, and is more unstable in the presence of proteolytic
enzymes in the nasal mucosa, so addition of HP-b-CD may
lead to insulin deaggregation from hexamers to dimers and
protect insulin from degradation by proteolytic enzymes in
the nasal mucosa.
Phospholipids can bring about enhanced delivery of polar
compounds administered nasally by inhibiting the apical
membrane sodium channels and causing structural changes
in tight junctions[26]. However, in our experiment a
significant decrease in FITC-rHV2 absorption was observed when
lecithin was added to the chitosan solution. This was
probably because lecithin is a negatively charged compound,
whereas chitosan has positively charged amino groups, so
the two oppositely charged molecules might interact and
produce no increase in FITC-rHV2 absorption.
Menthol is a monocyclic terpene. The mechanism of its
enhancing effect is mainly due to it forming a eutectic with
the penetrating compound, thereby increasing its solubility,
and also enhancing the fluidity of the local lipid
bilayers[27]. In the present study, the co-administration of menthol with
chitosan markedly increased FITC-rHV2 absorption compared
with chitosan solution alone, suggesting that the
co-administration of the two agents cause the FITC-rHV2 molecule to
more easily penetrate the cell membrane.
Sakai et al found that dipotassium glycyrrhizinate
decreased intracellular calcium ion levels and did not induce
any significant histomorphological changes in the actin
filaments[28]. In addition, dipotassium glycyrrhizinate enhanced
the cellular permeability of sodium fluorescein and
fluorescein isothiocyanate dextran by enhancing the activation of a
protein kinase C via sodium deoxycholate (an
enhancer)[29]. Also, the combined use of the two enhancers had fewer toxic
effects. In the present experiment we found that chitosan
with GAM exerted an obvious enhancing effect on
FITC-rHV2 absorption compared with chitosan alone, but it was
not clear whether this effect was related to the mechanism
described.
Because rat nasal epithelium covers an area that is too
small to fit the device used in the in
vitro experiment, we chose to use rabbit nasal epithelium instead. As shown in
Figure 3, there is considerable correlation
(R2=0.9059) between ER and Fr, suggesting a relationship between the
in vitro and in vivo studies. Therefore, in the present study, it
appears that using nasal mucosa from different animal
species in the in vitro and in vivo experiments did not influence
the correlation between the absorption-enhancing effects of
formulations in vitro and in vivo.
For most absorption enhancers, a direct relationship may
exist between the absorption-promoting effect and local
toxicity, hence it is important to evaluate the local toxic
effect for a prospective novel enhancer system. An
in situ toad palate model was used in the present study to evaluate
the ciliotoxicity of different formulations. Although
recording the duration of ciliary movement may be less objective
and less accurate than the ciliary beating frequency method,
using a microscope it is possible to directly examine the
quantity of fallen cilia, as well as the integrity of the mucosa at the
same time. This method is especially suitable for the initial
screening of drugs or formulations. Among the tested
enhancers, chitosan alone and chitosan with HP-b-CD, GAM
or Tween 80 were least ciliotoxic.
In conclusion, chitosan is an effective enhancer for
increasing the nasal absorption of FITC-rHV2, and
co-administration of chitosan with other enhancers can improve
absorption further. Some chitosan formulations were less
ciliotoxic than others. The chitosan formulation system could
be a useful approach for improving nasal absorption.
Acknowledgement
The authors are very grateful to Prof Sheng-geng ZHU
for kindly providing the rHV2.
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