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Introduction
Brucine is an alkaloid and exists mainly in the seeds of
Strychnos nux-vomica L
(Loganiaceae)[1], which is widely used in many southern Asian countries. The configuration
of brucine is shown in Figure 1.
Brucine itself is known as an anti-inflammatory and
analgesic drug for relieving arthritic and traumatic
pain[2]. Its main pharmacodynamic actions include relief of pain,
reduction of swelling, and the promotion of circulation.
Unfortunately, the potential use of brucine is severely
limited due to its high incidence of side-effects, including
violent convulsion and even lethal
poisoning[3]. Therefore, for a therapeutic application of this molecule, it is necessary to
find a suitable formulation to limit its dangerous side-effects
while maintaining or possibly enhancing its effectiveness.
Colloidal drug delivery systems, such as liposomes
represent a mature, versatile technology with considerable
potential for the entrapment of both lipophilic and hydrophilic
drugs[4]. Encapsulation or entrapment of drugs in liposomes
results in distinct changes of pharmacokinetic and
pharmacodynamic properties of free drugs, and in some cases,
causes an apparent decrease in toxicity and/or an increase in
therapeutic efficacy[5_7]. Meanwhile, liposomes have also
been suggested to be useful carriers for topical and
trans-dermal delivery[8,9].
Therefore, the objectives of our study are: (i) to develop
a safe and effective liposomal brucine (LB) which achieves
attenuation and synergia of brucine; (ii) to achieve topical
and transdermal administration of the drug; (iii) to assess
the acute dermal toxicity and skin irritation; and (iv) to
evaluate the analgesic and anti-inflammatory activities of LB
using acetic acid-induced writhing test and xylene-induced
mouse ear edema models.
Materials and methods
Materials Brucine was purchased from Fluka (Buchs
SG, Switzerland). Lecithin (from soy beans,
³92%), cholesterol, sodium deoxycholate, and mannitol were
obtained from Yuanju Biotech (Shanghai, China). Yunnan Baiyao
tincture was purchased from Yunnan Baiyao Group
(Kunm-ing, China), and xylene was obtained from Shanghai
Chemical Reagent (Shanghai, China). Acetic acid and Tween-80
were from Sinopharm Chemical Reagent (Shanghai, China).
All other reagents were of analytical grade.
Animals New Zealand albino rabbits of both sexes
weighing 2 kg, nude mice (BALB/c) of both sexes weighing
18_22 g, and ICR (Kunming) mice of both sexes weighing
18_22 g were used for the experiments. The animals were
housed in standard cages, maintained at a temperature of
22±2 °C with 12 h light/dark cycles, and provided with fresh
water and standardized diets ad libitum, unless otherwise
noted. The animal tests were performed according to the
Principles of Laboratory Animal Care and Use in Research
(Ministry of Health, Beijing, China).
Statistical analysis The results were expressed as mean±
SD. All data were processed with SPSS software (SPSS,
Chicago, IL, USA). P<0.01 was considered significant
difference.
Preparation of liposomes A modified ethanol-dripping
method was used to prepare LB[10]. An ethanol solution of
lipid (lecithin: cholesterol =6:1,
w/w), sodium deoxycholate, Tween-80, and brucine (16:4:4:1,
w/w) was dripped into mannitol solution (5.3 mg/mL). The ratio of the ethanolic-lipid
phase to the aqueous phase was 1:9
(v/v) in the final suspen-sion. The suspension was then sonicated (45 w, Shanghai
Kedao Ultrasonic Instrument Co, Shanghai, China) with an
ice water-bath for 20 min and freeze-dried for 72 h. The dry
powder was stored at -18 °C in a refrigerator. The dry
powder could be rehydrated and sonicated for 3 min prior to
application.
Characterization of liposomes Non-encapsulated
brucine was separated from the liposomes by gel filtration
(Sephadex G-25 column, Shanghai, China), and encapsulated
efficiency (EE) was calculated as the following equation:
EE=(total drug-unencapsulated drug)/total drug×100%.
The average diameters of liposomes were determined by
dynamic light scattering using a laser lightscattering
instrument (Nicomp, 380/ZLS, Santa Barbara, USA). The samples
were diluted in distilled water in order to avoid multiple
scattering. This measurement was performed at 25±0.1 °C, at
an angle of 90 degrees between laser and detector.
The morphology of the liposomes was studied with an
atomic force microscope (AFM). One sample was diluted
with distilled water. One drop of that liquid was dripped
onto a sheet of mica and characterized after it was dried by
AFM (Multimode, Veeco, Santa Barbara, USA).
In vitro release A volume of 2 mL of LB or free brucine
(drug content: 1 mg/mL) was taken in a dialysis tube
(Poly-carbonate, molecular cut-off, 3500) with both the ends tied.
The dialysis bag was suspended in phosphate buffer
solution (PBS, 100 mL, pH 7.4), maintained at 37 °C, and the PBS
was kept stirring. Periodically, 2 mL of PBS sample was
drawn, and then the absorbance was noted at 263 nm using
the spectrophotometer (756MC, Shanghai Yukang
Instrument Co, Shanghai, China). The volume of PBS was
maintained at 100 mL throughout the experiment.
In vitro skin permeation The in
vitro skin permeation study was based on a previously reported
method[11]. The skin fragment used for the experimentation was excised from
nude mice and stored at 4 °C 1 d before the experiment.
Subcutaneous fat was removed before the experiment started.
The skin samples were mounted on Franz diffusion cells
(diffusion surface: 3.14 m2). The receiver compartments were
filled with 15 mL of (PBS; pH 7.4), kept at 37 °C, and
continuously agitated by a magnetic stirrer. The donor chamber
was charged with 1 mL LB and free brucine (drug content: 1
mg/mL). At different periods of time (1, 2, 4, 6, 8, and 12 h),
0.5 mL samples were withdrawn from the receptor
compart-ment, replaced with an equal volume of fresh medium, and
HPLC was employed for the drug content. The HPLC
apparatus was Agilent 1100 (Santa Clara, USA) with a wavelength
detector. The detection wavelength was 260 nm. Analysis
was performed with a mixed solution (55% methanol and 45%
acetonitrile), with a flow rate of 1.0 mL/min on a reverse
column (Hypersil ODS C18 column, Shanghai, China) at 30±0.1 °C.
After a 12 h permeation period, the residual formulation
from each skin fragment was cleaned with distilled water.
Each skin sample was weighed, placed in 2 mL ethanol
solution (50%), and disintegrated by an ultrasonic processor.
After agitation for 2 min and centrifugation for 5 min at 1370
(×g), the supernatant was removed and filled with 2 mL
ethanol solution (50%). After repeating the extractive process 5
times, all supernatants were collected. Then, after filtration,
the drug concentration was determined by HPLC. The drug
accumulation of brucine in the skin was calculated by the
following formula:
Drug accumulation in skin=(Drug content in skin)/(skin weight).
Acute dermal toxicity An acute dermal toxicity study in
rabbits was conducted in accordance with the study of
Isbrucker et al[12]. Rabbits of both sexes were divided into 2
groups: integrity skin group and broken skin group. Each
group had 3 subgroups: brucine group, LB group, and
negative control group (saline solution, 0.9% NaCl); each
subgroup consisted of 4 rabbits. Two sides of each rabbit's
back were clipped with an electric clipper to expose an area
of approximately 10% of the total body surface 24 h before
the experiment. Only those animals without injury or
irritation of the skin were used in the test. Signs of "#" were cut
on the 2 depilated parts of each animal in the broken skin
group. The vehicle and drugs were topically administered
within 24 h. Then the test sites were gently cleaned of any
residual test substance. The animals were observed for
mortality, signs of gross toxicity, and behavioral changes
after application at least once daily thereafter 7 days.
Skin irritation study A primary skin irritation test was
conducted on the rabbits to determine the potential irritation
reactivity according to a previous
method[13]. The groups of rabbits were the same as those in the acute dermal toxicity
test. Each animal was treated with vehicle and drug (1
mg/mL) which was applied to the skin of 1 flank. The patch was
held in place with a bandage for 24 h, after which the
dressing and patch were removed and the skin was cleaned of
residual test substance with water. Skin reactions and
irritation effects were assessed at approximately 1, 24, 48, and 72
h after the removal of the dressings. Adjacent areas of
untreated skin from each animal served as controls. Erythema
and edema were scored on a scale of 0_4, with 0 showing no
effect and 4 representing severe erythema or edema (Table
1). The specific degree of irritancy was obtained by
calculating the primary dermal irritation index (PDII,
PDII=S(value of evaluation n of dermal reactions)/4) and classified according
to the descriptive rating for mean primary dermal irritation
index (0<PDII£2.0: slight irritation;
2.0<PDII£5.0: moderate irritation; and PDII >5.0: severe irritation).
Xylene-induced mouse ear edema The assay of
xylene-induced ear edema in mice was based on a previously
reported method[14]. Male mice were divided into 8 groups and
each group consisted of 10 mice. The positive control group
received Yunnan Baiyao tincture (3 mg/kg), the negative
group received saline solution (0.9% NaCl), 3 groups received
free brucine (1.5, 3, and 6 mg/kg), and another 3 groups
received LB (1.5, 3, and 6 mg/kg, brucine content), respectively.
The vehicle and drugs were topically applied to both sides
of the right ear of the mice. The left ear received only saline
solution. Thirty minutes after the administration of vehicle
and drugs, 50 µL xylene was applied to both surfaces of the
right ear. After treatment for 30 min, the mice were killed by
cervical dislocation, and a plug (9 mm diameter) was removed
from each ear. The edematous response was measured as
the weight difference between the 2 plugs. The
anti-inflammatory activity was expressed as the percentage of the
inhibition of edema (PIE). PIE was calculated using the
following formula:
PIE=([control weight variation-treated weight
variation]/control weight variation)×100%
Acetic acid-induced writhing test The response to the
intraperitoneal injection of 0.9% acetic acid was induced by
the method proposed by Koster et
al[15]. The groups of mice were the same as those in the xylene-induced ear edema test.
The abdomens of the mice were clipped with an electric
clipper 24 h before the experiment. Only those animals without
injury or irritation of the skin were used in the test. The
vehicle and drugs were topically administered on the
abdomens of mice 30 min before the intraperitoneal injection of
1% acetic acid (10 mg/kg). The number of abdominal writhes
within 20 min after the acetic acid injection was counted and
the percentage protection (PP) was calculated using the
following formula: PP=([control mean-treated mean]/control
mean)×100%
Duration of analgesic and anti-inflammatory activities
The acetic acid-induced writhing test and xylene-induced
ear edema in mice were also used to investigate the duration
of analgesic and anti-inflammatory activities of free brucine
and LB. Each test had 3 groups: free brucine group (3
mg/kg), LB group (3 mg/kg), and the negative control group
(saline solution). In the writhing test, the mice received an
intraperitoneal injection of 1% acetic acid at 30, 120, and 240
min after the administration of vehicle/drugs. In the
xylene-induced mouse ear edema test, after treatment with
vehicle/drugs for 30, 120, and 240 min, respectively, 50 µL xylene was
applied to both surfaces of the right ear. PP and PIE were
calculated according to the formulas defined in
xylene-induced mouse ear edema and acetic acid-induced writhing
test sections.
Results
Characterization of liposomes The particle size
distribution of the liposomes is shown in Figure 2. The average
size of the liposomes was between 25 and 110 nm, and the
mean diameter was 55.4 nm, indicating a narrow population
distribution. The AFM image of liposomes is presented in
Figure 3. It shows that the liposomes were discrete particles
with round structures and sharp boundaries and ranged from
20 to 100 nm, identical to Figure 2. The EE of LB determined
by gel filtration was 71.95%±2.65% (n=4).
In vitro release and skin permeation
studies The drug release profile of LB and free brucine through dialysis are
listed in Figure 4. It could be observed from Figure 4 that
before encapsulated, brucine diffused quickly and the
diffusion amount was up to 90% in 2.5 h. But after encapsulated
in liposome, the release rate of brucine was significantly
decreased and only 68% of drug released into PBS buffer after
10 h. This result showed that the LB exhibited an obvious
effect of slow release.
The amount of brucine permeation through skin is
plotted in Figure 5. In this Figure, almost no detectable brucine
was permeated through skin in the free brucine group in
12 h. But for the LB group, the cumulative drug permeated
increased with the increasing of the time and reached
8.7
µg/cm2 after 12 h. The drug accumulation of LB and free
brucine in skin is indicated in Figure 6. After 12 h permeation,
only 46.8 µg/g brucine accumulated in the skin in the LB
group. In the free brucine group, the accumulation reached
213 µg/g, which is over 5 times that of the LB group. These
results showed that LB exhibited an obvious promotion of
skin permeation.
Acute dermal toxicity The results of the acute dermal
toxicity test are presented in Table 2. It can be found that as
for the LB group and negative control group following the
dermal application of LB (100 mg/kg body weight), the
animals whether with integrity skin or broken skin, all survived,
gained weight and appeared active and healthy. There were
no signs of gross toxicity, dermal irritation, adverse
pharmacologic effects or abnormal behaviors. But in the case of the
free brucine groups, slight hyperaemia in integrity skin and
hyperaemia and cyanosis in broken skin group were noted
following the dermal application after removal of the dressing.
Thereby the acute dermal toxicity LD50, to rabbits of both
sexes was greater than 100 mg/kg. These results showed
that the LB exhibited the attenuation of brucine.
Skin irritation study The objective of this study was to
determine the potential of LB and brucine to produce
irritation from a single topical application to the integrity and
broken skin of New Zealand albino rabbits. The results of
this experiment showed that after application for 1, 24, 48,
and 72 h, no irritation was observed in the integrity group.
While in the broken group, 24 h after the application of
brucine, 1 case of erythema was observed and the severity
of irritation increased with time. The specific PDII of the
brucine subgroup in the broken skin group was 0.25, 0.5, and
1 after 24, 48, and 72 h, respectively. The results also showed
the attenuation of brucine, which was similar to the results
of the acute dermal toxicity study.
Xylene-induced mouse ear edema The
anti-inflammatory effects of brucine and LB on xylene-induced mouse ear
edema are presented in Table 3. Statistical analysis
demonstrated that both LB and brucine showed significant anti-inflammatory effects (P<0.01). The maximal PIE of LB was
34.3% at the dose of 6 mg/kg, even exceeding that of the
positive control (27.8%). Furthermore, LB, with doses
ranging from 1.5 to 6 mg/kg, caused significant stronger
inhibitory effects than the free brucine (P<0.01). The PIE of LB
was 2_4 times that of free brucine in the same doses, and LB
exerted its anti-inflammatory effects in a dose-dependent
manner.
The results of the duration of brucine and LB on
xylene-induced mouse ear edema are presented in Table 4. The
results demonstrated that both brucine and LB exerted their
maximal anti-inflammatory effects at 30 min after drug
administration, 11.2% for brucine at a dose of 3 mg/kg, and
24.3% for LB. After 30 min, the anti-inflammatory effects of
free brucine quickly decreased. At 120 and 240 min after
administration, the PIE of free brucine were only 2.4% and
4.6%, while the PIE of the LB groups were 8 and 4 times that of free
brucine, reaching 20.6% and 18.5%, respectively. This
result demonstrated that LB has longer lasting
anti-inflammatory effects than free brucine.
Acetic acid-induced writhing test The results of the
dose-dependent response effects of brucine and LB on
acetic acid-induced writhing in mice are presented in Table 5. It
was observed that the LB and brucine groups significantly
inhibited the writhing response of mice caused by the
intraperitoneal administration of acetic acid with the exception of
the free brucine at a dose of 1.5 mg/kg. The maximal
inhabitation of the nociceptive response was 40.7% for LB at the
dose of 6 mg/kg, which exceeded that of the positive control
at 3 mg/kg (36.4%). Compared with the corresponding
brucine groups at 1.5 and 6 mg/kg, the LB groups exhibited
significantly better analgesic efficacy. At 6 mg/kg, the pain
inhibition intensity of LB was 1.5 times higher than that of
free brucine, which suggested that after being encapsulated,
the analgesic efficacy of brucine was increased. Also, LB
exerted its pain-relieving effect in a dose-dependent manner.
The results of the duration of the analgesic effects of
brucine and LB are presented in Table 6. It showed that 30
min after drug administration, the analgesic effects of
brucine and LB at the dose of 3 mg/kg all peaked, and the PP of
brucine and LB were 25% and 27.5%, respectively. The PP
of free brucine at 240 min after administration was 18.2%,
while the PP of LB was still as high as 26.3%. The decrease
of the analgesic effects of LB and brucine showed that LB
exerted a more lasting analgesic effect.
Discussion
Local application of drugs is promising as it allows a
controlled and continuous delivery of drugs, and avoids or
reduces systemic side-effects. The greatest limitation to the
transdermal route is the skin barrier, which prevents the
uptake of drugs. Our strategy to overcome the skin barrier is
the encapsulation of drugs into liposomes, vesicles
consisting of a lipid bilayer that act as carriers. Liposomes as drug
delivery systems for drugs have great potential in providing
controlled release, decreasing the toxicity of drug, and
altering the bioavailability[5_7]. The following explanations will
support this conclusion.
In the present study, a liposomal brucine formulation for
transdermal delivery was prepared. The mean diameter of
the prepared liposomes (Figure 2), ranging from 25 to 110
nm, was 55.4 nm, and the AFM micrograph (Figure 3) was
further proof of the narrow size distribution of liposomes.
The influence of the liposome size on the properties seemed
to be contradictive. Verma and his co-workers reported that
small liposomes were more suitable to penetrate skin than
the large one with the same
composition[16]. But small particles always led to low encapsulation efficiency, thereby
decreasing the drug amount loaded. EE, standing for the
efficiency of encapsulating brucine, in our study was
71.95%±2.65% (n=4), which was turned out to be a proper
efficiency by the following in vitro and
in vivo evaluation experiment. A high EE value was the foundation for
achieving sustained-release, attenuation of toxic drugs and
effectively transporting drugs.
In vitro release (Figure 4) exhibited an obviously
sustained release of liposomes because only 68% brucine was
released in 10 h in LB, while 90% of the drug was released in
2.5 h in the free brucine group. Our data for the duration of
analgesic and anti-inflammatory activities (Tables 4, 6) also
revealed that compared with the groups administrated by
free brucine with the same dosage, the groups of LB
exhibited 4_8 folds increase of PIE and significant higher PP after
the same administration time, indicating that LB had longer
pharmacodynamic action than that of the free brucine.
Controlled/sustained release was a significant property of
colloidal drug delivery systems. Our in
vitro and in vivo studies demonstrated that liposomal formulation exerted an
adequate controlled/sustained release, which led to
long-lasting pharmacodynamic action and enhancing effects. These
results were significant in improving bioavailability and
decreasing the administering frequency of the drug.
It has been accepted that the most barrier of the
trans-dermal administration route is the skin, especially the
keratose layer, which greatly prevents the permeation and
uptake of the drug. In vitro permeation test indicated that
there was no detectable brucine permeation through the skin
into receiver compartments and drug accumulation in the
skin was 213 µg/g after 12 h for the free brucine group. In
contrast, the cumulative drug permeation of the LB group
reached 8.7 µg/cm2 while drug accumulation was just
one-fifth of that of free brucine. Brucine, due to skin barrier and
poor solubility of itself, scarcely penetrates through skin
layers and might accumulate in skin. Liposomes, as useful
carriers for topical drug delivery, can effectively improve the
drug permeation through the skin, thereby decreasing the
drug accumulation in the skin tissue. This mechanism is
important and useful for some toxic drugs to reduce their
dermal toxicity.
Because of the violent toxicity of brucine, the acute
dermal toxicity and skin irritation of LB, as a dermal formulation,
should certainly be investigated. Our study demonstrated
the acute dermal LD50 of prepared liposomal formulation to
New Zealand albino rabbits was greater than 100 mg/kg
(brucine content) and there was no skin irritation to integrity
and broken skin of New Zealand albino rabbits, while free
brucine exhibited acute toxicity to skin and slight irritation to
broken skin. It is clear that the toxicity of brucine was
reduced after being encapsulated in the liposomes and the
prepared LB was a safe formulation, even used on broken
skin, which was common in traumatic pains. The
in vitro high permeation through the skin and low drug
accumulation in skin, which are the inherent advantage of transdermal
liposomal delivery system, might contribute to this low
dermal toxicity.
The safety evaluation of LB is the prerequisite for the
application of brucine, but the question remains as to whether
LB keeps the pharmacodynamic actions of brucine after
being encapsulated. Previous studies have shown that
brucine was mainly responsible for the analgesic and
anti-inflammatory effects of the seeds of
Strychnos nux-vomica[2]. Our present study reevaluated the analgesic and
anti-inflammatory activities using the acetic acid-induced writhing test
and xylene-induced mouse ear edema models after brucine
was encapsulated in liposomes. The acetic acid-induced
writhing test and xylene-induced mouse ear edema are valid
and reliable models of nociception and edema. The trends of
analgesic and anti-inflammatory activities of LB and free
brucine, as shown in Table 3_6, are similar. The two
medicaments, whether encapsulated or not, all have
analgesic and anti-inflammatory effects. But in contrast, the
analgesic and anti-inflammatory efficiency of the LB at dosage
ranging from 1.5 mg/kg to 6 mg/kg are obvious better than
that of the free brucine. Additionally, LB was exhibited in a
dose-dependent manner in the 2 experiments and maintained
significantly longer analgesic and anti-inflammatory efficacy.
These results illustrated that LB not only keeps the
pharmacodynamic effects of brucine, but also pronouncedly
increases its bioavailability, which may be attributable to the
sustained-release and enhanced-penetration of liposomes.
From these findings, LB, prepared in our study, can be
confirmed as a transdermal formulation, which exerts a potential
topical and pantosomatous therapeutical effect.
In conclusion, the transdermal preparation of LB
succeeded in achieving attenuation, synergia and
sustained-release of brucine. It represents a novel, safe and effective
transdermal therapeutic formulation for relieving acute and
chronic pains such as arthritic and traumatic pain. These
encouraging results exhibit a good basis for the further
development of poisonous traditional Chinese medicine or
Western medicine.
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