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Introduction
Huperzine A (Hup A), extracted from a club moss
(Huperzia serrata), is an unsaturated sesquiterpene
alkaloid with a pyridone moiety and primary amino group. Its
empirical formula is
C15H18N2O, and its molecular weight is
242. Chemically, Hup A is
9-amino-13-ethylidene-11-methyl-4-azatricyclo [7.3.1.0(3.8)] trideca-3(8), 6, 11-trien-5-one, and
its structure is shown in Figure 1. Hup A is a powerful and
reversible inhibitor of acetyl cholinesterase. The agent
easily penetrates the blood brain barrier (BBB) and it is a
promising therapeutic agent for Alzheimer's disease. There are
several forms of Hup A, including tablet, capsule, transdermal
delivery system, injection and sustained release injectable
microsphere. Because Hup A can influence the cholinergic
system and results in side effects to peripheral tissues, it is
important to improve Hup A brain-targeting efficiency by
targeting routes[1].
Intranasal drug administration offers rapid absorption
to the systemic blood avoiding first-pass metabolism, and it
has been shown to present a safe and acceptable alternative
to the parenteral administration of a lot of drugs. Several
studies have shown a direct route of transport from the
olfactory region to the central nervous system in animal models,
without prior absorption to the circulating
blood[2].
Gel formulations can increase the contact time with the
mucosa and thereby facilitate the uptake extent of the drug.
On the other hand, in order to target the drug to the olfactory
mucosa, higher deposition of the drug in the nasal area is
crucial, so low viscosity of the formulations is required.
In situ gel was designed to meet the requirement of the above
purposes. In this study, in situ gel of gellan gum was used.
In an ion-free environment, the solution of gellan gum
exhibits a low viscosity, which forms a strong gel at physiological
cation concentration. It is liquid-like in
vitro and can be administered easily as a drop or by a spray device and
become semi-solid as soon as there is contact with the mucosa.
The aim of this paper was to study the drug brain
distribution in the rats following unilateral intranasal
administration of the Hup A in situ gel. Intravenous administration of
Hup A injection and administration of the oral formulation
(Hup A tablets dispersed in distilled water) were compared
with intranasal administration.
Materials and methods
Chemicals Hup A in situ gel (2 g/L) was obtained from
the Division of Pharmaceutics of Shanghai Institute of
Pharmaceutical Industry (Shanghai, China). The Hup A injection
(0.2 g/L) was purchased from Zhejiang Wanbang
Pharmaceutical Limited Cooperation (Taizhou, Zhejiang, China). The
oral formulation (0.15 g/L) was made by dispersing Hup A
tablets in distilled water. Hup A tablets (50 µg per tablet)
were produced by Shanghai Fudan Fuhua Pharmaceutical
Limited Cooperation (Shanghai, China). HPLC-grade
methanol was purchased from No 1 Zhenxing Chemical Industry
Factory (Shanghai, China). Analytical grade chloral hydrate
was purchased from Shanghai Chemical Agent Cooperation
(Shanghai, China), and triethanol amine was from Shanghai
Lingfeng Chemical Agent Limited Cooperation (Shanghai,
China). (S)-9,10-difluoro-3-methyl-7-oxo-2,3-dihydro-7
H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid methyl
ester (used as an internal standard [IS]), its structure shown in
Figure 2, was obtained from the Division of Chemical
Synthesis of Shanghai Institute of Pharmaceutical Industry
(Shanghai, China).
Animals Male Sprague-Dawley rats weighing about 300
g were purchased from Shanghai Laboratory Animal Center,
Chinese Academy of Sciences (Shanghai, China).
For the intranasal administration, the rats were
anesthetized with an ip injection of 10% chloral hydrate solution,
and 25 µL of the nasal formulation (Hup A in
situ gel) was administered via a PE 10 tube attached to a microlitre syringe
inserted 1 cm into left nostril of rats at a 166.7 µg/kg dose.
For the iv administration, the Hup A injection was delivered
(166.7 µg/kg) through the caudal vein. Oral gavage of Hup A
(500 µg/kg) was performed by attaching a stainless steel
feeding needle to a 1 mL syringe containing the oral
formulation. At 0.08, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4 and 6 h after
the intranasal or oral dose, and at 0.03, 0.08, 0.25, 0.5, 1, 1.5, 2,
3, 4, and 6 h after the iv dose, the animals were decapitated
and the blood was collected from the trunk. Then the skull
was cut open and the olfactory bulb, hippocampus,
cerebrum and cerebellum were carefully excised. The brain
tissues were quickly rinsed with saline and blotted with filter
paper to remove the blood taint and macroscopic blood
vessels as much as possible. After weighing, the olfactory bulb,
hippocampus, cerebrum and cerebellum samples were
homogenized with 0.4, 0.4, 0.5, and 1 mL water in tissue
homogenizers, respectively. Blood samples were
anticoagulated with heparin and centrifuged at 5000
×g for 10 min to obtain the plasma.
Both plasma and brain tissue homogenates were stored
in a deep freezer at -20 °C until HPLC analysis.
Measurements were repeated on 4 rats at each time point.
Sample preparation For the 175 µL plasma samples
or 0.2 g brain tissue homogenates, 10 µL IS methanol solution
(2 mg /L for the plasma sample and 1 mg/L for the brain tissue
sample), 100 µL
NaCO3-Na2BO4 buffer (pH 11.5) and 2 mL
chloroform were added. The mixture was vortexed for 5 min
and centrifuged at 5000×g for 10 min. Then the organic phase
was transferred to a conical tube and evaporated to dryness
under a gentle fluid of nitrogen at 40 °C. For the plasma
sample, the residue was reconstituted in the 50 µL mobile
phase, and then 20 µL supernatant was injected onto the
HPLC system. For the brain tissue samples, the residue was
reconstituted in 100 µL 0.01 mol/L acetic acid solution, and
then 40 µL supernatant was injected onto the HPLC system
after centrifugation at 20 000×g for 5 min. Samples were
quantified using peak area ratio of Hup A to IS.
High performance liquid chromatography The HPLC
system consisted of the LC-10AD VP delivery system,
RF-10AXL fluorescence spectrophotometric detector, and
CLASS-VP chromatographic integrator (Shimadzu, Japan).
The separation was performed on a Kromasil C-8 column
(5 µm×4.6 mm×15 mm). The mobile phase consisted of
methanol:water:triethanol amine (45:55:0.05). Briefly, a flow
rate of 1 mL/min, running time of 12 min, detector excitation
at 310 nm and emission at 370 nm were
used[3].
Pharmacokinetic calculations and statistics
The Cmax and
tmax values were read directly from the
concentration_time profile. The area under the concentration-time curve
(AUC0¡út) was calculated by the trapezoidal rule. The
variance for the AUC0¡útwas estimated using the method of
Yuan[4]. The absolute oral or nasal bioavailability of Hup A was
calculated as the ratio of the AUCin
(AUCoral) to the AUCiv:
Fin=(AUCin×Dose
iv)/(AUCiv×Dose
in)×100%
The ratio of the AUCbrain to the
AUCplasma (drug targeting efficiency) was calculated to evaluate the brain targeting of
the drug via 3 administration routes. The statistical
differences were assessed using the unpaired Student's
t-test.
Foral=(AUCoral×Dose
iv)/(AUCiv×Dose
oral)×100%
Results
Determination of Hup A in rat plasma and brain tissue
by HPLC
Separation and specificity Blank samples were
chromatographically screened and there was no chromatographic
interference with Hup A or IS; the retention times of Hup A
and IS were approximately 6.8 and 8.8 min, respectively. The
chromatograph is shown in Figure 3.
Calibration and linearity The calibration curves of Hup
A were prepared with drug-free plasma and brain tissue samples
spiked with known amounts of the drug, utilizing the peak
area ratio of Hup A to IS. The linear range of Hup A was
2.86_285.71 ng/mL for the plasma, and 1.25_125 ng/g for the
brain tissue.
Precision and accuracy The inter- and intra-day
precisions [relative standard deviation (SD)] and accuracy [relative
deviation (RD)] are summarized in Tables 1 and 2.
Recovery For the plasma samples, the average
extraction recoveries of Hup A for the low, medium and high QC
were 84.59%, 79.10%, and 73.52%, respectively. For brain
tissues samples, they were 71.47%, 68.21%, and 68.18%,
respectively. The average extraction recoveries of IS were
86.64% and 65.38% in the plasma and brain samples.
Pharmacokinetic analysis and brain tissue distribution
of Hup A
Rats plasma and brain tissue distribution of Hup
A The mean brain tissue and plasma concentration-time
profiles of Hup A in male rats following a single dose of the
nasal in situ gel, the iv injection and the oral formulation are
illustrated in Figure 4.
Following in administration of the nasal in
situ gel at the dose of 166.7 µg /kg, the
AUC0→6 h of Hup A in the cerebrum,
hippocampus, cerebellum, left olfactory bulb, right olfactory
bulb and plasma were 113.45±10.59
ng·h·g-1, 101.69±9.20
ng·h·g-1, 101.52±9.47
ng·h·g-1, 376.58±36.81
ng·h·g-1, 322.51±31.49
ng·h·g-1, and 202.30±18.86
µg·h·L-1, respectively.
The AUC0→6 h of Hup A in the cerebrum,
hippocampus,cerebellum, olfactory bulb and plasma following iv dose of
166.7 µg /kg were 76.83±7.76
ng·h·g-1, 76.55±7.02
ng·h·g-1, 99.88±9.64
ng·h·g-1, 326.39±40.53
ng·h·g-1, and 209.71±18.52
µg·h·L-1, respectively; and those after oral formulation at the
dose of 500 µg /kg were 124.21±10.95
ng·h·g-1, 141.38±18.74
ng·h·g-1, 161.56±21.34
ng·h·g-1, 364.06±25.52
ng·h·g-1, and 256.48±29.42
µg·h·L-1, respectively.
The absolute nasal bioavailability in the cerebrum,
hippocampus, cerebellum, left olfactory bulb, right olfactory
bulb and plasma were 147.7%, 132.8%, 101.6%,
115.4%, 98.8%, 96.5%, respectively. The absolute oral bioavailability
in the cerebrum, hippocampus, cerebellum, olfactory bulb
and plasma were 53.9%, 61.6%, 53.9%, 37.2 %, and 40.8%
respectively.
The AUC0→6 h of HupA in the plasma and all brain tissue
samples after intranasal administration of the Hup A
in situ gel were significantly higher
(P<0.01) than those administered with the oral formulation.
The uptake extent of Hup A into the cerebrum and
hippocampus 6 h after intranasal administration of the Hup A
in situ gel to the rats was significantly higher
(P<0.01) than that after iv administration of the injection. And there was no
difference in the AUC0→6 h in the cerebellum, olfactory bulb
and blood samples from rats receiving the drug in the form of
iv injection and nasal in situ gel.
AUCbrain/AUCplasma of Hup A after iv administration of
the injection and intranasal administration of the
in situ gel to male rats The AUC
brain/AUCplasma of Hup A, as the drug targeting efficiency (DTE), after a single dose of the iv
injection and the nasal in situ gel are illustrated in Figure 5.
Compared with intravenous injection, the intranasal
administration of the in situ gel produced significantly higher
(P<0.05) levels of the
AUCbrain/AUCplasma in the left olfactory
bulb over 0.083_6 h, and in the right olfactory bulb over
0.083_1 h. The AUCbrain/AUCplasma
of Hup A in the cerebrum and hippocampus following intranasal dose is significantly
higher (P <0.05) than the iv dose, at 0.083, 3, 4, and 6 h.
Discussion
In situ gel is liquid-like in
vitro which can be administered easily as a drop or by a spray device and become
semi-solid as soon as there is contact with the mucosa. Low
viscosity of the formulations is required for targeting the
drug to the olfactory mucosa, and the gel can prolong the
time of the drug in the nasal cavity so that it enhances the
drug absorbance. Thus, the nasal in situ gel should have a
prospective application.
Intranasal administration of the Hup A in situ
gel significantly increased the distribution of the drug into the rat brain
tissue, especially into the cerebrum and hippocampus. The
absolute oral bioavailability of Hup A in the plasma and
different brain regions were all lower than the absolute nasal
bioavailability (P<0.01). The nasal delivery of the Hup A
in situ gel, therefore, showed a viable alternative to the oral
formulation.
Figure 4 shows that the decrease of Hup A in the brain
tissue was slower than that in the plasma, which was the
same as the results reported by Liu X et
al[5].
It is believed that drugs uptake into the brain from the
nasal cavity via 2 different pathways. One is that drugs
administered by the nasal route may enter the systemic
circulation and subsequently reach the brain by crossing the
BBB. The other is that drugs may permeate the brain directly
via the olfactory region[6]. We can deduce that the amount
of drugs in the brain tissue after nasal application attributes
to these 2 parts. The AUC0→6
h after the intranasal administration of the Hup A
in situ gel was 1.5 times higher than that after iv administration of the injection in the cerebrum and
1.3 times higher than that in hippocampus. There were no
differences in the AUC0→6
h in the cerebellum, olfactory bulb and blood samples from rats receiving the drug by either
route. The absolute nasal bioavailability of Hup A in the
plasma was 96.5%. For the lipophilic and small molecule
weight character of the drug, intranasal application of the
Hup A in situ gel resulted in similar
AUCplasma to the iv injection and considerable quantity of Hup A delivered to the
brain along the nose-blood-brain pathways. Animals
receiving iv Hup A therefore provided a measure of Hup A
penetration into the central tissues expected from the bloodstream
after the intranasal administration of the in
situ gel. The difference in brain tissues was ascribed to targeted delivery
with intranasal administration of the Hup A in
situ gel. The excessive part of the drug in the brain tissues following
intranasal, rather than iv doses, represented the brain AUC
fraction contributed by the direct nose to brain pathway.
The direct nose to brain pathway could also be
demonstrated by comparing the
AUCbrain/AUCplasma of Hup A after
iv administration of the injection and that after intranasal
administration of the in situ gel to male rats. The
AUCbrain/AUCplasma of Hup A in the left olfactory bulb following the
intranasal dose was significantly higher (P<0.05) than the iv
dose at 0.083_0.75 h, implying the potential for Hup A into
the central tissue through the nose-brain pathway. The
AUCbrain/AUCplasma of Hup A in the cerebrum and
hippocampus following intranasal dose was significantly higher
(P<
0.05) than the iv dose, at 0.083, 3, 4, and 6 h. We could
deduce that Hup A could be absorbed in the brain tissue
through the olfactory bulb in the nose to brain pathway, but
it could not be explained that the
AUCbrain/AUCplasma of Hup A in the cerebellum following iv dose was significantly higher
(P<0.05)than intranasal dose, at 0.5, 0.75, and 1 h.
Additionally, it was necessary to anesthetize the rats in
order to ensure the exact administration dose. However, the
rat intranasal administration method still needs improvement,
and further research on the effects of the anesthetized state
on brain distribution of Hup A in in administration is needed.
The present study implicated a direct pathway for a
fraction of the drug into brain following intranasal
administration of the Hup A in situ gel. The intranasal delivery showed
a viable, non-invasive strategy for delivering the drug into
the brain.
References
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