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Introduction
Paclitaxel (PTX) is a naturally occurring taxane extracted from the needles and bark of the Pacific yew tree,
Taxus brevifolia, which has shown significant antitumor activity against various
tumors[1]. PTX has been used clinically in the treatment of
metastatic breast cancer, ovarian cancer, and several other
malignancies[2,3].
Because the water solubility of PTX (approximately 0.3
µg/ml) is very low, the commercial preparation of PTX is
formulated in a vehicle composed of a 50:50
(v/v) mixture of Cremophor EL (polyethoxylated castor oil) and dehydrated alcohol,
which is diluted with 5-20 fold in normal saline or dextrose solution (5%) before administration. However, serious side
effects, such as hypersensitivity reactions, nephrotoxicity and neurotoxicity, attributable to Cremophor EL have been
reported[4,5]. Lately, a number of alternative vehicles have been developed for solubilizing PTX, including liposomes, lipid
emulsions, mixed micelles, cyclodextrin complexes, and paclitaxel
conjugates[6-10], which are safer intravenous formulations
devoid of Cremophor EL.
Amphiphilic block copolymer micelles are effective vehicles for the solubilization of hydrophobic
drugs[11]. Recently, interests have been raised in the application of polymeric micelles as a novel carrier system because of its high drug-loading
capacity of the inner core as well as of the unique disposition characteristics in the
body[12].
Pluronics are block copolymers consisting of ethylene oxide (EO) and propylene oxide (PO) blocks with the A-B-A
structure. Preliminary studies showed that Pluronic molecules displayed important special biological activities, besides the
feature of spontaneously forming micelles in aqueous media. The relatively hydrophobic polymers, such as L61 and P85, are
promising agents for use in formulations to treat drug-resistant tumors by energy depletion, cellular membrane fluidization
and inhibition of the P-gp ATPase
activity[13,14].
Most of previous studies on the application of Pluronic as a carrier focused on hydrophilic block polymers, such as
Pluronic F68[15], which do not sensitize multidrug resistant (MDR) cells. Furthermore, most of them have high critical micellar
concentration (CMC), and, as a result, do not form stable micelles in the injection
solutions. Pluronic P123 is a relatively hydrophobic block copolymer, which has a relatively low CMC
(4.4×10-6mol/L)[16]. The hydrophilic-lipophilic balance of P123
(HLB=8) is situated between L61 (HLB=3) and P85 (HLB=16). So it has potential in the sensitization of MDR cells. In this
work, we prepared PTX-loaded micelles with Pluronic P123 and evaluated the effect of micellization of PTX on
pharmacokinetics and tissue distribution.
Materials and methods
Drugs and reagents Paclitaxel was purchased
from XiĄŻan Sanjiang Bio-Engineering (XiĄŻan,
China). PTX injection (Taxol, Anzatax Injection Concentrate, 30 mg/mL) was produced by FH Faulding trading as David Bull Lab (Australia). Diazepam
was obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Pluronic
P123 was kindly donated by BASF (Ludwigshafen, Germany). All reagents for
high performance liquid chromatography (HPLC) analysis, including acetonitrile and methanol were of HPLC grade. Other reagents were of analytical grade.
Animals Sprague-Dawley rats (220±30 g) and female Kunming strain mice (20±5 g) were supplied by
Laboratory Animal Center of Fudan University, Shanghai, China.
The animals were used following the guideline of the Ethical Committee for
Animal Experiments of Fudan University. The animals were acclimatized at a temperature of 25±2
°C and a relative humidity of 70%±5% under natural light/dark conditions for at least 24 h before dosing.
Preparation of paclitaxel-incorporated micelles
The polymeric micelles of PTX with Pluronic P123 were prepared
by a solid dispersion method[17,18]. Accurately weighed
amounts of PTX and Pluronic P123 were added to acetonitrile. After complete
dissolution, the solvents were evaporated under reduced pressures at 37 °C and the residual solvents remaining in the matrix
were removed under vacuum at room temperature for 24 h. Dissolution of the solid PTX/copolymer matrix was carried out by
preheating the matrix in a warm water bath (60 °C) to obtain a transparent gel-like sample. Adding water at the same
temperature to the gel-like matrix and stirring by a glass paddle (600 rpm), the clear PTX-incorporated micellar solution was
obtained. The solution was filtered through a 0.22
mm filter membrane and freeze-dried.
Characterization of micelles The mean diameter and particle size distributions of the polymeric micelles were determined
by dynamic light scattering measurement using a NICOMP 380 ZLS Zeta Potential/Particle Sizer (PSS Nicomp, Santa Barbara,
CA, USA) equipped with a 5 mW helium-neon laser at 632.8 nm. Sample solutions filtered through a
0.22 µm filter membrane were transferred into the light scattering cells. The intensity autocorrelation was measured at a scattering angle of 90° at room
temperature.
The morphological examination of micelles was performed using a transmission electron microscope (TEM, Philips CM120,
Netherlands). In practice, a drop of micellar solution containing 0.1%
(w:v) phosphotungstic acids was placed on a carbon
film coated on a copper grid and observed at 80 kV in the electron microscope.
In vitro release of PTX from
micelles The in vitro release properties of PTX from polymeric micelles were investigated
in an aqueous medium containing 1 mol/L sodium salicylate by a dialysis
method[19]. The freeze-dried PTX-incorporated
micelles were redissolved in water, and 1 mL micellar solution was introduced into a dialysis bag (MWCO=5 kDa, Green Bird
Science & Technology Develop-ment, Shanghai, China). The end-sealed dialysis bag was immersed into 50 mL 1 mol/L
sodium salicylate solution at
37 °C. The release medium was stirred at the speed of 75 rpm for 24 h. While the solubility of PTX in the release medium was
23.1 µg/mL, the maximum concentration of PTX in the medium was 2.0 µg/mL in this release experiment. So the sink condition
was assured. Samples 0.5 mL were withdrawn at different time intervals (0, 10, 20, 30, 45 min, and 1, 2, 4, 6, 9, 12, 24 h) and
replaced with an equal volume of fresh medium. The concentration of PTX in the samples was determined by the HPLC
method described below. PTX release from stock
solution and Taxol placed in a dialysis bag was conducted under the same
conditions as the control.
Pharmacokinetic studies Twelve Sprague-Dawley rats were used to investigate the effect of formulation on the
pharmacokinetics of PTX after intravenous administration. Rats were divided into 2 groups at random, and given a single 3 mg/kg
dose of Taxol or PTX micelles by tail-vein injection. As the control, Taxol was diluted 12-fold to 0.5 mg/mL with 5% glucose
solution shortly before administration. And the concentration of PTX in Pluronic P123 micelle solution for pharmacokinetic
study was 0.3 mg/mL.
Blood samples (0.5 mL) were collected into heparinized tubes from the femoral artery at 0 min (predose), 0.083, 0.25, 0.5, 1,
2, 3, 4, 6, and 8 h after intravenous administration. Blood was immediately processed for plasma by centrifugation at
900×g for 10 min. Plasma samples were frozen and maintained at -20 ºC until analysis.
Tissue biodistribution studies Seventy-two female Kunming strain mice were used in the experiment of accessing the
effect of formulation on the tissue distribution of PTX after intravenous administration. All mice were divided into 2 groups
at random and the administration protocol of tissue distribution study was the same as in the pharmacokinetic study. At
0.083, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h after drug injection, each animal
(n=4 for each time point) was killed and heart, spleen, lung,
liver, kidney, uterus/ovaries, brain as well as blood samples were collected. Tissue samples were washed in ice-cold saline,
blotted with paper towel to remove excess fluid, weighed and stored at -50 ºC until assessed for drug concentration by HPLC.
HPLC analysis The analyses of PTX levels
in vitro and in vivo were carried out using an RP-HPLC method on a system
equipped with an LC-10ATVP pump, a
SPD-10AVP UV-Vis detector (Shimadzu, Kyoto, Japan) and a HS2000 interface ( Hangzhou
Empire Science & Tech, Hangzhou, China) operated at 230 nm. A reversed-phase column (Gemini 5
mm C18, 150×4.6 mm, Phenomenex, California, USA) was used at room temperature. The mobile phase consisted of acetonitrile, and ammonium
acetate buffer solution (10 mmol/L, pH 5.0) (50:45,
v/v) was freshly prepared for each run and degassed before use. Samples
20 µL were injected into the HPLC column for all the analyses. With a flow rate of 1.0 mL/min for the mobile phase, the
retention time of PTX was approximately 8.2 min.
Tissue samples were homogenized in the mixed solution of acetonitrile and water (50:50,
v/v). Diazepam (1 µg/mL, 50 µL ) as internal standard was added into 200 µL of plasma or tissue samples and vortexed for 1 min. The drug and internal
standard were then extracted into 3 mL of diethyl ether anhydrous by vortex mixing for 2 min. After centrifugation at
6000×g for10 min, the clear supernatant was removed and evaporated under a gentle stream of nitrogen. The residue was then
dissolved by 100 µL acetonitrile and centrifuged at
1400×g for 5 min before HPLC analysis.
Statistical analysis The compartment of model was simulated by 3p87 program (Practical Pharmacokinetic Program, 1987,
China) and the parameters of pharmacokinetics were obtained. The calculation of AUC was based on statistical moment
theory. The pharmacokinetic parameters were analyzed for statistical significance by unpaired StudentĄŻs
t-test. For this purpose the level of significance was set at
a=0.05. In the tissue distribution studies, the AUC could not be determined in
individual mice because of the destructive study design.
Results
Characterization of micelles The mean diameter and particle size distribution of the Pluronic P123 micelle were
determined by dynamic light scattering measurement. The mean size of blank micelles, PTX-loaded micelles and freeze-dried
PTX-loaded micelles were 20.8±2.9 nm, 25.2±2.9 nm, and 28.5±2.1 nm, respectively. It could be seen that the size distribution was
relatively narrow (Figure 1). There was no significant difference in particle size of these three micelles
(P>0.05).
The freeze-dried PTX-incorporated micelles were redissolved in water and the morphology was investigated by TEM. All
these micelles had a spherical shape. The particle surface was very smooth and no drug crystal was visible (Figure 2).
In vitro release of PTX from
micelles The result of the cumulative PTX release profile from Pluronic P123 micelle is
shown in Figure 3. PTX release from stock solution and Taxol were also investigated as control. The maximum
concentration of PTX in the medium in this release experiment was 2.0 µg/mL, while the solubility of PTX in the release medium was
23.1 µg/mL. And this system could provide a good sink condition for PTX. PTX was continuously released from the block
copolymer micelles in the aqueous medium containing 1 mol/L sodium salicylate for 24 h at 37 ºC. The dialysis bag may
become the rate-limiting step in the in
vitro release of PTX. In order to examine this possibility, release of PTX from the
stock solution across the dialysis membrane was carried out. Only 41.2% PTX was released from the micelles in 4 h, while
95.7% PTX in the stock solution and 90.5% PTX in Taxol were released during the same time period. There was no
significant difference in the PTX release from Taxol and stock solution
(P>0.05). The Pluronic P123 micelles released
approximately 87.8% PTX during 24 h.
Pharmacokinetics of polymeric micellar paclitaxel
The HPLC method for analysis was validated. Linearity in the
standard curves was demonstrated over the concentration range studied, and endogenous components had no interference
in the chromatograms. The mean plasma concentra
tion-time profiles of PTX after iv administration of PTX micelles and Taxol injection at a single dose of 3 mg/kg are shown in
Figure 4. The PTX concentration versus time date was analyzed by two compartmental model analyses with
1/C weighted using computer program 3p87. The pharmacokinetic parameters are shown in Table 1 and analyzed for
statistical significance by unpaired StudentĄŻs
t-test. The concentration showed a rapid decline in distribution phase for the first 1
h after dosing. The t1/2a was less than 0.2 h and there was no significant difference
(P>0.05) between the two preparations. The
t1/2b of micelles and control was 2.50 h and 5.85 h, respectively. The analysis of variance of the
t1/2b, K10, clearance (Cl)
showed significant difference among groups
(P<0.05). These data indicated that the formulation with micelle increased the
systemic circulation time of PTX. And this result was in agreement with the
in vitro release of PTX from micelles. Meanwhile,
plasma concentrations of PTX in Pluronic P123 micelles were higher than that in Taxol. Pluronic P123 micelles provided
significantly higher (2.9-fold) AUC compared to Taxol.
Tissue distribution of polymeric micellar paclitaxel in healthy animal models
In vivo behavior of PTX after intravenous
administration of micelles to mice was investigated with Taxol as control (Figure 5). The blood PTX concentration-time
profiles observed in mice were similar to the pharmacokinetic study in rats. The time of distribution phase was short and the
concentration decreased quickly in this phase. The AUC of two preparations in different tissues, including plasma, heart,
spleen, lung, liver, kidney, ovary and uterus, and brain were calculated (Table 2). The PTX AUC of polymeric micelles was
lower in liver and higher in plasma, ovary and uterus, lung, and kidney compared to the control. In those tissues, the order
in AUC from highest to lowest for Taxol was liver>lung>spleen>kidney>ovary and uterus>heart>plasma>brain. In contrast,
the corresponding order for the Pluronic P123 micelles was liver>ovary and uterus>kidney>lung>spleen>heart>plasma>
brain.
Discussion
By a solid dispersion method, PTX was effectively loaded in Pluronic P123 micelles. The PTX-loaded micelles had a
narrow particle size distribution and a spherical shape.
PTX is a poorly water-soluble drug. Its solubility in water is approximately 0.3 µg/mL. Inclusion of surfactants in release
media is the most popular method for in
vitro PTX release. But the addition of surfactants in release media might have a
significant effect on the micellar structure and distort the release profiles. So maintaining a good sink condition for PTX is
one of the challenges in designing in vitro release experiments of polymeric micelles. Hydrotropic agents, sodium salicylate
solution, which can solubilize PTX, were used to maintain the sink condition of the aqueous release media by Huh
et al[20]. Their work showed that sodium salicylate did not significantly affect the physical stability of micelles up to a concentration
of 1.0 mol/L. So in this experiment we used 1 mol/L sodium salicylate solution for drug release. In the
in vitro release experiment, almost all PTX in stock solution was rapidly released in 4 h, which indicates that the hindrance effect of the
dialysis bag was minimal. The release profile of PTX from Taxol was similar with the stock solution. The cumulative release
amount of PTX from Taxol in 4 h was around 90%. Pluronic P123 micelles released only 41.2% PTX in 4 h and 87.8% PTX in
24 h, which was much slower than Taxol. This result showed that the micellar carrier can not only solubilize the
water-insoluble drug but also sustain the release of it.
With a
EO20-PO70-EO20 structure, Pluronic P123 spontaneously form micelles in aqueous media with the hydrophobic core
of poly(propylene oxide) (PPO) and hydrophilic shell of poly(ethylene oxide) (PEO) at concentrations of the block copolymer
above the CMC (4.4×10-6 mol/L). The water insoluble drug, PTX, was entrapped in the hydrophobic core. The hydrophilic
shell of PEO made the micelles avoid the recognition and uptake by the reticuloendothelial systems and prolong the time of
blood circulation of PTX. This effect of Pluronic P123 micelles resulted in an increase in
t1/2b, AUC and a reduction of Cl of
PTX compared to Taxol. The higher AUC in plasma and lower AUC in liver of PTX-loaded micelles than the control in
biodistribution investigation were also a result of this effect.
In general, amphiphilic polymeric micelles can reduce PTX uptake by the kidney and liver. But in our work,
the AUC of PTX in kidney of Pluronic P123 micelles was
higher than the control. This result may be explained by the
biological property of Pluronic P123. It was reported earlier that Pluronics were excreted primarily through the
kidneys[21]. The clearance in rats, dogs, and humans was shown to be almost entirely by renal excretion. Studies also showed that the formation of micelles had
no effect on the elimination clearance of the block
copolymer[22]. The biodistribution study of
Pluronic P 105 micelles also showed that the concentration of this material accumulated in kidney cells was higher than the other organs in mice after ip
injections[23]. It is acceptable that Pluronic P123 micelles leading to an increase in AUC of PTX in kidney because PTX had
no toxic side effect to the kidney in the range of clinical
dosage[24].
In tissue distribution studies, the higher concentrations of PTX following iv administration in mice were found in the liver,
lung, kidney, and spleen than in plasma. The PTX concentration in the brain was very low; many brain samples couldnĄŻt be
accurately measured because the concentration was lower than the
limit of quantitation. These results were consistent with
the work reported by Rowinsky and
Donehower[25].
Both the ovaries and uterus are too small to determine
singlely, so in this study, we placed them together. An interesting
result in this work was that Pluronic P123 micelles significantly increased the distribution of PTX in the ovaries and uterus,
while PTX had been proved effective in the treatment of ovarian cancer.
In summary, these studies have shown that Pluronic P123 micelles may efficiently load, protect and retain PTX in the
biological environment. As a result, the pharmacokinetics and tissue distribution parameters of PTX are changed.
Incorporating PTX in Pluronic P123 micelles can increase the drug residence time in the blood circulation and the distribution
in blood, kidney, ovaries and uterus, and lung. At the same time they can reduce the distribution in the liver.
These results suggest that polymeric micellar formulation can provide useful alternative dosage forms for intravenous administration of
PTX. A study on the potential of sensitization of MDR
cells of Pluronic P123 is in progress in our lab.
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