Preparation, Pharmacokinetic Profile, and Tissue Distribution Studies of a Liposome-Based Formulation of SN-38 Using an UPLC–MS/MS Method
Preparation, Pharmacokinetic Prof ile, and Tissue Distribution Studies of a Liposome-Based Formulation of SN-38 Using an UPLC-MS/MS Method
Kai Li 0
Shujun Wang 0
0 Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University , 103 Wenhua Road, Shenyang, 110016 , China
The application of 7-ethyl-10-hydroxycamptothecin (SN-38) in cancer treatment is limited by its low solubility. This study is to develop a liposome-entrapped formulation of SN-38 (LE-SN38) to solve the obstacle and to evaluate its pharmacokinetic profile in dogs and tissue distribution in mice. LE-SN38 which is more likely to be suitable for large-scale production was prepared by the carrier-deposition method. An UPLC-MS/MS method was used to determinate the concentration of SN-38 in this study. LESN38 was cleared rapidly from dog plasma within 1 h, and the AUC0−∞ values of three dosages of LESN38 indicated an apparent dose-dependent manner. As for the distribution study, the peak of SN-38 levels in most tissues were detected within 10 min after LE-SN38 administration. In addition, concentration of SN-38 in most tissues except kidney and heart in LE-SN38 group was higher than that in irinotecan hydrochloride (CPT-11) group generally, whereas the administrated CPT-11 had 20 times dosage compared to LE-SN38. LE-SN38 was rapidly eliminated from dog plasma and manifested linear dynamics in dose range of 0.411-1.644 mg/kg. The distribution behavior of SN-38 is altered in a liposome-based delivery system. At the same time, LE-SN38 has lower toxicity compared to CPT-11 in some degree.
carrier-deposition method; LE-SN38; pharmacokinetic study; tissue distribution; UPLC-MS/MS
7-Ethyl-10-hydroxycamptothecin (SN-38) is the
biological active metabolite of irinotecan hydrochloride (CPT-11).
CPT-11 has shown clinical activity against colorectal cancer
and other malignances, but it just acts as a prodrug and has to
be transported to its active metabolite, SN-38, by various
enzyme systems (
). And the metabolic conversion rate is
only 2–8% in spite of the fact that CPT-11 could be converted
to SN-38 in both liver and tumor tissues (
). In addition, it
not only poses significant life threatening toxicity risks but also
complicates the clinical management of patients, as the
transformation from CPT-11 to SN-38 is full of variable between
patients (6). Moreover, SN-38 is approximately 100–1000-fold
more cytotoxic than the parent drug (CPT-11) (
). Due to
its high anticancer activity without any conversion, the direct
use of SN-38 would have a very broad application prospects.
And it is more possible to be recognized by doctors and
patients. However, SN-38 undergoes pH-dependent
conversion of pharmacologically active lactone ring to inactive
carboxylate form at pH >6 (
). And the active lactone ring has
poor solubility in aqueous solutions and is practically insoluble
in most pharmaceutically acceptable excipient, as it was
reported that the solubility of SN-38 in water, pH 3.5 and 7.4
buffers were assessed as 11–38, 7.2, and 36 μg/ml, respectively
). Hence, the further use of SN-38 in clinic is limited by its
low solubility and stability, as well as its severe toxicity (
These drawbacks above can potentially get over by
loading the drug into delivery system, particularly
liposome. By using a liposome system, drug solubility could
be enhanced and the toxicity of loaded anticancer agents
would be reduced as well. What is more, it could also
improve stability of loaded drugs by protecting the
compound from in vivo conditions like acid or alkaline
). These characters are exactly what the
optimization of SN-38 delivery needs. Therefore, here, it
would be a wise choice to develop a liposome-based
formulation of SN-38.
It is well-known that a good understanding of drugs’ in
vivo behavior and the proper knowledge on the distribution is
vital to investigate the action mechanism and the major target
sites of the product (
The objective of this study is to develop a liposome-based
formulation of SN-38 and to evaluate its pharmacokinetic
profile and biological distribution using UPLC–MS/MS. It is
expected that this experiment could provide helpful
information for preclinical and clinical studies.
MATERIALS AND METHODS
Materials and Animals
SN-38 was provided by Jingmen Shuaibang Chemical
Science and Technology Co., Ltd, China. Camptothecin was
purchased from Sichuang Chengxin Technology Co., Ltd,
China. Irinotecan hydrochloride injection was obtained from Qilu
Pharmaceutical Co., Ltd, China. Soybean phospholipid was
supplied by Shanghai Advanced Vehicle Technology Co., Ltd,
China. Cholesterol was provided by Tianjin Concord
Technology Co., Ltd, China.
The beagle dogs were purchased from the Research
Institute for Laboratory Animal in Kangping (Shenyang,
China; SCXK(Liao)2009-0005). And the SPF Kunming
mice weighing 18–22 g were obtained from the
Experimental Animal Center of Shenyang Pharmaceutical University
(Shenyang, China; SCXK(Liao)2010-0001). All the animal
experiments mentioned in this study were in accordance
with the Guidelines for the Care and Use of Laboratory
Animals, and they were approved by the University Ethics
Preparation of Liposome-Encapsulated SN-38
T h e c a r r i e r - d e p o s i t i o n m e t h o d w a s u s e d f o r
liposome-entrapped formulation of SN-38 (LE-SN38)
). Briefly, Solutol HS® 15 was
solubilized in anhydrous ethanol and then transferred into a
round-bottom flask which contains SN-38, cholesterol,
and soybean phospholipid. After dissolution, the
resulting mixture and glucose powder were rotated
under ultrasonic condition for uniform mixing. Then the
solution was evaporated to a thin film and rehydrated
with phosphate-buffered saline (PBS) containing 10%
mannitol by magnetic stirring. The hydration process
was performed at 55°C. Ten minutes later, a
homogeneous dispersion was formed and immediately sonicated
at 400 W power for 5 min using probe sonicator
(S-4000-010, Misonix, USA), and the whole processes
was carried out in ice bath. The resulting was filtered
using a 0.22 μm filter before being transferred into 10
ml vials. Lyophilization was operated by a FD-1 freeze
drier, which comprised the following steps: pre-freezing
at −40°C for 12 h and then drying for 8 h under the
pressure of 30 Pa.
Characterization of LE-SN38
Drug entrapment efficiency was carried out on a
mini-column centrifugation method (
G-50 was filled to a column (1.0 × 10 cm) which was
applied to separate free drug from SN-38 liposome.
Briefly, 0.5 ml of the SN-38 liposome suspension was loaded
into the gel column. Subsequently, it was centrifuged for
2 min and then eluted by 0.5 ml PBS and centrifuged
again. The procedure was repeated twice. Then all the
eluent, the solution of liposomes enveloping SN-38, was
collected. After that, 0.5 ml of the eluent and uneluted
LE-SN38 were separately diluted by methanol. The
amount of SN-38 encapsulated into liposomes was
determined by UV spectrophotometer.
Wenveloped was the concentration of SN-38 enveloped in
liposomes; Wtotal was the concentration of SN-38 in liposome
The zeta potential and particle size of LE-SN38 were
performed on a laser light scattering Zetasizer (Nano-ZS90,
Malvern, UK) at 25°C.
The pharmacokinetic study was carried out on beagle
dogs. Before the experiment day, nine of these dogs were
fasted overnight but allowed free access to water. Then the
dogs were randomly divided into three groups and received
injections of 0.411, 0.822, and 1.644 mg/kg single dose of
LESN38, respectively. The intravenous drip was conducted for
about 30 min, then 2 ml of blood samples were collected into
heparinized centrifuge tube at 5 min, 10 min 15 min, 30 min,
1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration.
The plasma samples were centrifuged and then stored at
−40°C until determination.
Tissue Distribution Studies
The tissue distribution experiment was studied on mice.
There were eight groups with six mice in each group. For the
first four groups (LE-SN38 groups), 4 mg/kg of LE-SN38 was
given intravenously via the tail vein, and as for the other four
groups (CPT-11 groups), CPT-11 was injected at a dose of
80 mg/kg. At 10, 30, 120, and 360 min after administration,
the mice were immediately sacrificed, and organs including
heart, liver, spleen, lung, kidney, and intestine were collected;
then they were rinsed with saline and then stored at −40°C
Samples were thawed to room temperature before
dealing with them. Then the tissues were homogenized with
physiological saline at the ratio of 1:10 (g/ml) for the liver, spleen,
lung, and intestine and 1:2 (g/ml) for the heart and kidney to
get tissue homogenate samples. An aliquot of 100 μl IS
solutions (50 ng/mL) was added to 100 μl of matrix sample in
1.5 ml microcentrifuge tubes. The sample mixture was
vortexed for 30 s at room temperature. Next, 400 μl of
acetonitrile (containing 0.5% acetic acid) was added and vigorously
vortexed for 1 min (
). After centrifugation, the supernatant
was extracted and transferred to another clean centrifuge tube
and evaporated under N2 flow at 50°C. Then 100 μl 0.1%
formic acid–acetonitrile (70:30, v:v) was added and vortexed
for 30 s. After being centrifuged for 10 min, 10 μl of the
supernatant was injected for analysis.
For appearance: + + good appearance, + good appearance with slight shrinkage at the edge, − poor appearance, −−very poor appearance.
For reconstitution: + + ease, uniform size, no crystal; + large size, some crystal; − difficult to reconstitute uniformly
Statistical Analysis of Data Li and Wang
SN-38 and CPT (IS) w ere separated using an
ACQUITY UPLC®BEH C18 column (2.1 × 50 mm,
1.7 μm, Waters, USA) at 30°C. It is a gradient elution with
acetonitrile (A) and water (B, containing 0.1% formic
acid) as the mobile phase, and the flow rate was 0.2 ml/
min. The initial mobile phase composition was A–B (20:80,
v/v) with a 0.1 min held and followed by a linear increase
to A–B (99:1, v/v) over a 0.1 min period, then held for
0.8 min, and finally returned to A–B (20:80, v/v) during
0.5 min. The total time was 4 min with an injection 10 μl
for analysis (
The Waters Xevo TQD triple quadrupole mass
spectrometer (Waters Corp., Milford, MA, USA) equipped with an
electrospray ion source was used for mass detection. The
capillary voltage, cone voltage, and desolvation temperature
were 3.05 kV, 47 V, and 300°C, respectively. Nitrogen was used
as the desolvation gas (650 l/h) and cone gas (50 l/h). And the
multiple reaction monitoring (MRM) was used at the
transitions of m/z 393.3 → 349.2 for SN-38 and m/z 349.2 → 305.3
for IS, respectively.
The method was validated for the quantification of SN-38
in dog plasma and mice tissue samples. The calibration curves
were linear within 0.5–500 ng/ml in dog plasma and within 1–
500 ng/ml in mice tissues (r = 0.9974–0.9995). The lower limit
of quantification (LLOQ) was 0.5 ng/ml in plasma and the
precision was 9.53% with accuracy of 97.32%. In tissue
samples, the LLOQ was 1 ng/ml, and its precision was ranging
from 4.65 to 8.46% with accuracy from 94.9 to 105.5%. The
intra- and inter-day precision at three concentrations (1, 50,
and 500 ng/ml) was less than 10%. The recoveries were
accessed by comparing peak area ratios of extracted QC
samples with the blank plasma and blank tissue samples spiked
with SN-38 after protein precipitation at the three
concentrations (1, 50, and 500 ng/ml), having a result of 92.25–98.45 and
90.33–93.64%, respectively. The absolute matrix effect values
were in the range of 94.43–105.13% for plasma and 90.67–
103.23% for tissue homogenate. These results indicated that
there was no significant matrix effect for SN-38 in the matrix
sample for this UPLC–MS/MS determination.
All measurements were taken at least in triplicate.
Results were represented as mean ± SD. The non-compartmental
pharmacokinetic data were carried with Drug and Statistics
2.0 (DAS 2.0) software (Mathematical Pharmacology
Professional Committee of China, Shanghai, China). Statistical
analysis of the study was calculated with SPSS 11.5 software. It was
considered to be statistically significant with values of P < 0.05.
RESULTS Preparation of Liposome-Encapsulated SN-38
The carrier-deposition method was used to prepare
LESN38. The concentration of SN-38 for the optimized
formulation was 1 mg/ml, and the weight percent ratios of
drug-tolipids and SPC to cholesterol was 1:30 and 1:6, respectively.
Lyophilization was adopted to enhance the stability of
LESN38 in the formulation. However, vesicle fusion and leakage
of contents can appear for the liposome solution in the
absence of any protective agents (
). In this study, freshly
prepared LE-SN38 suspensions were lyophilized with
different excipients to assess for cryoprotective activity. And the
results were shown in Table I. The structure of the
freezedrying cakes was obviously different while mixing with
different protective agents. Both mannitol and dextran groups
could contain integrity freeze-drying cakes, but the dextran
group expressed with a larger size and some crystal. Hence,
mannitol was an ideal choice.
In another study, liposomes were freeze-dried at different
concentrations of mannitol, including 3, 5, 8, and 10% (w/v). It
showed that 10% mannitol (lipid to mannitol ratios of 1:3.3
(w/w)) could inhibit liposomal fusion or degradation during
lyophilization, and the results were assessed by the
appearance, reconstitution, and the particle size of the reconstitution
solution. And the entrapment efficiency has no significant
change. Hence, 10% mannitol tends to be an ideal choice to
effectively protect LE-SN38 during freeze-drying procedures.
The EE, particle size, and zeta potential of LE-SN38
were obtained and shown in Table II. The mean particle size
0.51 × 10−2
AUC area under the curve, t1/2 half-life, V apparent volume of distribution, CL clearance
was 130 ± 46 nm (<200 nm), with an EE of 91.7 ± 4.2% for
LESN38 before lyophilized. Because zeta potential of LE-SN38
was −0.387 mV, the low stability should be improved. Here,
lyophilization might help to this situation in some degree.
The pharmacokinetic study was performed on beagle
dogs by i.v. drip administration of 0.411, 0.822, and 1.644 mg/
kg LE-SN38. The non-compartmental pharmacokinetic
parameters resulting from DAS 2.0 software were listed in
The concentration–time profiles of SN-38 are shown in
Fig. 1. According to the figure, we can infer that the plasma
concentration of SN-38 was initially decreased rapidly for all
three dose groups, and then equilibrium was reached at 6 h.
The primary rapid decline indicates that SN-38 might have
drive away from the plasma and been distributed into the
other tissues, which was confirmed by our tissue distribution
study in mice.
Furthermore, we found that the fold increased in dosage
(0.411 versus 0.822 versus 1.644 mg/kg) led to an approximate
fold increase in AUC0−∞ (92.5 versus 165.3 versus 317.1 ng/ml
× h). No significant difference appeared (P > 0.05) in systemic
clearance and t1/2 at each dose, that is to say LE-SN38 may
have linear pharmacokinetics in dose range of 0.411–1.644 mg/
In addition, during the whole study, none of the nine
dogs exhibited gastrointestinal reaction, such as vomiting
and diarrhea. And there was no significant difference in
body weight in dogs (data not shown). This might indicate
that LE-SN38 has lower toxicity compared to CPT-11.
Thus, LE-SN38 may provide a potential therapeutic option
for cancer treatment.
Tissue Distribution Studies
Peak SN-38 levels were measured in most tissues within
10 min of treatments (Fig. 2); the results suggested that they
both underwent a rapid distribution to tissues. For LE-SN38
group, the AUC0–6 h in these tissues was detected in the
following order: liver > spleen > lung > intestine > kidney >
heart. It can be concluded that SN-38 might mainly
concentrate in the liver, spleen, and lung, as they accounted for 91%.
While it is a wide distribution in CPT-11 group, which
especially distributed in kidney (29%) and heart (1.4%). And
the concentrations of SN-38, with the exception of the kidney
and heart, were generally higher than those following
treatment with 20-fold doses of CPT-11 (Fig. 3). The AUC0–6 h of
LE-SN38 in the spleen, liver, and lung were significant higher
than that of CPT-11. The AUC0–6 h for LE-SN38 was
improved by 385% in the liver, 670% in the spleen, and 465%
in the lung compared to CPT-11 (Table IV). These results
demonstrated that LE-SN38 altered the distribution behavior
of SN-38 in tissues.
In this study, LE-SN38 was prepared by
carrierdeposition method, and it is a modified film dispersion
method. Using this method, drug and lipids were precipitated on
glucose to form solid dispersion during the evaporation. Then
it can be scraped from the sides of the bottle and milled to a
fine powder to obtain a more complete drying. On the other
hand, glucose that is soluble in water could accelerate the
hydration rate and shorten the hydration time. Moreover,
the operator could timely adjust the accident occurring during
the preparation (
). Therefore, the stability and quality of
LE-SN38 could be enhanced by this method, and the
preparation process could be more simple.
On the other hand, the product in this study would be
stored in solid form as freeze-dried powder, and it leaves no
contact with the hydration medium which could avoid the
hydrolysis of phospholipids. Therefore, it has no physical
stability and drug leakage problems which are found in the
storage of liposome solution (
). In summary, this method
solves the problem that restricted industrial production. So it
is more likely to be put into large-scale production.
Recently, several methods have been reported to be
applied for the quantification of SN-38 and CPT-11. But the
method for determination of SN-38 in dog plasma only
utilized HPLC with fluorescence detector, and it required over
6 min and 200 μl or more plasma as sample (
we would like to investigate a determination method by which
shorter analysis time and lower LLOQ could be achieved. To
the best of our knowledge, the bio-analytical method
described here is the first UPLC–MS/MS method for dog plasma
quantification of SN-38. A good separation was achieved
without using any ion-pairing agents. And it has lower LLOQ
(0.5 ng/ml) in dog plasma than the previous reports
determined with fluorescence detector (1 ng/ml). Moreover, it not
only shortens the analysis time but also uses a smaller sample
volume (0.1 ml). These results suggest that the UPLC–MS/MS
method develop here is suitable.
In the pharmacokinetic study, LE-SN38 was quickly
decreasing from dog plasma. It was reported that peak SN-38
levels were quickly measured in the liver after LE-SN38
), and our distribution study in mice also showed that
peak tissue levels were measured within 10 min in most tissues
after LE-SN38 treatment. Furthermore, the plasma
concentration in three dose groups followed the same eliminating
trend. And within the dose range of 0.411–1.644 mg/kg, the
AUC is proportional to dose. This suggested that the safety of
LE-SN38 administration is enhanced and it would also
provide helpful information on clinical medicine.
On the other hand, a comparative distribution
experiment was studied between LE-SN38 and CPT-11 by
measuring the concentration of SN-38 in mice tissues. Compared to
CPT-11, SN-38 has relatively high drug distribution in the
liver, lung, and spleen, and it still maintains a high
concentration in these tissues 6 h later. This means that it might have
good therapeutic effect for the treatment of these cancers (
and the retention time of SN-38 was prolonged in these organs
by a liposome-based formulation. The increased uptake in the
liver, lung, and spleen was probably mediated by the RES
system for the characterization of liposome. Furthermore,
concentration of SN-38 in the heart and kidney tended to be
much lower than that of CPT-11 group, providing important
evidence for reduced toxicity of LE-SN38. And this is also
consistent with our pharmacokinetic study. All the results
suggest that the distribution behavior of SN-38 is altered in a
liposome-based delivery system.
In this study, in order to surmount the solubility and
stability obstacle of SN-38, we have prepared a
liposomebased SN-38 formulation. SN-38 lyophilized powder was
prepared by carrier-deposition method, and it is stable during the
storage and more possible to be put into large-scale
production. Then we evaluate the pharmacokinetic profile of
LESN38 in beagle dogs as well as its biological distribution in
mice using an UPLC–MS/MS method. LE-SN38 was rapidly
eliminated from dog plasma and followed linear
pharmacokinetic pattern, and the distribution behavior of SN-38 was also
altered in the liposome-based formulation. It is expected that
this study could help to further clarify the in vivo behavior of
LE-SN38 and provide available information for clinical
We wish to acknowledge the support of the Pharmacy
Laboratory Centre and Animal Centre of Shenyang
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interest
The authors declare that they have no
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