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Introduction
Rabdosia rubescences (Chinese name "Donglingcao"), a herbal medicine, is traditionally used in China for the treatment
of tonsillitis and a variety of cancers. Oridonin, a diterpenoid extracted from
Rabdosia rubescences, is the marker compound
and one of the major antitumor components of this
herb[1,2]. Oridonin injection is used alone or in combination with other
drugs to treat human cancers, especially for the treatment of liver
cancer[3,4], esophageal carcinoma and carcinoma of gastric
cardia[5]. Oridonin injection and Rabdosia
rubescences can also extend the lives of advanced cancer patients as well as
improve their living quality. Due to the low toxicity of oridonin and
Rabdosia rube- scences, they can be used at a high dose
for a long period of time, and only a few patients suffer abdominal
discomfort[5]. Because of the abundance of
Rabdosia rubescences in China, recently, oridonin and
Rabdosia rubescences have attracted special attention.
Although Rabdosia
rubescences and oridonin have been used clinically for a long time, there is little information in
published literature on the pharmacokinetics of oridonin. To our knowledge, only the pharmacokinetics of oridonin after iv
administration in rabbits and iv and ip administration in mice have been
reported[6,7], and there is no published information
about the pharmacokinetics of oridonin following oral administration. In clinical trials,
Rabdosia rubescences is usually administered orally, so a better understanding of the pharmacokinetics and oral bioavailability of oridonin is very important
for explaining the therapeutical outcomes produced by the drug in clinical trials and to help establish a rational dosage
regimen.
In the present paper, we describe the pharmacokinetics behavior of oridonin after intravenous, oral and intraperitoneal
administrations to rats.
Materials and methods
Chemicals and reagents Oridonin (98.9%) was
extracted from the aerial parts of Rabdosia
rubescences and refined in our laboratory (identified by
1H-NMR, UV and MS). The internal standard, ethyl hydroxybenzoate (99.5%), was supplied by
Shenyang Dongxing Reagent Factory (Shenyang, China). HPLC-grade methanol was obtained from Concord Tech Co
(Tianjin, China) while HPLC-grade ethyl acetate and
n-butanol were from purchased from Tianjin Kermel Chemical Reagents
Development Centre (Tianjin, China). All other reagents were of analytical grade. Distilled water, prepared from deionized
water, was used throughout the study.
Oridonin solution For intravenous, oral and intraperitoneal administration, oridonin (5 mg/mL) solution was prepared in
0.9% (w/v) saline containing 30% ethanol
(v/v).
Animals and surgical
procedures Male Wistar rats (230_250 g) were supplied by the Lab Animal Center of Shenyang
Pharmaceutical University (Shenyang, China). All experimental procedures were carried out in accordance with the
guidelines of the Experimental Animal Care and Use Committee of Shenyang Pharmaceutical University. The animals were
maintained under standard laboratory conditions on a 12 h light/dark cycle and were fed standard rat chow and water
ad libitum. The rats fasted overnight before the experiments and food was returned 2 h after dosing. Water was available
ad libitum throughout the experiments.
Drug administration and sample
collection Three groups of rats (each group contained 6 rats) were given oridonin
solution as a single dose of 5, 10 and 15 mg/kg via the femoral vein (slightly anaesthetized by aether). The infusion time was
about 10 s. Blood samples (250 µL) were collected in heparinized tubes from orbit veins with an heparinized glass tube at
0.083, 0.167, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, and 48 h after administration; another 3 groups of rats (each group contained 6 rats)
received a single dose of 20, 40, and 80 mg/kg by oral gavage. Blood samples (250 µL) were collected in the heparinized tubes
from each rat at 0.05, 0.1, 0.167, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, and 48 h after administration. Six rats were given oridonin
solution as single dose of 10 mg/kg intraperitoneally and blood samples (250 µL) were collected in the heparinized tubes from
each rat at 0.083, 0.167, 0.5, 1, 2, 4, 6, 9, 12, 24, and 36 h after administration. Blood samples were immediately centrifuged and
stored at -20 °C until analysis.
Sample preparation and
analysis The concentrations of oridonin in rat plasma were determined by an HPLC/electrospray
ionization mass spectrometric detection (HPLC/ESI-MS) method developed and validated in our
laboratory[8]. Briefly, to 100 µL plasma in glass centrifuge tubes 50 µL ethyl hydroxybenzoate (internal standard, 80 ng/mL) and 50 µL mobile phase were
added. Samples were then vortex-mixed for 30 s and extracted with 3 mL ethyl
acetate-n-butyl alcohol (100:2,
v/v). After vortex-mixing for 1 min and shaking for 10 min, the organic and aqueous phases were separated by centrifugation at
2000×g for 10 min, then the upper organic layer was transferred to another tube and evaporated to dryness at 40 °C under a gentle
stream of nitrogen. The residue was reconstituted in 100 µL mobile phase followed by vortex-mixing and centrifugation at 2
000×g for 10 min. Then, 20 µL of an aliquot of the supernatant was
injected onto the HPLC/ESI-MS system.
The high-performance liquid chromatography was performed using a Waters 1525 Binary pump
(Framingham, Massachusetts, USA), which was controlled by Masslynx 4.0 Software (Waters
Corp, Framingham, Massachusetts, USA). The mobile phase consisted of methanol-water
(80:20, v/v) at a flow rate of 1.0 mL/min and the injection volume was 20 µL. The analytical column used was a
DiamonsilTM C18 column (200 mm×4.6 mm id, 5 µm) from Dikma Tech (Beijing, China) at a column temperature of 25 °C.
A ZQ2000 micromass spectrometer (Waters Corp, USA) fitted with a Z-Spray ion interface was used for all analyses.
Ionization was achieved by using electrospray in the negative mode. The following parameters were optimized for the
analysis of oridonin: capillary voltage, 3.0 kV; cone voltage, 25 V; source temperature, 105 °C; and desolvation gas (nitrogen)
heated to 350 °C and delivered at a flow rate of 350 L/h. Quantification was performed using the selected ion recording
of m/z 363 for oridonin and
m/z 165 for ethyl hydroxybenzoate. The lower limit of quantification of the
method was 10 ng/mL and the quantitation range was 10_4
000 ng/mL. For samples containing oridonin at a concentration higher than the upper limit of the
range in the standard curve, an aliquot of the sample was first diluted with blank rat plasma and then 100 µL of the diluted
sample was treated as described. The intraday and interday accuracy and precision of the assay were less than 9%.
Pharmacokinetic analysis All pharmacokinetic parameters were determined by non-compartmental analysis. The peak
plasma level (Cmax) and the time to reach the peak plasma concentration
(tmax) were obtained directly from the
concentration-time data. The elimination rate constant
(Ke) was calculated from the slope of the logarithm of the plasma concentration
versus time using the final 4 points. The apparent elimination half life
(t1/2) was calculated as
0.693/Ke. The area under the plasma concentration-time curve (AUC) and the area under the first moment curve (AUMC) were calculated by the
trapezoidal rule. Total body clearance was calculated as
X0/AUC. The extent of absolute bioavailability was estimated from the
dose-normalized ratios of
(AUC0-¥)poto
(AUC0-¥)iv (based on iv 10 mg/kg). The mean residence times after intravenous administration
(MRTiv) and oral administration
(MRTpo) were calculated by dividing the
AUMC by the AUC. The mean absorption time (MAT) was calculated by subtracting
MRTiv from
MRTpo. The values were calculated by Microsoft Excel (Microsoft, Seattle, Washington, USA) and each value
was expressed as mean±SD.
Results
Pharmacokinetic analysis of plasma concentrations
after intravenous administration After intravenous administra-tion, the plasma concentration of oridonin first decreased
rapidly and then more slowly, that is to say, the plasma concentration of oridonin after intravenous administration decreased
polyexponentially and the terminal elimination half life was relatively long (about 10 h). After intravenous administration, the
pharmacokinetic parameters of oridonin were dose-independent at 3 doses: 5, 10, and 15 mg/kg. These results show that
oridonin exhibits linear
kinetics following intravenous administration over the dose range studied (Table 1, Figure 1).
Pharmacokinetic analysis of plasma concentrations
after oral administration The mean plasma concentration versus time curve of oridonin increased rapidly after oral
administration reaching the maximum level less than 15 min after administration. Starting 9 h after administration, the profiles of oral
and intravenous administration declined in parallel. The mean plasma concentration versus time curves after intravenous
administration was much higher than that after oral administration at a higher dose. The extent of absolute bioavailability was
rather low and appeared to be dose dependent. At a high dose (80 mg/kg), the extent of absolute oral bioavailability was
about 2 times higher than those of low doses (20 and 40 mg/kg) (Table 1, Figure 2).
Pharmacokinetic analysis of plasma concentrations
after intraperitoneal administration After intraperitoneal administration, the plasma concentration-time curve was similar to
that after intravenous administration, except that the plateau was absent. Furthermore, the extent of absolute bioavailability
of oridonin was only 12.6% (Figure 3).
Discussion
The oral absolute bioavailability of oridonin was rather low (4.32%_0.8%) and appeared to be dose dependent.
Considering the amount of unchanged oridonin recovered from the gastrointestinal tract and feces 48 h after oral administration (the
mean value was approximately 6.52%; Figure 4), the low extent of absolute oral bioavailability values are most likely due to
hepatic, gastric and/or intestinal first-pass effects. After intraperitoneal administration of oridonin solution to rats at a single
dose of 10 mg/kg, the bioavailability of oridonin was 12.6%; Figure5). This result shows that hepatic first-pass effect may be
the main reason for the low oral bioavail-ability of oridonin. Oridonin is a water-insoluble drug; some researchers have tried
to enhance its oral bioavailability by simply increasing the dissolution rate of the drug from its dosage
form[9]. Although in vivo behavior of those dosage forms had not been performed, based on the result of our paper we can speculate that through
those meanings, the enhanced oral bioavailability is limited. Other drug delivery systems which can circumvent the liver
first-pass effect may work, such as the M cell drug delivery
system[10] (Table 1, Figure 3).
Many natural products display beneficial anticancer effects
in vitro, but only a few have been involved in clinical trials.
One of the main obstacles which prevent the development of natural anticancer drugs is that the concentration of drugs
in vivo can not reach the level used in
vitro. The in vitro anticancer effect of oridonin has been studied by several
groups[11_16]. These research show that the anticancer
effect of oridonin is time and dose-dependent; at a low µmol/L
concentration (about
1 µg/mL), oridonin has weak apoptosis-inducing effects, while at a concentration of 8_10 µmol/L, oridonin has strong
apoptosis-inducing effects on most cancer
cells[11,13]. According to the clinical dose of
Rabdosia rubescences[17] and the
content of oridonin in the herb[1], a patient receiving about 175 mg oridonin per time orally is equal to the dose of 13.5 mg/kg
to rats[18]. Obviously at this dose the plasma concentration of oridonin is too low to have a beneficial effect. In clinical trials,
patients receive Rabdosia rubescences solution 3 times per day. If the similar poor and dose-dependent oral bioavailability
phenomenon is found in men, a higher dose and a longer dosing interval may produce better curative effects.
In clinical trials, Rabdosia
rubescences and oridonin are usually used in combination with other drugs to enhance the
anticancer effects of chemical
drugs[17,19]. Since the hepatic first-pass effect may be the main reason for the low oral
bioavailability of oridonin, potential drug-drug interactions may occur: enhanced oral bioavailability of oridonin and
decreased drug metabolism, which may lead to better anticancer effects or more severe side effects.
The pharmacokinetic parameters of oridonin were dose independent at 3 doses, 5, 10, and 15 mg/kg after intravenous
administration. The dosage range was set according to the toxicity of the menstruum and the sensitivity of our analytical
method, but the dosage range is somewhat narrow to give more pharmacokinetic information. Considering that the length of
the plateaus of the 3 curves in Figure 1 look dose dependent, we wonder what will happen with a reduction of the dose.
Although an intravenous administration experiment at an even lower dose was not carried out, the intraperitoneal
administration experiment may be used as a reference. After intraperitoneal administration of oridonin solution to rats at a single dose
of 10 mg/kg, the plateau of plasma concentration-time disappeared. Since the percentages of the intravenous dose of
oridonin excreted in bile as unchanged drug was 16.0% in the rats (data not shown), the
enterohepatic circulation may contribute to the plateau in the plasma concentration-time curves of Figure 1 and Figure 2.
The plateaus of the curves in Figure 1 may also be accounted by saturable tissue uptake since the sharp decrease of the plasma oridonin concentration after
intravenous administration was due to tissue uptake.
In conclusion, we systematically investigated the pharmacokinetic behaviors of oridonin after intravenous, oral and
intraperitoneal administration in rats. The mean plasma concentration-time curve appeared to be polyexponential, and
oridonin exhibited linear kinetics at 3 doses following intravenous administration. The extent of absolute oral bioavailability
was rather low and dose-dependent.
Acknowledgments
The authors would like to thank Dr Feng QIU and Dr Liang CUI for the technical help in animal experiments.
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