1. have spent lot of efforts to identify

1. Introduction

Maoecrystal A (MC-A, Fig. 1)
is an ent-kaurane-type diterpene isolated from Rabdosia eriocalyx (Dunn)
Hara, a well-known herbal
material used in a few China Traditional Medicines. 1-3 The leaves and roots of this plant are
used by local folk for the treatment of enteritis, jaundice, hepatitis,
laryngopharyngitis, lepromatous leprosy, and ascariasis 4. Due to long
history of usage and promising efficacy, chemists have spent lot of efforts to
identify ent-kaurane-type diterpenes as the active components in this plant 5-7.
In addition, total synthesis and structure modifications of maoecrystals have
been conducted by medicinal chemists trying to develop this class of compounds
as drugs for the treatment of different types of diseases. 8, 9

Pharmacological
studies showed that ent-kaurane diterpenes possess different activities such as
anticancer, anti-diabetes, antibacterial, anti-inflammatory, and bacteriostatic
functions 10, 11. For example, Li et al reported that an ent-kaurane
isolated from isodon Phyllostacys was
highly active against a human leukemia cancer cell line K562 and its activity
was even higher than that of cisplatin. 12 Mechanism studies showed that
ent-kaurane diterpenes could selectively inhibit 11?-HSD1 11, activate Akt
signaling 13, or induce apoptosis through the mitochondrial pathway 14, 15.

Although the bioactivities of ent-kaurane
diterpenes are promising, their pharmaceutical properties have not been
well-studied. The reason could be because of lack of bioanalytical method as
this class of compounds is not sensitive in UV detector. In this paper, we
developed a sensitive UPLC-MS/MS method to quantify MC-A in biological samples
and apply the method in in vivo
sample analysis and determine the PK properties and oral bioavailability of
MC-A.

2. Experimental

2.1 Chemicals and reagents.

The
standard compound MC-A (purity >98%) was purchased
from Chengdu Must Bio-Technology Co., Ltd (Chengdu China). The standard oridonin
(purity > 98%), which was used as the internal standard, was obtained from the
National Institutes for Food and Drug Control (Beijing, China). LC-MS grade acetonitrile
and methanol were from Merck and formic acid was from Sigma. DD water was
prepared in our lab using a GWA-UN ultra-pure water apparatus (Purkinje
General, China).

2.2 Instrument and conditions.

The separation was performed using a Waters HSS T3 column
(50 mm× 2.1 mm, 1.8 µm) in an AcquityTM ultra performance liquid
chromatography (UPLC) system. Methanol and water containing 0.1% formic acid was
used as mobile phase A (A) and mobile phase B (B), respectively. Elution
gradient was: 0-0.5 min (95-95% B), 0.5-3.0 min (95-5% B), 3.0-3.5 min (5-5%
B), 3.5-5.0 min (5-95% B) and 5.0-5.5 min (95-95% B). The flow rate was 0.3
mL/min. The temperature for the auto-sampler and the column was 18°C and 40°C,
respectively.

The analyte was
quantified using a waters XEVO TQ triple quadrupole mass spectrometer equipped
with an electro-spray ionization (ESI) source. Multiple reactions monitoring
(MRM) scan type was used to increase the specificity of the analysis. The MS
parameters were listed in Table 1. Masslynx 4.1 was used to control the
instrument and data anlaysis.

2.3 Preparation of stock solution, working
solution, calibration curve in rat plasma, quality control (QC) samples, and PK
samples.

The stock solutions of MC-A
and oridonin (1,000 µg/mL) were prepared in 50% methanol (containing 0.1%
formic acid) by accurately weighing appropriate amounts of MC-A or oridonin and
dissolved the powder into the solvent in a volumetric bottle. The working
solution were prepared by diluting the stock solution of MC-A into 50% methanol
at final concentrations of 4,000.00, 2000.00, 1000.00, 500.00, 250.00, 125.00,
62.50, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0.24 ng/mL.

The standard curve samples in rat plasma were prepared by
spiking the each of the above working solution (20 µL) and I.S. solution (20 µL
in methanol, 200 ng/mL) into blank rat plasma (100 µL) and extract with 1.0 mL
of ethyl acetate. After centrifugation at 14,000 rpm for 15 min at 4 ?C, the supernatant
was transferred into a clean microcentrifuge tube and the solvent was evaporated
under N2 flow. The residue was re-constituted in 50% methanol (100
µL) for injection after centrifugation (14,000 rpm, 15 min, 4 ?C). The QC
samples were prepared at 0.80, 40.00, 500.00, and 1,500.00 ng/mL following the
same procedure.  

The PK plasma samples were prepared by spiking the blank
solvent (50% methanol, 20 µL) and I.S. (20 µL in methanol, 200 ng/mL) into the plasma
samples (100 µL) and were extracted using ethyl acetate as described above.

2.4 Method validation.

The method was validated according to the guidance from
the FDA by evaluating the specificity, linearity, lower limit of detection (LLOD), recovery, matrix effect, accuracy, precision,
and stability.

2.4.1 Specificity, linearity, and LLOD. The specificity of the
method was determined by injecting the samples prepared from blank plasma
(pooled from 6 rats), blank plasma spiked with the I.S., blank plasma spiked
with MC-A (at the LLOQ level), and post-dosing plasma samples. The linearity of the standard curve
was determined by injecting the standard curve samples prepared in the plasma
according to the method described above. The LLOD was determined by checking
the signal to noise (S/N) ratio in the chromatogram. 

 

2.4.2 Recovery and matrix effect. The extraction recovery was
determined by comparing the peak areas obtained from samples prepared from blank
plasma spiked with the QC samples at 0.80, 40.00, 500.00, and 1,500.00 ng/mL with those from samples prepared
from water spiked with the same concentrations. Matrix effect was determined by
comparing the peak areas of samples prepared from residues of pooled blank
plasma spiked with QC samples at 0.80, 40.00, 500.00, and 1,500.00 ng/mL with those from residues of mobile
phase A spiked with the same volume of QC samples.

 

2.4.3 Accuracy and
precision.
The accuracy was calculated using QC samples at 0.80, 40.00, 500.00, and 1,500.00
ng/mL. The intra-day and inter-day precisions were determined by injecting the
QC samples at these four concentrations on the same day or on three continuous
days.

 

2.5 Pharmacokinetic study using SD rats.

Male Sprague-Dawley rats (250 – 300 g) were supplied by
the Animal Center at Hubei University of Medicine. Animals were maintained in
an environment control animal facility (22 ± 2°C and 55 ± 5% relative humidity
on a 12 h light/12 h dark cycle) for at least 5 days before the experiment.

Before the PK studies, the rats were fasted overnight with
free access of water. Blood samples (~ 0.2 mL) were collected into heparinized microcentrifuge
tubes from the fossa orbitalis vein at 10, 30, 60, 90, 105, 120, 150, 180, 210,
240, 300 and 480 min after a single oral administration of MC-A (10 mg/kg in
oral suspending vehicle). Similarly, after I.V. administration (2 mg/kg in saline),
blood samples were collected at 0, 5, 15, 30, 60, 120, 240, 360, 480, and 1440
min. The samples were centrifuged at 14,000 rpm for 10 min immediately after
collection to afford the plasma. All the samples were stored at -80°C until
analysis. The pharmacokinetic parameters were calculated with DAS (Drug and
Statistics) version 2.1.1 software (edited by the Chinese Mathematical
Pharmacology Society) using non-compartment model.

3 Results and discussions

3.1 Optimizing the UPLC-MS/MS conditions

To
optimize the LC condition, different mobile phases including methanol, acetonitrile,
ammonia acetate, and formic acid, and different types of columns including C18,
HSS T3, BEH Amide columns, were tested. Based on the shape of the peaks and the
signal response in MS, methanol (containing 0.1% formic acid)/water (containing
0.1% formic acid) and HSS T3 column were selected as the mobile and the
stationary phases. A gradient
elution was established based on the shape of the MC-A peak to increase the
through-put of the method. In addition, both positive and negative scan mood
were tested. The results showed that positive scan was more
sensitive. Compound dependent parameter and instrument dependent parameters
were optimized by infusing the compound solution into the MS directly using a
syringe pump. MRM scan type was used to improve the specificity. The MS/MS fragmentation
patterns of MC-A and the I.S. are shown in Fig. 2A and 2B.

3.2. Specificity, linearity, LLOD

The specificity of the method was evaluated by analyzing blank plasma,
blank plasma spiked with MC-A and I.S., and plasma samples from the PK study.
The results showed that there is no interference at the retention times of MC-A
and I.S. (S/N>3, Fig. 3), indicating the specificity of this method is
acceptable. The standard curves were linear in the
concentration range of 0.49-2,000.00 ng/mL in the plasma. The LLOD was 0.24
ng/mL.

3.5. Recovery and matrix effect

The extraction recoveries were
determined using three replicates of QC samples at four concentrations as
described above. The recovery was > 78.1% (Table 2), suggesting that the
extraction procedure is suitable for MC-A. The matrix effect at four
concentrations were <15%, indicating that matrix effect of this extraction is in the acceptable range. 3.4. Accuracy and Precision The quantification accuracy and inter/intra-day precisions of this method was determined using the QC samples at four different concentrations. All the results of the tested samples were within the acceptable criteria (RSD% < 15%, Table 3) according to the FDA guidance, suggesting that this method is accurate and precise. 3.6. Stability in the plasma The bench, short-term, long-term, and freeze-thaw stabilities of MC-A in rat plasma were evaluated. The results showed that MC-A was stable (variation<15%) in the plasma at these different conditions (Table 4), indicating this method was suitable for bioanalysis of MC-A.   3.7 PK studies using SD rats The validated method was used to quantify MC-A in the plasma in PK studies. The mean plasma concentration-time profiles of MC-A are shown in Fig. 4 after oral and i.v. administration. The main PK parameters are listed in Table 5. In the i.v. injection, the half-life (t1/2) of MC-A was 57.73 ± 2.43 min, suggesting the clearance was rapid. The AUC(0-t) of MC-A in the i.v. administration (44875.52 ± 3806.47 µg/L*min) is ~ 10-fold higher than that (4558.096 ± 979.556 µg/L*min) of the p.o. administration. The absolute oral bioavailability is only 2.9 %. These data showed that it is a challenge to develop MC-A as an drug administrated through oral route. Since there is an acetyl in the structure (Fig. 1), hydrolysis could be one of the possible metabolism causing rapid clearance and low oral bioavailability. Further studies are needed to verify the mechanism that lead to low oral bioavailability. 4. Conclusion. In conclusion, an accurate, precise, sensitive, and rapid UPLC-MS/MS method was developed and validated to quantify MC-A in rat plasma. The method was successfully used to quantify MC-A in PK studies using SD rats. The main PK parameters and the oral bioavailability of MC-A were calculated. Since the oral bioavailability of MC-A is extremely low, efforts on absorption/metabolism are needed in order to develop this compound as a drug administered through oral route. Other ent-kaurane-type diterpenes may also suffer from the same challenge.

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