602 Original Article

Gender Differences in the Toxicokinetics of Triptolide after Single- and Multiple-dose Administration in Rats

Authors

L. Liu1, 2, J. Zhang3, Z. Wang2, D. Xu2, Z. Jiang1, T. Wang1, W. Ju3, L. Zhang1

Affiliations

1

Key words ▶ gender difference ● ▶ triptolide ● ▶ toxicokinetics ●

Abstract

Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1395676 Published online: February 3, 2015 Drug Res 2015; 65: 602–606 © Georg Thieme Verlag KG Stuttgart · New York ISSN 2194-9379 Correspondence Prof. L. Zhang Jiangsu Center of Drug Screening China Pharmaceutical University 24 Tong Jia Xiang Nanjing 210038 China Tel.:  + 86/25/83271 500 Fax:  + 86/25/83271 500 [email protected]



Triptolide is a natural compound extracted from the traditional Chinese medicine Tripterygium wilfordii Hook F with distinguishing pharmacological activities and evident toxicities. We reported previously that 28 continuous days of oral administration of triptolide in rats resulted in gender dimorphic profiles in toxicities. To better understand this issue, the toxicokinetics of triptolide was observed in this study. Rats of both sexes were administered 400 μg/kg triptolide either as a single dose or multiple doses for 28 days. Triptolide concentrations in rat plasma were determined using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The plasma concentration-time curve and toxicokinetic parameters revealed gender differences after single and repeated triptolide administration, including significantly

Introduction



Gender differences in pharmacokinetics and metabolic patterns have long been recognized, mainly due to the increasing data on gender variation in drug efficacy and toxicity profiles. Extensive attention has been paid to this field recently with the belief that gender may be an important variable in the processes of absorption, distribution, metabolism, and excretion. Sex-based differences in these 4 major factors are conceived to originate from physiological variations between males and females, such as body weight, plasma volume, gastric emptying time, plasma protein levels, cytochrome P450 activity, drug transporter function, and excretion activity [1, 2]. Studying gender specific pharmacokinetics has gained momentum with the increased emphasis on individualized therapies and the need to prevent adverse drug reactions.

Liu L et al. Gender Dimorphic TK of Triptolide …  Drug Res 2015; 65: 602–606

higher area under the plasma concentrationtime curve (AUC0–∞) and peak plasma concentration (Cmax), lower clearance rate (CL) and longer terminal elimination half-life (t1/2) of triptolide in females, and lower drug exposure levels and greater CL in males. The gender differential disposition of triptolide may be the cause of increased toxicity in females. Moreover, autoinhibition of metabolism and the resulting increase in drug exposure were observed after repeated dosing. The AUC0–∞ of triptolide was increased 6-fold in females and 2-fold in males, while the CL of triptolide was significantly decreased by 84 % in females and 55 % in males. These results indicated that gender-related differences existed in the toxicokinetics of triptolide and long-term oral administration of triptolide resulted in drug accumulation, which might account for the gender differences in the toxicities of triptolide.

▶  Fig. 1), a major active diterpenoid Triptolide ( ● triepoxide isolated from the roots of Tripterygium wilfordii Hook F (TWHF), possesses multiple distinguishing pharmacological activities such as anti-inflammatory, anti-fertility, anti-neoplastic and immunosuppressive properties [3–6]. It has been reported to be effective in therapies for arthritis, lupus erythematosus, diabetic nephropathy, cancer, etc. [4, 6–8]. However, triptolide has a narrow therapeutic window and serious toxicities effecting the digestive, urogenital and blood circulatory systems [9, 10]. The dosage range in clinical use is strictly defined in order to maximize efficacy while minimizing toxicity or sideeffects. Studies have shown that the metabolism, bioavailability and toxicity of triptolide are heavily dependent on hepatic CYP450 activities in mice and in rats [10, 11], indicating that metabolism may play an important role in the disposition of

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received 14.08.2014 accepted 20.11.2014

 Jiangsu Center of Drug Screening, China Pharmaceutical University, Nanjing, China  School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, China 3  Department of Clinical Pharmacology, Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, Nanjing, China 2

Original Article 603

Materials and Methods



Chemicals and reagents

Triptolide and prednisolone (Internal Standard, I.S.) were purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). HPLC grade methanol and ethyl acetate were obtained from Tedia (Fairfield, OH, USA). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA). The other chemicals and solvents used were of analytical grade.

Toxicokinetic studies

Male and female 6-week-old Sprague-Dawley (SD) rats weighing 180–220 g, purchased from the SIPPR-BK Experimental Animal Company (Shanghai, China), were housed in an air conditioned room with 12/12 h light/dark cycles and acclimated to the laboratory for 1 week before the start of the experiments. Standard rat chow and water were available ad libitum. Animal experiments were carried out in compliance with the standard ethical guidelines and under the control of the faculty on the Ethical Committee. For the single-dose toxicokinetic study, male and female SD rats (6 animals per sex group) were administered an oral dose (400 μg/ kg body weight) of triptolide. For multiple-dose toxicokinetic

Fig. 1  Structures of triptolide a and prednisolone (I. S.) b.

study, male and female SD rats (6 animals per sex group) were administered triptolide (400 μg/kg body weight) by gavage once daily for 28 days. Blood samples of 0.25 mL were collected in heparinized eppendorf tubes via the posterior orbital venous plexus before and subsequently at 0.083, 0.167, 0.25, 0.3, 0.5, 0.75, 1, 1.5, 2 and 3 h after the final administration. The samples were immediately centrifuged at 3 000 rpm for 10 min and the aliquots of plasma were frozen at − 70 °C until bioanalysis.

LC-MS/MS analysis of toxicokinetic study samples

Plasma concentrations of triptolide were determined using a validated LC-MS/MS method that was accurate, precise, specific, sensitive and reproducible. A 100 μL aliquot of plasma sample, spiked with I.S. working solution (10 μL), was vortex-mixed for 30 s and extracted with ethyl acetate (2 mL) after 3 min of vortex mixing. The tubes were then centrifuged at 3 000 rpm for 10 min. The upper organic phase was quantitatively transferred into a 5 mL glass tube and evaporated to dryness under a gentle stream of nitrogen at 37 °C. The residues were then reconstituted with 50 μL of the mobile phase and centrifuged at 12 000 rpm for 10 min after vortex mixing, and 10 μL supernatant was injected onto the analytical column. The LC-MS/MS analysis was carried out using an Agilent 1200 series liquid chromatographic system (Wilmington, DE, USA), interfaced to an API4000 triple quadruple mass spectrometer (Applied Biosystems-SCIEX, Concord, Canada) and coupled with atmosphere pressure chemical ionization (APCI). Triptolide and prednisolone were separated on a ZorbaxSB-C18 column (3.0 mm × 100 mm, 3.5 μm, Agilent Technologies, USA). The mobile phase consisted of methanol (B) and 4 mM ammonium acetate containing 0.2 % acetic acid (D). The gradient elution was: 0 min, B:D 65/35 (v/v); 1 min, B:D 95/5 (v/v); 6 min, B:D 65/35 (v/v). The flow rate was 0.4 mL/min. The column temperature was maintained at 40 °C. The mass spectrometer was operated at APCI positive ion mode, and the detection of the ions was performed in the multiple reaction monitoring (MRM) mode, monitoring the transition of the m/z 378.2 precursor ion [M + H] +  to the m/z 361.3 product ion for triptolide and the m/z 361.2 precursor ion [M + H] +  to the m/z 147.2 product ion for I.S.. Nitrogen was used as the collision gas, and the nebulizer (GS1), curtain, and collision gases were set to 80, 10 and 8 psi, respectively. Compound parameters, viz., declustering potential (DP), entrance potential (EP), collision energy (CE) and collision exit potential (CXP) were optimized at 40 V, 5 V, 20e V, 8 V and 45 V, 10 V, 29e V, 9 V for triptolide and I.S., respectively. Data acquisition and quantitation were performed using analyst software version 1.4.2 (Applied Biosystems, MDS Sciex Toronto, Canada). The retention times for triptolide and I.S. were 2.3 and 3.0 min, respectively.

Table 1  Accuracy and precision of LC/MS/MS method for determining triptolide concentrations in rat plasma samples. Intra-day (n = 5)

Nominal concentration

Concentration found

Precision

(ng/ml)

(mean ± SD; ng/ml)

( %)

1.64 8.22 41.1

1.596 ± 0.05 8.132 ± 0.446 43.34 ± 1.95

3.15 5.48 4.49

Inter-day (n = 3) Accuracy ( %) 97.32 98.93 105.45

Concentration found

Precision

(mean ± SD; ng/ml)

( %)

1.557 ± 0.097 7.713 ± 0.465 43.1 ± 1.572

6.24 6.03 3.65

Accuracy ( %) 94.92 93.84 104.87

Precision = (SD/mean) × 100 Accuracy = (mean concentration found/nominal concentration) × 100 %

Liu L et al. Gender Dimorphic TK of Triptolide …  Drug Res 2015; 65: 602–606

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triptolide. Triptolide has been reported to be mainly biotransformed into hydroxytriptolide by CYP3A in rat microsomes and CYP3A4 in human liver microsomes [12]. Furthermore, the metabolites of triptolide show less toxicity and fewer side effects than the parent compound [13]. Our previous study showed that male-predominant CYP3A2-mediated in vitro faster metabolic rate of triptolide might result in less triptolide toxicity in male rats [11]. In this study, the toxicokinetics of triptolide were evaluated after single- and multiple-dose oral administration to male and female rats to (1) observe the sex-dependent in vivo metabolism of triptolide in rats and (2) predict the effect of triptolide on CYP by comparing the changes in triptolide concentrations by sex after 4 weeks of daily administration.

Toxicokinetic analysis

The main toxicokinetic parameters, including the area under the plasma concentration-time curve (AUC0–∞), peak plasma concentration (Cmax), clearance rate (CL), and terminal elimination half-life (t1/2), were calculated by non-compartmental methods using DAS Software 2.0 (Chinese Pharmacological Society).

Statistical analysis

A value of p  10). The mean extraction recovery of triptolide was 89.15 ± 5.4 %. The intra- and

Table 2  Stability of triptolide in rat plasma under different storage conditions. Storage

Concentration

Concentration found

found Remaining

conditions

added (ng/ml)

(mean ± SD; ng/ml)

Percentage ( %)

Freeze-thaw

At 4 °C 4 h

In autosampler for 24 h At  − 20 °C 15 days

1.64 8.11 41.1 1.64 8.11 41.1 1.64 8.11 41.1 1.64 8.11 41.1

1.6 ± 0.10 8.53 ± 0.19 44.8 ± 0.69 1.62 ± 0.15 8.38 ± 0.47 42.4 ± 1.5 1.55 ± 0.10 7.81 ± 0.38 39.83 ± 2.80 1.50 ± 0.13 9.02 ± 0.31 43.1 ± 0.99

97.56 103.81 109.00 98.78 101.95 103.16 94.72 95.01 96.92 91.16 109.73 104.87

Remaining percentage ( %) = (concentration found)/(concentration added) × 100 (n = 3)

inter-day precisions were less than 7 % with accuracies ranged ▶  Table 1). Triptolide plasma samples from 93.84 % to 105.45 % ( ● were stable after 3 freeze-thaw cycles, stored at 4 °C for 4 h, in an ▶  Table 2). The autosampler for 24 h and at  − 70 °C for 2 weeks ( ● developed method was successfully used for determining triptolide concentrations in rat plasma. The mean plasma concentration vs. time profiles of triptolide after single and multiple dose (400 μg/kg) administration in rats are shown in ●  ▶  Fig. 2, 3, and a comparison of the toxicokinetic parameters of triptolide are presented in ●  ▶  Table 3. At each time point on the plasma concentration-time curve, the concentrations of triptolide in individual female rats were higher than those in males. The AUC0–∞ and Cmax of triptolide in females were also found to be significantly greater than males after single as well as repeated administrations. The higher drug exposure level of triptolide in female rats explained the female susceptible toxicological properties of triptolide in our previous finding [11]. The greater AUC0–∞ of triptolide in females was supported by both a slower CL and a longer t1/2. The CL of triptolide in males was 2.34 and 6.46 times higher as compared to females following single and multiple doses, respectively. These observations are consistent with our previous report that male rat microsomes showed greater activity in triptolide metabolism than females [11]. Based on the significant differences in triptolide elimination, it may be inferred that the activity of the enzymes that metabolize triptolide differs between the sexes. It has been reported that triptolide is mainly metabolized to hydroxytriptolide by CYP3A in rats [12]. CYP3A is an abundant component of CYPs in rats. CYP3A1 and 3A2 are the main isoforms of CYP3A subfamily in rats. Notwithstanding they share a high degree of structural homology, a distinct sex-specific expression pattern happens in CYP3A2, not in 3A1. Male-predominant CYP3A2 has been thought to be a possible candidate responsible for the sex-dependent metabolism and toxicity of triptolide in rats [11, 17]. In addition, triptolide was also the substrate of P-gp and P-gp, as well as CYP3A, contributed to the disposition and detoxification of triptolide [18]. Furthermore, P-gp activity also appears to be greater in males vs. females [19]. Thus, the sex-dependent differences in the toxicokinetics of triptolide could be explained by sexually dimorphic CYPs and transport proteins. Triptolide has long been involved in traditional Chinese medicine (e. g., Lei Gong Teng Duo Dai Pian and Lei Gong Teng Pian, conventionally available dosage forms of TWHF in China) and has been used clinically for the treatment of chronic diseases that require long-term medication, such as arthritis, lupus erythematosus, diabetic nephropathy and tumors. It has been reported that Lei Gong Teng Pian inhibited the CYP3A activity in

Fig. 2  Plasma concentration-time curve of triptolide (400 μg/kg) after a single oral administration to male a and female b rats.

Liu L et al. Gender Dimorphic TK of Triptolide …  Drug Res 2015; 65: 602–606

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604 Original Article

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Fig. 3  Plasma concentration-time curve of triptolide (400 μg/kg) after 28 days oral administration to male a and female b rats.

Table 3  Toxicokinetic parameters of triptolide (400 μg/kg) after single and multiple oral dose administration to rats (n = 6 per sex group). single dose administration male AUC(0–t)/ng/mL · min AUC(0–∞)/ng/mL · min t1/2/min Tmax/min CL/F/L/min/kg V/F/L/kg Cmax/ng/mL

340.39 ± 90.06 376.19 ± 104.04 28.18 ± 12.49 17.00 ± 7.58 1.17 ± 0.50 49.92 ± 13.03 7.00 ± 2.43

multiple doses administration

female

male

801.12 ± 220.93 * *  848.80 ± 248.20 * *  47.44 ± 24.31 18.75 ± 7.5 0.50 ± 0.14 *  32.40 ± 11.13 14.07 ± 4.46 * 

768.26 ± 198.03# 789.31 ± 196.44# 30.59 ± 6.33 27.5 ± 6.12 0.53 ± 0.12# 26.82 ± 9.34# 13.52 ± 5.97#

female 3 987.08 ± 1 352.67 * * ## 5 207.49 ± 1 591.28 * * ## 83.13 ± 33.34 * * # 27.00 ± 6.71 0.082 ± 0.21 * * ## 11.40 ± 7.63 * # 52.14 ± 15.41 * * ##

 * p 

Gender Differences in the Toxicokinetics of Triptolide after Single- and Multiple-dose Administration in Rats.

Triptolide is a natural compound extracted from the traditional Chinese medicine Tripterygium wilfordii Hook F with distinguishing pharmacological act...
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