Pharmacokinetics and safety of the multiple constituents of Shuanghua Baihe tablets in healthy subjects

Abstract

Shuanghua Baihe tablets (SBT) are a traditional Chinese medicine formula which is used for treatment of oral mucositis (OM) for more than 30 years in China. So far, there has been no report of the quantification of the major components in SBT and their pharmacokinetics in humans. In this study, high performance liquid chromatography coupled to triple-quadrupole mass spectrometry was used to determine the content of the major compounds in SBT and to evaluate the pharmacokinetics of nine major constituents (berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline) following single and multiple oral administrations of SBT in healthy subjects. This study was an open-label and 2-period clinical trial. It was conducted in 12 Chinese healthy subjects. Each subject received a single dose in period 1 and multiple doses in period 2. Blood samples were collected and determined over 96 h. Subjects underwent safety assessments and were monitored for adverse events. There was no serious adverse event, death or withdrawal. Gender had a significant effect on the pharmacokinetics of the active alkaloids except for magnoflorine, berberrubine, corynoline and acetylcorynoline. Compared with the single dosing, the exposure of the alkaloids increased significantly except for berberrubine after multiple dosing. Single and multiple oral doses of SBT were safe in healthy Chinese subjects. The accumulation of the alkaloids to different extents was observed with repeated dosing except for berberrubine. This work provides useful information for clinical application in the treatment of OM, and a scientific basis for the study on the material foundation of the medicinal effectiveness of SBT and its mechanism of action.

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Introduction

Oral mucositis (OM) is a particularly severe and common acute side effect of chemotherapy or radiotherapy.^1,2 In patients undergoing high-dose myeloablative therapies, the incidence rate of OM is almost 100%,³ and in cancer patients undergoing standard-dose chemotherapy the rate is 40 to 70%.⁴ The pathogenesis of the OM is related to the inflammation.^1,5 OM lesions are often serious enough to delay, reduce or halt cancer therapy, ultimately leading to higher mortality in cancer patients, because there is no effective treatment. To date, there are few FDA approved drugs to treat OM in cancer patients. Several drugs are known to reduce the severity of OM,^1,6 but the therapeutic outcome is often not satisfactory.⁴ Hence, there is an urgent need for new therapeutic interventions for the prevention and treatment of OM.

Traditional Chinese medicines (TCMs) have been used for thousands of years in Asia and attracted increasing research and application in Western countries.^7,8 Most herbal medicines are prescribed in combination based on the theory of TCM to obtain synergistic effects or diminish the possible adverse reactions. These multiple herbs are called “TCM formula”.⁹ Shuanghua Baihe tablets (SBT) is a TCM formula that has been clinically employed for the prevention and treatment of OM for more than 30 years in China.^10,11 Because of the long-term clinical practices and good therapeutic outcomes, it had been approved by the China Food and Drug Administration (CFDA) in 2012 (approval number Z20123033) for treating OM. SBT is composed of ten crude herbs, consisting of Coptidis Rhizoma, Corydalis Bungeanae Herba, Isatidis Radix, Arnebiae Radix, Lonicerae Japonicae Flos, Lophatheri Herba, Rehmanniae Radix, Lilii bulbus, Asari radix et rhizoma and Snake Bile.¹² In this formula, Coptidis Rhizoma is the monarch drug. Corydalis Bungeanae Herba, Isatidis Radix and Arnebiae Radix are the minister drugs, and others are adjuvant and courier herbs. Coptidis Rhizoma and Corydalis Bungeanae Herba are well-known TCMs widely used in many prescriptions. They have pharmacological effects such as heat-clearing, damp-drying and detoxification.^13,14 SBT have the functions of invigorating blood circulation, antibiotic, anti-inflammatory, anti-ulcer and regulation of immune function.¹⁵

Currently, the studies on the clinical efficacy of SBT for treating OM have been reported in China.^10,15 Generally, the therapeutic and pharmacological effects of TCM are usually attributed to synergism among multiple herbs and constituents, termed ‘‘TCM formula compatibility’’.¹⁶ In order to indicate the active ingredients in SBT and its mechanism of action and promote the internationalization and modernization of SBT, well studies are needed to be done. In our previous study,¹² the chemical compounds in SBT and constituents in rat plasma following oral administration of SBT were initially identified using liquid chromatography-high resolution mass spectrometry techniques. Besides, pharmacokinetic studies are useful to explain and predict a variety of events related to the efficacy and toxicity of drugs, thus it is valuable to perform pharmacokinetic studies for evaluating the rationality and compatibility of herbs or prescriptions.^17–20 Until now, there has been no report about the pharmacokinetic study of SBT. As multiple compounds exist in herbal medicines, we should choose representative active compounds and be able to explain pharmacokinetic behaviors and drug–drug interactions in herbal medicines.²¹ According to our previous study,¹² nine major bioactive alkaloids (Fig. 1; berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine and berberrubine derived from Coptidis Rhizoma; corynoline and acetylcorynoline derived from Corydalis Bungeanae Herba) were selected as indicative compounds for the pharmacokinetic study.

Fig. 1 Chemical structures of the nine active alkaloids in the traditional Chinese medicine formula Shuanghua Baihe tablets.

Additionally, the prevalence of use of TCMs is high and continues to increase in the world.²² Moreover, global interests in the safety of herbal medicines have grown.²³ Since producers may omit safety information on product labels at their own discretion, labels themselves may lack clinically pertinent information.²⁴ Hence, it is very important to be aware of the safety issues associated with the administration of SBT. Therefore, the objective of our study was to evaluate the pharmacokinetics and safety of the nine major bioactive ingredients following single and multiple oral administrations of SBT in healthy subjects.

Materials and methods

Chemicals and materials

Shuanghua Baihe tablets (0.6 g per tablet) were provided by Yangtze River Pharmaceutical Group Co. Ltd. (Taizhou, China). The reference standards of berberine, palmatine, jatrorrhizine, corynoline and donepezil (internal standard, IS) were purchased from the National Institutes for Food and Drug Control (Beijing, China). Epiberberine, coptisine, magnoflorine, berberrubine were obtained from Shanghai Youxuan Biotechnology Co. Ltd. (Shanghai, China). Acetylcorynoline came from Chengdu Ruifensi Biotechnology Co. Ltd. (Chengdu, China). Methanol was HPLC grade and purchased from Merck (Darmstadt, Germany). Formic acid (analytical grade) was purchased from Nanjing Chemical Reagents Co., Ltd. (Nanjing, China). Deionized water was purified using a Milli-Q system (Millipore, Milford, MA, USA).

Content determination of the major constituents in SBT

The content of nine major constituents in the SBT was quantified by the liquid chromatography with tandem mass spectrometry (LC-MS/MS). The liquid chromatography (LC) was performed on an Agilent 1200 Series LC (Agilent Technologies, Palo, Alto, CA, USA), which included an Agilent 1200 binary pump (model G1312B), vacuum degasser (model G1322A), Agilent 1200 autosampler (model G1367C), and temperature controlled column compartment (model G1330B). Chromatographic separation was carried on a Hedera ODS-2 C₁₈ analytical column (150 × 2.1 mm, 5 μm; Hanbon Science and Technology, Huai'an, China) with a security Guard-C₁₈ column (4 mm × 2.0 mm, 5 μm; Phenomenex, Torrance, CA, USA). The chromatographic separation was conducted on the column mentioned above at a flow rate of 0.3 mL min⁻¹ with a mobile phase of methanol (solvent A) and water containing 0.5% formic acid (solvent B) in a linearly gradient program, under the following conditions: 0 to 11 min, 35% B; 11 to 11.1 min, 35 to 70% B; 11.1 to 14 min, 70% B; 14 to 14.1 min, 70 to 35% B; 14.1 to 20 min, 35% B. The column temperature was maintained at 30 °C. The injection volume was 10 μL.

The LC system was coupled to an Agilent 6410B triple quadrupole mass spectrometer (USA) equipped with an ESI source.† The mass spectrometer was operated in the positive ESI† mode with the drying gas temperature of 300 °C with N₂ gas flow at 10 L min⁻¹, nebulizer pressure of 40 psi, and capillary voltage of 4000 V. The specific other mass parameters for each analyte are displayed in Table 1. The signal acquisition and peak integration were performed using the MassHunter Qualitative Analysis Software (B.03.01) supplied by Agilent Technologies.

Table 1 Optimized mass parameters for the analytes

Analytes	Precursor ion (m/z)	Product ion (m/z)	Fragmentor voltage (V)	Collosion energy (eV)
Coptisine	319.8	291.9	160	25
Berberrubine	322.1	307.1	130	25
Berberine	336.0	292.0	140	30
Epiberberine	336.0	292.0	140	30
Jatrorrhizine	338.0	323.0	145	20
Magnoflorine	341.9	296.9	130	15
Palmatine	352.0	336.0	140	25
Corynoline	368.0	288.9	140	25
Acetylcorynoline	410.0	288.9	130	25
Donepezil (IS)	380.2	91.2	105	46

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SBT (ten tablets) were pulverized in a mortar. Then, 0.1 g of SBT powder was accurately weighed into a volumetric flask and subjected to ultrasonic treatment at room temperature with 10 mL methanol/water (70 : 30, v/v) for 30 min. The methanol extraction was centrifuged at 16 000 rpm for 10 min. The supernatant was collected and filtered through a 0.22 μm membrane. Then the solutions of SBT were diluted 200 times with methanol/water (30 : 70, v/v). An aliquot of 10 μL supernatant was then used for the LC-MS/MS analysis.

Ethics and study subjects

The clinical trial was performed at a single center (Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College) in Nanjing, China. The study was approved by the Ethics Committee at this study center (the approval number LS201410) and complied with the Declaration of Helsinki and Good Clinical Practice guidelines. Subjects provided written informed consent prior to undergoing any procedure for this study.

Subjects were screened 2 weeks before the initialization of this study. Eligibility criteria for male and female subjects included age 18–45 years, weight not less than 50 kg and a body mass index (BMI) in the normal range (19–24 kg m⁻² inclusive). Subjects were in good health as evidenced by their medical history, physical examination, vital signs, 12-lead electrocardiogram (ECG) and laboratory profile. The following exclusion criteria were applied for subjects in this clinical trial: a history of clinically significant cardiovascular, renal, urinary tract, hepatic, pulmonary, gastrointestinal diseases; a history of known allergy or intolerance to any drugs; a history of tobacco, alcohol or drug abuse; those with abnormalities in clinical laboratory parameters; those who had received an investigational drug, or donation of blood in the preceding 3 months, or had received any drug within 4 weeks before the study start date, or was considered by the investigator, for any reason, to be an unsuitable candidate for receiving SBT. Females who were lactating or who had a positive pregnancy test were also ineligible. A total of 12 subjects, 6 male and 6 female, participated in this study.

Study design and sample collection

This study was a single-center, open-label, randomized, one sequence, 2-period, phase I clinical trial. It was aimed to evaluate the pharmacokinetics of the nine alkaloids in SBT after single dose and repeated doses in healthy Chinese subjects. The day before the trial, all subjects were hospitalized in the national drug clinical trial institution phase I ward of Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College.

In the first period, all the eligible subjects received a single dose (4 tablets) of SBT with 250 mL water at 7:00 am on day 1 after an overnight fast at least of 10 h. Blood samples were collected from each subject at pre-dose, 0.25, 0.5, 0.75, 1, 1.33, 1.67, 2, 2.5, 3, 4, 5, 6, 8, 12, 24, 48, 72 and 96 h after dosing. Water intake was allowed 2 h after administration of the drug, and standard meals were provided 4 h after administration of the drug.

After an 8 day wash out period, the same subjects participated in the multiple dose design. In this second period, all subjects received multiple doses (4 tablets at once, 3 times a day) of SBT at 7:00 am, 1:00 pm and 7:00 pm from day 9 to day 13 about half an hour before the meals and a single dose (4 tablets) at morning of day 14 under fasted condition. Blood samples used to confirm the steady-state were drawn before administration (pre-dose) on day 12 to day 14. Besides, blood sample collection on day 14 was the same as that on day 1. All doses were administered with 250 mL of water. Morning doses were administered after at least a 10 h overnight fasting.

In both periods of the study, approximately 5 mL of blood were drawn from a suitable forearm vein using an indwelling catheter into heparin containing tubes. These samples were centrifuged at 1700g for 10 min at 4 °C. The separated plasma was transferred to labelled tubes and stored at −80 °C until analysis.

Safety assessments

Safety was assessed throughout the study based on all adverse events (AEs), physical examinations, 12-lead ECG evaluations, vital signs measurements (systolic and diastolic blood pressure, heart and respiratory rate and axillary body temperature) and laboratory safety tests (hematology, blood chemistry, urine analysis). Details of AEs were obtained and recorded in the source data record by the study physicians following questioning and spontaneous reporting, and shown on the case report form.

Sample analysis

Plasma sample analysis was conducted in an independent laboratory (pharmaceutical analysis department of China Pharmaceutical University, Nanjing, China). The nine alkaloids content in the SBT were quantified by the liquid chromatography tandem mass spectrometry (LC-MS/MS; Agilent 6410B, Agilent Technologies, Palo Alto, CA, USA). The nine alkaloids concentrations in plasma were measured, after protein precipitation, by a validated LC-MS/MS method (API4000 AB Sciex, Foster City, CA, USA) using donepezil as the internal standard. The calibration ranges of the assay were 15–5000 pg mL⁻¹ for magnoflorine and berberrubine, 15–800 pg mL⁻¹ for berberine, coptisine and acetylcorynoline, 15–1500 pg mL⁻¹ for corynoline, 15–300 pg mL⁻¹ for epiberberine, and 1.5–100 pg mL⁻¹ for palmatine and jatrorrhizine, respectively. The intra- and inter-batch precisions (% CV, coefficient of variation) for the quality control (QC) samples ranged from 2.4% to 14.4%, and the accuracy (nominal value) of the QC samples ranged from 90.5% to 107.4% for all the analytes. Dilution integrity was established up to 5 times for the analytes.

Pharmacokinetic analysis

The plasma concentration–time profiles of the nine alkaloids of each subject were constructed. The pharmacokinetic parameters were calculated by non-compartmental methods using DAS 3.2 software (professional edition version 3.2, Drug and Statistics, Shanghai, China). The following pharmacokinetic parameters were obtained: maximum plasma concentration (C_max); time to reach C_max(T_max), C_max at steady state (C_max,ss); minimum plasma concentration at steady state (C_min,ss); average value of the steady-state plasma concentration (C_av); area under the plasma concentration–time curve (AUC) from time zero to the last measurable sampling time point (AUC_0–96); AUC from time zero to infinity (AUC_0–∞); AUC at steady state from time 0 to τ (AUC_ss, τ was the dosing interval 6 h); terminal elimination half-life (t_1/2); mean residence time (MRT) from time zero to the last measurable sampling time point (MRT_0–96); apparent clearance (CL/F); apparent volume of distribution (V/F); accumulation ratio (R); the degree of fluctuation of drug concentrations (DF). All parameters except R and DF were calculated using DAS software. R_AUC, accumulation ratio calculated based on AUC, calculated by the equation: AUC_ss/(AUC_0–τ, day 1); R_Cmax, accumulation ratio calculated based on C_max, calculated by the equation: C_max,ss/(C_max, day 1); DF calculated by the equation: (C_max − C_min)/C_av. All results were expressed as arithmetic mean with standard deviation.

Statistical analysis

Statistical analyses were performed using SPSS software version 11.5 (SPSS, Inc., Chicago, IL, USA). Subjects who completed the study according to the protocol were included into the statistical evaluation. The arithmetic mean, the standard deviation, coefficient of variation of the parameters were reported. Mean plasma concentration–time curves of all the analytes following a single dose and multiple doses of SBT were plotted. Prior to analysis, concentration related parameters (C_max and AUC) were transformed using natural logarithms of individual values. The statistical comparisons of pharmacokinetic parameters between male and female subjects were performed by the independent t-test (ITT). The differences of pharmacokinetic parameters between single and multiple doses were evaluated using the paired t-test (PTT). Non-parametric test (NPT) was used to compare T_max between male and female subjects, and between single and multiple doses. An analysis of variance (ANOVA) was performed for the last three values of C_min at days 12, 13 and 14 to assess whether steady-state had been reached. P < 0.05 was considered statistically significant difference.

Results and discussion

Content determination of the major constituents in SBT

In this work, the content of the major compounds in SBT was determined, which is indispensable for the related studies of SBT. The analytical methodology of content determination of the major constituents in SBT was validated according to the International Conference on Harmonization (ICH) guideline.²⁵ The specificity, linearity, limit of detection, limit of quantification, precision, accuracy, solution stability, system suitability and robustness were evaluated accordingly, and the results demonstrated that they met the criteria given in the guideline. The calibration curves were linear over the concentration ranges of 100–2000 ng mL⁻¹ for coptisine, jatrorrhizine and magnoflorine, 1000–5000 ng mL⁻¹ for berberine, 100–1500 ng mL⁻¹ for epiberberine and palmatine, 50–1000 ng mL⁻¹ for berberrubine, 20–200 ng mL⁻¹ for corynoline, and 2–20 ng mL⁻¹ for acetylcorynoline, respectively. The method was simple, accurate and reproducible. Representative MRM chromatograms of the nine alkaloids are shown in Fig. S1 (see ESI†). The results of the calibration curves and content of the major constituents in Shuanghua Baihe tablets are listed in Table 2.

Table 2 Calibration curves and content of the major constituents in Shuanghua Baihe tablets

Constituents	Calibration curve	Regression coefficient (r^2)	Content (mg per tablet)
Coptisine	y = 0.001464x + 0.1946	0.9976	5.108
Berberrubine	y = 0.02828x + 1.255	0.9990	1.041
Berberine	y = 0.01127x + 6.624	0.9993	18.01
Epiberberine	y = 0.003179x + 0.1311	0.9990	5.378
Jatrorrhizine	y = 0.005598x + 0.6717	0.9978	2.423
Magnoflorine	y = 0.0005506x + 0.1376	0.9961	1.903
Palmatine	y = 0.005624x + 0.1811	0.9994	4.922
Corynoline	y = 0.005519x + 0.006205	0.9991	0.4646
Acetylcorynoline	y = 0.002698x − 0.001770	0.9991	0.06112

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Subjects demographics

A total of 12 healthy Chinese subjects, 6 males and 6 females, were enrolled in this study after signing the informed consent form. The age of the volunteers who participated in the study ranged from 19 to 25 years and their BMI ranged from 19.1 to 23.7 kg m⁻². No subject dropped out of the study. Demographic and baseline characteristics of study subjects are presented in Table 3.

Table 3 Demographics and baseline characteristics of 12 subjects in the study^a

Subject	Gender	Age (years)	Height (cm)	Body weight (kg)	BMI (kg m^−2)
a M = male; F = female; BMI = Body Mass Index; data are presented as mean with standard deviation (SD).
A	M	20	186	82	23.70
B	M	24	175	65	21.22
C	M	21	170	60	20.76
D	M	19	176	60	19.37
E	M	20	160	60	23.44
F	M	20	165	52	19.10
G	F	21	163	55	20.70
H	F	22	165	55	20.20
I	F	22	163	54	20.32
J	F	24	157	53	21.50
K	F	25	160	52	20.31
L	F	23	155	50	20.81
Mean	—	22	166	58	20.95
SD	—	2	9	9	1.39

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Pharmacokinetics

All plasma samples from the drug-treated subjects were analyzed and reported.

Single dose study. Pharmacokinetic parameters of the nine alkaloids (berberine, epiberberine, coptisine, palmatine, jatrorrhizine, magnoflorine, berberrubine, corynoline and acetylcorynoline) obtained in the single-dose study of SBT are summarized in Table 4. The mean plasma concentration–time curves of the nine alkaloids are shown in Fig. 2 and 3. Also, pharmacokinetic parameters for male and female subjects in the single-dose study are presented in Table 5.

Table 4 Main pharmacokinetic parameters of nine alkaloids following single oral dose of Shuanghua Baihe tablets (n = 12)^a

Parameters/ingredients	Cmax (pg mL^−1)	Tmax (h)	t1/2 (h)	MRT0–96 (h)	CL/F (×10^3 L h^−1)	V/F (×10^3 L)	AUC0–96 (pg h mL^−1)
a Data are presented as mean ± SD (standard deviation). The dose represents the content of each alkaloid in four tablets of Shanghua Baihe tablets. Abbreviations are as follows: Cmax, maximum plasma concentration; Tmax, time to reach Cmax; t1/2, terminal elimination half-life; MRT0–96, mean residence time from 0 to 96 h; CL/F, apparent clearance; V/F, apparent volume of distribution; AUC0–96, area under the plasma concentration–time curve from time 0 to 96 hours after administration.
Berberine	281.1 ± 88.6	1.6 ± 0.9	48.5 ± 35.7	22.3 ± 13.2	28.51 ± 26.88	1105 ± 687	3133 ± 1838
Coptisine	296.2 ± 143.5	2.0 ± 0.6	46.3 ± 28.3	23.3 ± 11.5	7.554 ± 9.708	261.3 ± 128.9	4333 ± 3214
Epiberberine	135.6 ± 37.2	1.5 ± 0.7	33.5 ± 22.0	12.3 ± 7.2	15.34 ± 17.42	444.3 ± 212.6	1266 ± 887
Jatrorrhizine	25.74 ± 12.96	2.1 ± 1.1	31.6 ± 17.8	20.2 ± 8.5	26.64 ± 14.04	971.1 ± 421.3	355.2 ± 186.4
Magnoflorine	2106 ± 654	2.7 ± 1.3	18.7 ± 13.4	14.1 ± 5.6	0.389 ± 0.156	9.425 ± 6.141	21 063 ± 7894
Berberrubine	2961 ± 1171	1.1 ± 0.5	19.8 ± 13.1	8.7 ± 2.8	0.336 ± 0.111	9.190 ± 6.579	13 059 ± 4239
Palmatine	53.37 ± 22.55	1.8 ± 0.9	37.5 ± 21.1	24.2 ± 7.8	33.01 ± 19.47	1428 ± 481	616 ± 253
Corynoline	473.9 ± 442.1	1.5 ± 0.5	3.0 ± 2.3	3.2 ± 1.3	2.958 ± 2.570	7.680 ± 4.483	1625 ± 1887
Acetylcorynoline	369.9 ± 146.6	1.4 ± 0.5	6.8 ± 5.5	3.8 ± 1.1	0.217 ± 0.072	1.814 ± 1.007	1086 ± 1887

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Fig. 2 Mean plasma concentration–time curves of berberine (A), coptisine (B), epiberberine (C), jatrorrhizine (D), magnoflorine (E) and berberrubine (F) in healthy human volunteers following single dose and multiple doses of Shuanghua Baihe tablets. Data are presented as mean with standard deviation.

Fig. 3 Mean plasma concentration–time curves of palmatine (A), corynoline (B) and acetylcorynoline (C) in healthy human volunteers following single dose and multiple doses of Shuanghua Baihe tablets. Data are presented as mean with standard deviation.

Table 5 Main pharmacokinetic parameters of nine alkaloids in male and female subjects following single oral dose of Shuanghua Baihe tablets (n = 6)^a

Parameters/ingredients	Gender	Cmax (pg mL^−1)	Tmax (h)	t1/2 (h)	MRT0–96 (h)	CL/F (×10^3 L h^−1)	V/F (×10^3 L)	AUC0–96 (pg h mL^−1)
a Data are presented as mean ± SD (standard deviation). Abbreviations are as follows: M, male; F, female; Cmax, maximum plasma concentration; Tmax, time to reach Cmax; t1/2, terminal elimination half-life; MRT0–96, mean residence time from 0 to 96 h; CL/F, apparent clearance; V/F, apparent volume of distribution; AUC0–96, area under the plasma concentration–time curve from time 0 to 96 hours after administration; asterisks (*) indicate significant difference (P < 0.05) between male and female subjects.
Berberine	M	230.0 ± 94.4	1.2 ± 0.4	33.3 ± 37.8	14.6 ± 14.5*	45.39 ± 29.91*	1218 ± 928	1762 ± 1254*
	F	332.3 ± 45.6	2.0 ± 1.1	63.6 ± 28.9	30.0 ± 5.4*	11.63 ± 3.36*	991.8 ± 381.8	4503 ± 1163*
Coptisine	M	304.3 ± 185.8	1.7 ± 0.6	38.9 ± 32.6	15.3 ± 8.8*	11.55 ± 12.70	295.3 ± 153.5	3027 ± 3232
	F	288.0 ± 103.0	2.2 ± 0.6	53.9 ± 23.6	31.4 ± 7.6*	3.559 ± 2.764	227.3 ± 100.9	5639 ± 2860
Epiberberine	M	126.4 ± 49.1	1.4 ± 0.6	32.6 ± 30.4	7.2 ± 2.8*	21.60 ± 23.50	483.6 ± 288.3	693.9 ± 282.0*
	F	144.7 ± 20.9	1.7 ± 0.9	34.4 ± 11.7	17.3 ± 6.8*	9.086 ± 4.565	404.9 ± 112.4	1839 ± 930*
Jatrorrhizine	M	15.53 ± 4.84*	1.9 ± 1.1	23.9 ± 14.1	16.7 ± 6.5	36.88 ± 12.49*	1108 ± 494	219.3 ± 52.5*
	F	35.94 ± 9.82*	2.2 ± 1.2	39.4 ± 18.8	23.7 ± 9.3	16.41 ± 5.13*	834.1 ± 319.2	491.1 ± 171.4*
Magnoflorine	M	2232 ± 359	2.6 ± 1.1	15.3 ± 6.1	13.7 ± 5.7	0.368 ± 0.165	7.201 ± 2.567	22 521 ± 8325
	F	1981 ± 881	2.8 ± 1.6	22.1 ± 18.2	14.5 ± 6.0	0.410 ± 0.159	11.65 ± 8.032	19 606 ± 7917
Berberrubine	M	2670 ± 851	0.8 ± 0.5	21.3 ± 18.1	9.4 ± 3.9	0.393 ± 0.124	10.93 ± 8.83	11 035 ± 3652
	F	3252 ± 1445	1.3 ± 0.4	18.3 ± 6.9	8.0 ± 0.9	0.279 ± 0.061	7.449 ± 3.166	15 083 ± 4046
Palmatine	M	40.87 ± 21.88	1.4 ± 0.4	27.3 ± 10.7	18.7 ± 6.3*	46.35 ± 19.20*	1680 ± 507	406.7 ± 144.2*
	F	65.88 ± 16.27	2.2 ± 1.2	47.7 ± 24.8	29.7 ± 4.8*	19.67 ± 6.20*	1176 ± 317	825.0 ± 124.5*
Corynoline	M	373.7 ± 388.3	1.3 ± 0.5	1.8 ± 0.8	2.7 ± 0.7	3.766 ± 2.863	7.825 ± 5.545	1071 ± 1144
	F	574.1 ± 505.1	1.6 ± 0.4	4.2 ± 2.8	3.8 ± 1.5	2.150 ± 2.184	7.534 ± 3.662	2178 ± 2406
Acetylcorynoline	M	374.4 ± 186.1	1.4 ± 0.7	5.2 ± 2.9	3.4 ± 1.0	0.235 ± 0.089	1.541 ± 0.423	1052 ± 427
	F	365.4 ± 112.2	1.4 ± 0.2	8.4 ± 7.2	4.1 ± 1.2	0.198 ± 0.052	2.087 ± 1.368	1120 ± 250

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Following single oral administration of SBT to human volunteers, most of the alkaloids were rapidly absorbed and exhibited good oral exposure. Peak plasma concentrations were observed within 1.1–2.7 h post-dose, and t_1/2 ranged from 3.0 h (corynoline) to 48.5 h (berberine). The differences of pharmacokinetic parameters between male and female subjects in the first period of the study were estimated. After single dose administration, no significant differences (P > 0.05) were observed in T_max, t_1/2, MRT_0–96, CL/F, V/F, ln AUC^*_0–96 (AUC had been body weight-normalized) and ln C^*_max (C_max had been body weight-normalized) of the four alkaloids (magnoflorine, berberrubine, corynoline and acetylcorynoline) for male and female subjects. However, the ln AUC^*_0–96, MRT_0–96 and CL/F of berberine for female subjects were 1–2 times, 2–3 times and 3–4 times higher than those of the male subjects, respectively; the MRT_0–96 of coptisine for female subjects was about 2 times that of male subjects; the ln AUC^*_0–96 and MRT_0–96 of epiberberine for female subjects were both 2–3 times higher than those of male subjects, respectively; the ln C^*_max and ln AUC^*_0–96 of jatrorrhizine for female subjects were both 1–2 times more than those of the male subjects, respectively; the ln AUC^*_0–96 and MRT_0–96 of palmatine for female subjects were both 1–2 times more than those of male subjects, respectively.

Multiple doses study. Pharmacokinetic parameters of nine alkaloids from the non-compartmental analysis of measured plasma concentrations in the multiple-dose study are provided in Table 6. The mean plasma concentration–time curves of nine alkaloids after receiving the SBT 3 times daily for 5 consecutive days are illustrated in Fig. 2 and 3.

Table 6 Main pharmacokinetic parameters of nine alkaloids following multiple oral doses of Shuanghua Baihe tablets (n = 12)^a

Ingredients/parameters	Berberine	Coptisine	Epiberberine	Jatrorrhizine	Magnoflorine	Berberrubine	Palmatine	Corynoline	Acetylcorynoline
a Data are presented as mean ± SD (standard deviation). Abbreviations are as follows: Cmax, maximum plasma concentration; Cmin,ss, minimum plasma concentration at steady state; Cav, average value of the steady-state plasma concentration; Tmax, time to reach Cmax; t1/2, terminal elimination half-life; MRT0–96, mean residence time from 0 to 96 h; CL/F, apparent clearance; V/F, apparent volume of distribution; AUCss, AUC at steady state from time 0 to τ (τ was the dosing interval 6 h); AUC0–96, area under the plasma concentration–time curve from time 0 to 96 hours after administration; DF, the degree of fluctuation; RAUC, accumulation ratio calculated based on AUC; RCmax, accumulation ratio calculated based on Cmax; asterisks (*) indicate significant difference (P < 0.05) between multiple doses and single dose.
Cmax (pg mL^−1)	389.5 ± 125.1*	483.1 ± 171.3*	279.3 ± 109.3*	56.23 ± 19.63*	2902 ± 872*	2620 ± 1129	110.3 ± 48.0*	1818 ± 1146*	659.9 ± 296.8*
Css,min (pg mL^−1)	193.7 ± 71.7	201.7 ± 125.2	102.5 ± 39.4	27.77 ± 9.91	741.2 ± 347.3	330.7 ± 112.8	57.42 ± 20.29	423.0 ± 338.4	141.7 ± 39.0
Cav (pg mL^−1)	261.1 ± 77.7	280.4 ± 102.1	171.0 ± 56.4	38.09 ± 12.07	1978 ± 504	1251 ± 404	78.62 ± 24.85	944.9 ± 610.8	345.7 ± 128.2
Tmax (h)	1.9 ± 0.8	1.6 ± 0.6	1.5 ± 0.5	2.0 ± 1.1	2.1 ± 1.1	1.0 ± 0.5	1.8 ± 0.9	1.7 ± 0.9	1.4 ± 0.6
t1/2 (h)	56.3 ± 18.0	50.5 ± 22.9	49.0 ± 21.3	45.2 ± 16.3*	15.6 ± 12.0	35.5 ± 26.9	53.3 ± 9.5	31.4 ± 9.7*	56.7 ± 20.5*
MRT0–96 (h)	36.5 ± 3.4*	35.0 ± 3.2*	31.2 ± 7.3*	33.6 ± 2.7*	14.4 ± 5.1	14.0 ± 1.8*	35.8 ± 3.0*	22.3 ± 5.7*	32.1 ± 4.0*
CL/F (×10^3 L h^−1)	50.92 ± 18.74*	14.08 ± 6.16*	23.49 ± 8.87	47.20 ± 17.86*	0.688 ± 0.203*	0.614 ± 0.223*	45.78 ± 15.30*	0.480 ± 0.306*	0.134 ± 0.048*
V/F (×10^3 L)	367.2 ± 176.8*	102.5 ± 66.0*	197.9 ± 92.2*	327.7 ± 136.4*	5.908 ± 6.238	12.64 ± 7.99	345.1 ± 137.9*	6.718 ± 5.277	1.730 ± 0.486
AUCss (pg h mL^−1)	1567 ± 466	1683 ± 613	1026 ± 338.3	228.5 ± 72.4	11 870 ± 3027	7508 ± 2421	471.7 ± 149.1	5669 ± 3665	2074 ± 769
AUC0–96 (pg h mL^−1)	12 895 ± 4746*	13 247 ± 6367*	6458 ± 2808*	1675 ± 680*	32 023 ± 8781*	15 999 ± 4445*	3635 ± 1283*	19 084 ± 15 241*	9191 ± 2950*
DF	0.750 ± 0.261	1.046 ± 0.568	1.017 ± 0.246	0.737 ± 0.243	1.089 ± 0.264	1.771 ± 0.393	0.666 ± 0.280	1.581 ± 0.679	1.442 ± 0.369
RAUC	1.92 ± 0.35	1.72 ± 0.39	2.51 ± 0.79	2.87 ± 0.55	1.50 ± 0.37	0.89 ± 0.11	3.10 ± 0.81	6.26 ± 3.01	2.38 ± 0.48
RCmax	1.45 ± 0.43	1.86 ± 1.06	2.10 ± 0.72	2.45 ± 0.83	1.45 ± 0.48	0.88 ± 0.20	2.38 ± 1.15	5.38 ± 3.16	1.81 ± 0.45

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To evaluate whether steady-state had been reached, a statistical analysis of the C_min values obtained at days 12, 13 and 14 was performed. The results showed that there were no significant differences in the C_min values of the active alkaloids expect epiberberine, jatrorrhizine and palmatine at the administered doses (P > 0.05). Therefore, it could be proposed that steady-state had been reached for berberine, coptisine, magnoflorine, berberrubine, corynoline and acetylcorynoline after 5 days of administration of SBT (4 tablets at once, three times a day). Small fluctuations between maximum and minimum concentrations for all the nine ingredients were observed following repeat dosing of SBT with the DF ranging from 0.75 to 1.77. No significant differences (P > 0.05) were observed in T_max of the nine ingredients after single- and multiple-dose administration of SBT. Median T_max did not change after multiple oral administration of SBT for 5 days. Compared with the single dosing, the C_max and AUC of the alkaloids increased significantly except berberrubine after 5 days of multiple dosing. The accumulation index calculated based on AUC (R_AUC) and C_max(R_Cmax) indicated that the exposure of eight ingredients (except berberrubine) increased in different extent with 1.5–6.3 times after repeated doses. Compared with the single dosing, the C_max and AUC_ss of epiberberine, jatrorrhizine, palmatine, corynoline and acetylcorynoline were significantly increased by more than 2-fold after multiple dosing with SBT, especially corynoline, which increased by 5–6 times, but they could not be detected from human body essentially 96 h after the last administration.

The statistical results of other pharmacokinetic parameters (t_1/2, MRT_0–96, CL/F, V/F) of the nine alkaloids after single- and multiple-dose administration of SBT were as follows: except for jatrorrhizine, corynoline and acetylcorynoline, no significant differences (P > 0.05) were observed in t_1/2 of nine alkaloids; there were significant differences (P < 0.05) in MRT_0–96 of the nine alkaloids except magnoflorine; significant differences (P < 0.05) were observed in CL/F of nine alkaloids except epiberberine; no significant differences (P > 0.05) were observed in V/F of nine alkaloids except magnoflorine, berberrubine, corynoline and acetylcorynoline.

Pharmacokinetics is one of the key properties for the design of a safe and effective drug. Data and information obtained from the pharmacokinetic study could help clarify the complex interactions between clinically used medicines. The studies described in this report represent the initial first in man studies to examine the pharmacokinetic and safety profiles after single and multiple oral doses of SBT in healthy volunteers.

In the present study, the most significant differences of the main pharmacokinetic parameters (C_max, AUC_ss and t_1/2) of corynoline were observed between the single- and multiple-dose studies. The exposure of corynoline was increased significantly, and t_1/2 was obviously prolonged without affecting T_max noticeably by comparison to those of single dose. These results could be attributed to compatible effects of the ten herbs composed of SBT and auto-induction of metabolism. Distinct from chemical drugs, herbs medicines are multi-components and may exert a holistic treatment to diseases and show potential clinical benefits.²⁶ The co-existence of multiple compounds may lead to metabolic and pharmacokinetic interactions.^27,28 Those are the current hot topics of herb–drug and herb–herb interactions. Cytochrome P450 (CYP450) inhibition or induction is probably the most common mechanism for the pharmacokinetic interactions of herbs and drugs. Similarly, herbal constituents can be substrates, inhibitors, or inducers of CYP450 and have an impact on the pharmacokinetics of any co-administered drugs.²⁷ One study demonstrated that corynoline could be metabolized by CYP2C9 and CYP3A4, and corynoline was also demonstrated to inhibit CYP2C9 (competitively) and CYP3A4 (noncompetitively).²⁹ Besides, we identified that CYP2D6 also participated in the metabolism of corynoline (data not shown). Rhizoma Coptidis is the monarch (main) drug in SBT, and berberine is the highest and most effective constituent. Another study showed that repeated administration of berberine decreased CYP2D6, CYP2C9, and CYP3A4 activities, and herb–drug interactions should be considered when berberine is co-administered.³⁰ Consequently, the C_max and AUC of corynoline were increased, as less parent drug was metabolized by CYP3A4, CYP2D6 and CYP2C9. Half-life of corynoline was prolonged and the eliminated rate of corynoline was reduced because of the same reason mentioned above, which was consistent with the results of the study on corynoline and its potential interaction in SBT in rats.³¹

In addition, compared with the pharmacokinetic parameters of berberine reported in the literature³² after oral administration of single berberine in healthy volunteers, the T_max of berberine was obviously shortened from 9.8 to 1.6 h after oral administration of the formula SBT, the C_max and AUC (dose-normalized) of berberine were significantly increased by 2–3 times, and t_1/2 was significantly prolonged from 28.6 to 48.5 h. The results indicated that other ingredients in the formula SBT could enhance the adsorption rate and bioavailability of berberine. The metabolism of berberine is mainly mediated by CYP1A2, 3A4, 2D6 enzymes.³³ It implied that corynoline or other ingredients in the formula SBT might possess an inhibitory effect on the metabolism of berberine, which would be studied in our later research. Further studies on the herb–drug or herb–herb interactions are needed and more research on the clinical pharmacology mechanism of action should be conducted.

Safety and tolerability

SBT was safe and well tolerated when administered as a single dose (4 tablets, once daily) and multiple doses (4 tablets at once, three times daily) for one course (5 days). The safety was assessed and recorded by study investigators according to the good clinical practice (GCP) and guidance for industry and investigators about safety reporting requirements for investigational new drugs (INDs) and BA/BE Studies set by Food and Drug Administration (FDA).³⁴ No death or serious adverse events (SAEs) were reported during the study and all subjects were in good compliance.

Several AEs occurred in two female subjects during the multiple doses study. One subject experienced two events with the symptoms of hiccups and heartburn, 20 minutes after taking the drug on the evening of day 13, which was considered unlikely to be related to the study drug by investigators according to the judgment rules of AEs. The other subject experienced two events with the symptoms of repeated abdominal pain and diarrhea after taking SBT on the evening of day 13 and morning of day 14, which was considered to be related to the study medication. An abnormality was found in the ECG of the latter subject when comparing baseline and end of study evaluations, which was considered to be possibly related to study drug. All the observed AEs were generally classified as mild events, and resolved without dose interruption, treatment or sequelae. No other individual clinically significant changes from baseline in laboratory evaluations, physical examinations, or vital signs were observed during the study.

It was observed that the incidence of AEs was higher in subjects receiving multiple doses than the single dose, and it was also higher in female subjects than male subjects. It was interesting that the phenomenon seems to be related to the results of the pharmacokinetic parameters of SBT, in which the exposure (AUC and C_max) of some alkaloids increased significantly after multiple dosing and they were higher in female subjects. However, further research with a larger number of subjects is needed to see whether the exposure of the alkaloids has any link to the incidence of AEs. Besides, future investigations to evaluate the effect of long-term doses of SBT on the OM, abdominal pain, diarrhea and ECG are warned.

Conclusion

In summary, this is the first clinical study to investigate the pharmacokinetics and safety of SBT in healthy subjects and the first attempt to systematically reveal the pharmacokinetics of SBT. SBT is safe and well tolerated in healthy subjects at single and multiple doses for 5 days with no serious adverse events observed. The results indicated that interactions could alter pharmacokinetics among compounds in SBT. The data from the study form a basis for further investigation of the safety and efficacy of SBT in patients suffering from OM. SBT is currently being evaluated in a series of phase IV clinical studies as the therapeutic agent for OM in China. This study provides the important scientific basis for guiding rational clinical applications of SBT and research on its mechanism of action, and also promotes the modernization and internationalization of SBT.

Acknowledgements

This study was supported financially by the National Natural Science Foundation of China (Grant No. 81273482) and the Graduate Innovation Fund of Zhejiang Huahai Pharmaceuticals Co., Ltd. The authors also thank Ernest Simpemba for his kind help in revising the English language of this article.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra18665j

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