Journal of Ethnopharmacology 156 (2014) 210–215
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Safety, tolerability, and pharmacokinetics of a single ascending dose of baicalein chewable tablets in healthy subjects Min Li a, Aixin Shi a,n, Hongxian Pang a, Wei Xue a, Yang Li a, Guoying Cao a, Bei Yan a, Fan Dong a, Kexin Li a, Wei Xiao b, Guorong He c, Guanhua Du c, Xin Hu a a Beijing Key Laboratory of Drug Clinical Risk and Personalized Medication Evaluation, Department of Clinical pharmacology, Beijing Hospital of the Ministry of Health, Beijing 100730, China b StateKey Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, JiangsuKanion Parmaceutical CO. LTD., Lianyungang, Jiangsu 222001, China c Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 May 2014 Received in revised form 11 August 2014 Accepted 24 August 2014 Available online 8 September 2014
Ethnopharmacological relevance: The root of Scutellaria baicalensis Georgi has been used extensively in traditional Chinese medicine for the treatment of inflammation, fever, cough, dysentery, and hypertension. Baicalein is a flavonoid isolated from the root of Scutellaria baicalensis Georgi and is a novel neuroprotective agent under development for the treatment of Parkinson's disease. We aimed to investigate the pharmacokinetic (PK) properties of baicalein and its main metabolite, bacalin, after single-dose administration in healthy Chinese subjects. The safety and tolerability of baicalein were also assessed. Materials and methods: This was a Phase I, randomized, double-blind, single-dose trial of baicalein (100– 2800 mg) in 72 healthy adults. Samples of blood, urine and feces were collected at regular intervals up to 48 h after administration of the study drug. Baicalein and baicalin were then analyzed using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The maximum concentration that the drug achieved after dosing (Cmax), time to Cmax (Tmax), terminal half-life (t1/2), area under the curve from time zero to time of last quantifiable concentration (AUC(0, t)), area under the curve from time zero to infinity (AUC(0, 1)), apparent total plasma clearance (CL/F), and apparent total volume of distribution (V/F) were determined using non-compartmental models. Dose proportion was tested using a method combining the equivalence criterion and power model. Physical examinations, vital signs, ECG findings, hematology, and urinalysis were monitored before and at regular intervals after administration of the study drug. Results: The PK profile of baicelein and baicelin was characterized by a median Tmax of 0.75–3.5 h and 0.5–3 h, respectively, followed by a multiphasic profile with a t1/2 of 1.90–15.01 h and 4.22–10.80 h, respectively. The estimates of the proportionality coefficient (90% CI) for Cmax, AUC0–t and AUC0–1 were 0.83 (0.70–0.96), 0.91 (0.81–1.00) and 0.92 (0.82–1.02), respectively. All values overlapped within the pre-specified range of (0.89–1.11), (0.93–1.07), and (0.93–1.07), respectively. Dose proportionality was inconclusive for a baicalein dose range of 100–2800 mg. The total urinary clearance of baicalein and baicalin was o 1%. Approximately 27% of baicalein was eliminated as unchanged drug in feces. Baicalein was well tolerated. Eleven treatment-related adverse events were observed, and all were rated as “mild” and resolved without further treatment. No serious adverse events occurred. Conclusions: Single oral doses of 100–2800 mg of baicalein were safe and well tolerated by healthy subjects. Clinical laboratory assessments showed no signs of toxicity in the liver or kidney. The favorable safety profile and PK properties warrant further clinical studies for baicalein. & 2014 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Baicalein (PubChem CID: 5281605) Baicalin (PubChem CID:64982) Keywords: Baicalein Baicalin Parkinson's Disease Safety Tolerability Pharmacokinetics
1. Introduction
n
Correspondance to: Beijing key laboratory of drug clinical risk and personalized medication evaluation, Department of clinical pharmacology, Beijing Hospital of the Ministry of Health, Beijing, 100730, China Tel:þ 86 10 85133632. E-mail address: [email protected] (A. Shi). http://dx.doi.org/10.1016/j.jep.2014.08.031 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Parkinson's disease (PD) is an age-related neurodegenerative disorder caused by the disturbance of the function of the extrapyramidal system. The prevalence of PD in people aged 460 years is E1% (Nutt and Wooten, 2005). The precise pathogenesis of PD is not known, but environmental factors, genetic factors,
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2. Methods 2.1. Study design
Fig. 1. Chemical structures of baicalein (A) and baicalin (B).
inflammatory responses, oxidative stress, and mitochondrial dysfunction might be involved in the development of PD (Dauer and Przedborski, 2003; Von Bohlen und Halbach et al., 2004; Niranjan, 2014). Several drugs have been developed for the treatment of PD, but one that can retard the progression of PD has not been identified (Koller and Tse, 2004; Pedrosa and Timmermann, 2013). Moreover, the long-term utility of these drugs (e.g., levodopa) are often limited by their unavoidable side effects. New drugs with novel mechanisms and fewer side effects are needed urgently. Recently, the concept of “neuroprotective therapy” has emerged as a potential treatment of PD (Olanow and Jankovic, 2005; Hart et al., 2009). “Skullcap”, the root of Scutellaria baicalensis Georgi, has been used extensively in traditional Chinese medicine for the treatment of inflammation, fever, cough, dysentery, and hypertension (Lin and Shieh, 1996). More than 50 compounds have been isolated and identified from skullcap. Flavonoids are the principal components contributing to its bioactivity (Gao et al., 1999). Baicalein (5,6,7-trihydroxyflavone) is a flavonoid originally isolated from skullcap (Fig. 1). Among its multiple biological activities, anti-oxidant, anti-inflammatory and neuroprotective activities have been demonstrated in various in vitro and in vivo models (Shao et al., 2002; Shen et al., 2003). Studies have demonstrated that baicalein exerts neuroprotective effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6hydroxydopamine-induced experimental models of PD (Lee et al., 2005; Cheng et al., 2008; Mu et al., 2009). Studies on molecular mechanisms have suggested that baicalein exerts its neuroprotective effects by regulating the function of the dopaminergic system and anti-oxidative damage. The anti-inflammatory effects and improved mitochondrial dysfunction of baicalein have also been observed (Mu et al., 2011; Li et al., 2012). Those results suggest that baicalein could have multiple mechanisms of actions on Parkinson's disease (Yu et al., 2012). An application for a clinical trial using baicalein to treat PD has been approved by the China Food and Drug Administration. The primary objective of our study was to investigate the pharmacokinetic (PK) properties of baicalein and its primary metabolite baicalin after administration of single escalating oral doses. The secondary objective was to characterize the safety and tolerability of baicalein in healthy male and female Chinese volunteers.
This trial was a single-center, randomized, double-blind, placebo-controlled, single dose-escalation Phase I study with healthy males and females. Research was conducted in accordance with Good Clinical Practice procedures and the principles of the Helsinki Declaration. The study protocol was approved by the Ethics Committee of the Beijing Hospital of the Ministry of Health (Beijing, China). Written informed consent was obtained from each subject before commencement of any protocol-specific procedures and after full explanation of the study design. The dose started at 100 mg, then escalated to 200, 400, 800, 1200, 1600, 2200, and 2800 mg. Six participants received 100 mg of β-form baicalein in the preliminary study. Then, treatment allocation was undertaken in sequential groups of 8–10 participants per dose group. Among the 8–10 subjects within each group, 2 subjects were assigned randomly to receive the placebo and 6–8 assigned randomly to receive the β-form active drug. Evaluations (safety, PK) were conducted before escalation to the next dose level. Chewable doses were administered with 200 mL of water after an overnight fast of Z10 h and standardized meals were started 4 h after dosing.
2.2. Subjects The study population comprised healthy male and female subjects (18–40 years; body mass index (BMI), 19–25 kg/m 2). Exclusion criteria were clinically significant abnormal medical history or physical examination; excessive alcohol intake or smoking; participation in any other clinical trial o 4 weeks before the first dose or consumption of any prescription/over-the-counter drug o2 weeks before administration; aiming to becoming pregnant in o6 months. Laboratory tests, vital signs and cardiac parameters (electrocardiography (ECG)) had to be within normal parameters. Subjects had to refrain from consumption of alcohol, cigarettes, brassicaceous vegetables, tomato, soya, apples, oranges or grapefruits for 3 days before dosing and during hospital admission.
2.3. Investigational products Baicalein chewable tablets and placebo were manufactured in accordance with applicable GMP and complying with applicable regulatory requirements. Batch sample analyses and characteristics records were maintained. The active ingredient was β-form baicalein. The inactive ingredients included mannitol, microcrystalline cellulose, aspartame, citric acid, sodium hydrogen sulfite, sodium calcium edentate, and other ingredients.
2.4. Safety evaluation Safety assessments comprised recording of all adverse events (AEs) and serious adverse events (SAEs) along with their severities and their relationship to the study drug. Safety assessments also included regular monitoring by: hematology; analyses of serum chemistry; coagulation assays; urinalysis; determination of vital signs; 12-lead ECG; physical examinations. The medications that the subjects were taking concomitantly were noted. Safety and tolerability results were analyzed descriptively.
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2.5. Sampling of plasma, urine and feces
2.7. Statistical analyses
2.5.1. Plasma collection A series of time points was chosen at which to collect the blood samples of subjects over a period of 48 h after dosing. Samples were obtained at time zero (pre-dosing) 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, and 48 h for the preliminary study (100 mg); and at time zero (pre-dosing), 10, 20, 30 min, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, and 48 h for the remainder of the groups (200–2800 mg). At each time for each subject, 3 mL of blood was collected. The blood sample was centrifuged at 1811g for 10 min at 4 1C within 30 min of collection. Plasma was divided into 3 parts and transferred to centrifuge tubes containing Vitamin C (10 mg mL 1) as a stabilizer. These tubes were labeled and stored at 70 1C for subsequent analyses.
The PK parameters were the maximum concentration that the drug achieved after dosing (Cmax); time to Cmax (Tmax); terminal half-life (t1/2); area under the curve from time zero to time of last quantifiable concentration (AUC(0–t)); area under the curve from time zero to infinity (AUC(0–1)); apparent total plasma clearance (CL/F); and the apparent total volume of distribution (V/F). These parameters were derived by non-compartmental analysis using WinNonlins v6.1 (Pharsight, St Louis, MO, USA) and summarized as the mean 7SD. Dose proportionality was assessed using a method combining the equivalence criterion and power model (Gough et al., 1995; Smith et al., 2000).
3. Results 2.5.2. Urine collection Blank urine samples were collected at –2 h to 0 h (pre-dosing). Subsequently, urine samples were collected over six intervals (0–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h) after administration of the study drug. Urine samples were collected at each time point, mixed well with the stabilizer Vc (10 mg mL 1) and volumes measured accurately. 10 mL of each sample was retained in a test tube, divided into three parts, sealed immediately, and stored at – 70 1C for subsequent analyses. 2.5.3. Feces collection Blank feces samples were collected at –12 h to 0 h (pre-dosing). Subsequently, feces samples (if any) were collected over a period of 48 h after dosing. Feces samples were weighed, homogenized with an aqueous solution of 0.1% formic acid, and the volume measured accurately. 10 mL of each sample was retained in a test tube, divided into three parts, mixed with the stabilizer Vc (10 mg mL 1), sealed immediately, and stored at –70 1C for subsequent analyses. 2.6. Bioanalytical methods Samples of plasma, urine and feces were analyzed for concentrations of baicalein and baicalin. Briefly, liquid–liquid extraction was employed for pretreatment of plasma and urine samples, and a protein precipitation method was used for pretreatment of feces samples. Chromatographic separation was undertaken using a C12 column with gradient elution. That is, A: acetonitrile–methanol (50:50, v/v) solution containing 0.5% formic acid; and B: aqueous solution of 3 mM ammonium acetate containing 0.5% formic acid. The gradient flow rate was 0.30 mL min 1 and the entire analysis lasted for 12 min. Concentrations of baicalein and baicalin were measured using a triple quadrupole tandem mass spectrometer in multiple re-action monitoring (MRM) mode via a positive electrospray ionization source. The m/z ratios of monitored parent ions and daughter ions of baicalein were 271.1 einf . Also, 447.1-271.0 were monitored for baicalin in plasma and feces, whereas 447.1lasma were monitored for baicalin in urine. The calibration range in plasma samples was 1–400 ng mL 1 for baicalein and baicalin. The range in urine samples was 1–400 ng mL–1 for baicalein and 2–800 ng mL 1 for baicalin. The range in feces samples was 100–20,000 ng mL 1 for baicalein and 10–4000 ng mL 1 for baicalin. The precision (expressed as the coefficient of variation) of the assay was shown to be 3.4–6.9% for plasma baicalein and ranged between 4.4% and 9.6% for the metabolite. Accuracy was 96–109% for plasma baicalein and 92–103% for baicalin. Similar precision and accuracy were demonstrated for the analysis of samples of urine and feces.
3.1. Subjects Seventy-two healthy male (n ¼46) and female (n ¼26) volunteers (mean age, 29.2 (range, 21–39) years; mean weight, 62.2 (range, 50–80) kg) were enrolled. Two subjects discontinued before drug administration: one because of high blood pressure and the other because of an ECG abnormality. Another subject withdrew 24 h after drug administration because of fear of blood sampling. All 70 subjects who were administered with baicalein were included in the pharmacokinetic analysis. 3.2. Safety There were 34 AEs in 24 subjects after administration of the study drug or placebo. All AEs were rated as “mild” and resolved without further treatment. Of the 34 reported AEs, 11 were determined by the study investigators to be probably or possibly related to the study drug (Table 1). The overall prevalence of AEs was the highest in the 1600-mg group (6/10 subjects, 60%) and ranged from 0% to 50% in the other groups. There was no evidence of a dose-related increase in AEs with baicalein administration. There were no SAEs in this study. None of the subjects withdrew from the study or were discontinued by the investigators because of AEs. There were no clinically relevant changes in blood pressure or ECG in individuals during the study. 3.3. PK The mean plasma concentration–time curves of baicalein and its metabolite baicalin after drug administration are shown in Fig. 2. The PK parameters derived by non-compartmental analysis of baicalein and baicalin are shown in Tables 2 and 3. The mean plasma concentration–time profiles of baicalein administration were characterized by a median tmax that varied from 0.75 h to 3.5 h across the dose groups, with mean Cmax values
Table 1 Summary of treatment-related adverse events. Adverse event
No. (%) of subjects
Hyperactive bowel sounds Abdominal distention Constipation Dizziness Somnolence Blurred vision Plasma fibrinogen decreased Blood leukocyte decreased
1 2 1 1 1 2 2 1
(1.8) (3.6) (1.8) (1.8) (1.8) (3.6) (3.6) (1.8)
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ranging from 5.82 ng mL–1 (100 mg) to 108.17 ng mL–1 (2800 mg). The mean t1/2 of baicalein varied from 1.9 h to 15.01 h. The shape of the plasma concentration–time profiles for baicalin resembled that of the parent compound: it had multiple peaks. The median tmax varied from 0.5 h to 3.0 h across the dose groups. The mean t1/2 varied from 4.22 h to 9.65 h. However, exposure to the metabolite baicalin was much higher compared with bacalein, with mean Cmax values ranging from 75.47 ng mL–1 L (100 mg) to 2264.50 ng mL–1 (2200 mg). The dose proportionality constant, β for baicalein was 0.83 (90% confidence interval (90% Cl), 0.70–0.96), 0.91 (0.81–1.00), and 0.92 (0.82–1.02), for Cmax, AUC(0–t), and AUC(0–1), respectively. These values overlapped with the acceptance interval (0.89–1.11), (0.93– 1.07), and (0.93–1.07), for Cmax, AUC(0–t), and AUC(0–1), respectively, suggesting that dose proportionality of baicalein for Cmax, AUC(0–t), and AUC(0–1) was inconclusive. The dose proportionality constant, β for baicalin was 0.82 (90% confidence interval (90% Cl), 0.69–0.95), 0.89 (0.78–1.00) and 0.88 (0.78–0.99) for Cmax, AUC(0–t) and AUC(0–1), respectively. These values overlapped within the pre-specified range, suggesting that
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dose proportionality of baicalin for Cmax, AUC(0–t), and AUC(0–1) was also inconclusive. The amount of drug excreted in urine from 0 h to 48 h after dosing was 0.20–4.51 mg of baicalein at a dose range of 100– 2800 mg, which corresponded to 0.10–0.27% of the administered dose, respectively. The amount of baicalein excreted in feces from 0 h to 48 h after dosing was 7.72–898.13 mg at a dose range of 100–2800 mg, which corresponded to 7.72% and 40.82% of the administered dose, respectively (Table 2). The amount excreted in urine from 0 h to 48 h after dosing was 1.32 mg to 22.76 mg of baicalin at a dose range of 100 mg to 2800 mg, respectively. The amount of baicalin excreted in feces from 0 h to 48 h after dosing ranged between 0.01 mg and 36.43 mg at a dose range of 100 mg to 2800 mg, respectively (Table 3).
4. Discussion The present first-in-human study was designed to investigate the PK, safety and tolerability of baicalein after oral administration in healthy subjects of both sexes. Previous studies have demonstrated that baicalein has two crystal forms, α-form and β-form. The crystalline pattern of naturally occurred baicalein was α-form with very low bioavailability, which was described in many studies. Therefore, therapeutic applications of naturally occurred baicalein were limited by its low bioavailability. On the other hand, the bioavailability of the newfound β-form was much higher than the α-form, which could achieve therapeutic concentrations in various animal models. For example, the absolute bioavailability of β-form in monkeys ranged from 13% to 23% at 50–500 mg/kg dose level (Tian et al., 2012). The application for the patent of two crystal substances of baicalin, and preparations, pharmaceutical composition and uses thereof has been granted (CN101434593 B, Du et al., 2013). The β-form baicalein used in the present study was expected to achieve higher body exposure in man compared with naturally occurred α-form. The single-ascending-dose design permitted the safety and PK data at a lower dose to be evaluated before proceeding to higher doses. Preclinical toxicology data fully supported the safety of the starting dose. Using the standardized algorithm recommended by FDA (Food and Drug Administration., 2005), 1200-mg was the upper limit of starting doses (12-fold for the 100-mg starting dose we used). Eight single dose levels were investigated from 100 mg to 2800 mg, resulting in a maximum 28-fold increase in dose. The plasma concentration–time profiles of baicalein showed that maximum plasma concentrations were achieved E0.75 h to E3 h after administration and, thereafter, baicalein concentrations decreased in a multiphasic way. The multiple peaks of baicalein in the plasma drug concentration–time profile observed in the present study and the predominant amount of baicalein compared
Fig. 2. Mean plasma concentration vs. time profiles of baicalein (A) and its metabolites baicalin (B) after a single dose (n¼ 6–8 per group) of 100–2800 mg baicalein.
Table 2 Main pharmacokinetic parameters of baicalein in healthy Chinese subjects after administration of a single dose of 100–2800 mg of baicalein. Values are the mean 7 SD for all parameters with the exception of tmax, which is the median (range). Parameter
100 mg
200 mg
400 mg
800 mg
1200 mg
1600 mg
2200 mg
2800 mg
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0-t ( ng/mL h) AUC0-1 (ng/mL h) Vd/F (L) CLz/F (L/h) U_Recovered (%) F_Recovered (%)
1.90 3.5 5.820 21.90 24.95 15,926 4796 0.20 10.30
8.03 3 11.54 69.15 74.94 32,723 2779 0.25 32.06
4.82 3 20.47 98.25 101.72 28,519 5072 0. 19 31.04
8.23 1.5 35.38 210.57 218.02 48,631 4078 0.15 32.18
10.16 2 51.62 307.91 323.48 62,778 4438 0.13 28.03
8.71 1.5 43.06 239.18 255.23 88,281 6892 0.10 26.81
7.29 1.25 131.36 478.45 497.45 59,743 5398 0.16 40.82
15.01 0.75 108.17 540.93 678.23 80,950 4929 0.16 18.15
(1.17) (0.5–5) (3.33) (10.98) (10.78) (18,345) (2491) (0.09) (13.70)
(3.07) (0.5–5) (4.10) (18.74) (17.87) (13,955) (536) (0.05) (22.04)
(2.95) (0.17–5) (11.81) (59.44) (60.90) (15,465) (2384) (0.10) (16.84)
(1.84) (0.17–4) (19.17) (77.41) (78.50) (18,968) (1393.46) (0.06) (28.65)
(4.07) (0.17–5) (46.50) (164.21) (164.63) (25,882) (1824) (0.04) (20.20)
(2.00) (0.5–4) (22.67) (71.22) (80.64) (50,158) (2397) (0.03) (18.97)
(4.16) (0.17–5) (140.25) (263.47) (260.39) (45,724) (2396) (0.10) (36.97)
(14.97) (0.33–4) (83.16) (175.71) (304.95) (47,692) (2153) (0.04) (17.76)
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Table 3 Main pharmacokinetic parameters of baicalin in healthy Chinese subjects after administration of a single dose of 100–2800 mg of baicalein. Values are the mean 7 SD for all parameters with the exception of tmax, which is the median (range). Parameter
100 mg
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0-t ( ng/mL h) AUC0-1 (ng/mL h) Vd/F (L) CLz/F (L/h) U_Recovered (mg) F_Recovered (mg)
8.58 1.00 75.47 556.44
200 mg
400 mg
800 mg
(5.06) 4.22 (0.42) 4.56 (0.5–4) 3 (0.33–5) 2.75 (26.43) 182.00 (90.53) 234.82 (136.54) 979.65 (369.72) 1314.66
(1.71) 4.84 (0.33–5) 1.5 (140.48) 428.58 (760.59) 3853.07
1200 mg (1.62) 10.80 (0.33–4) 0.5 (156.88) 770.00 (1756.89) 4887.99
580.24 (135.69) 982.69 (369.70) 1322.10 (766.82) 3877.79 (1777.29)
1600 mg
(4.97) 8.64 (0.33–2) 1.5 (749.68) 635.85 (2581.04) 3838.56
5177.42 (2586.50)
2200 mg
2800 mg
(2.59) 5.87 (2.85) 9.65 (0.5–4) 0.75 (0.5–5) 1.25 (230.74) 2264.50 (3022.58) 1847.21 (1186.55) 7391.79 (5118.18) 10,349.15
3917.97 (1181.40) 7500.35 (5106.65)
(5.78) (0.5–4) (1269.03) (3319.71)
11,961.53 (6016.22)
3692 (2588) 299 (74) 1.32 (0.58)
2298 (855) 377 (134) 2.37 (0.68)
4234 (2879) 661 (348) 3.37 (1.33)
2733 (1088) 423 (228) 8.08 (3.88)
6841 (3249) 447 (160) 11.58 (7.00)
8831 (3004) 718 (169) 8.81 (1.78)
7279 (7445) 725 (468) 15.09 (6.85)
5247 (515) 465 (202) 22.76 (7.56)
0.04 (0.04)
0.86 (0.97)
5.61 (2.97)
4.44 (3.83)
1.44 (3.00)
6.48 (6.07)
36.43 (58.38)
7.21 (8.14)
with its metabolite in feces are in accordance with the hypothesis that baicalein undergoes enterohepatic recirculation (Xing et al., 2005). This phenomenon has been reported to complicate absorption and recycling, and affect the PK of certain drugs by influencing t1/2, the area under the plasma concentration–time curve, and bioavailability (Tsai et al., 2000). Studies have indicated that baicalein is well absorbed from the stomach and small intestine with limited absorption from the colon, whereas baicalin is moderately absorbed in the stomach but poorly absorbed from the small intestine and colon. These two moieties maintain a balance of systemic levels in the body through dynamic reconversion. However, it was concluded that baicalein was the preferred species for oral absorption because of a more rapid and complete absorption of baicalein and restoration of baicalin in the systemic circulation by the hydrolysis of baicalein. Circulating levels of baicalin would be expected to re-enter the gastrointestinal tract via biliary excretion (Taiming and Xueha, 2006). In contrast to the relative low systemic levels of the parent compound baicalein, its most abundant metabolite conjugate, baicalin, was present in plasma at a Cmax concentration range of 0.075–1.8 μmol L–1. The neuroprotective effect in rodent models is thought to be mediated primarily via the parent compound. The extensive glucuronidation and sulfation of baicalein with consequent poor bioavailability of the parent compound, as described here and previously, calls into question the role of the metabolite baicalin in the mediation of efficacy. The pharmacologic properties of most baicalein conjugates are not known, but baicalin (the 7βglucuronidase-catalyzed conjugate) has been suggested to contribute to the pharmacologic activity of the parent molecule (Srinivas, 2010). In hepatoma cell lines, baicalin can induce apoptosis or inhibit proliferation (Chang et al., 2002). Baicalin also seems to retain (at least in part) the antioxidant properties of the parent molecule (Shieh et al., 2000). Therefore, the exposure of both baicalein and baicalin in the human body was studied. Preclinical data showed that baicalein is metabolized predominatly by glucuronidation via uridine 50 -diphospho-glucuronosyltransferase systems. The urinary excretion of unchanged baicalein was extremely small. Of all the administered oral doses, E0.7% baicalein was excreted unchanged in urine and 27.1% excreted unchanged in feces within 48 h. These findings suggested that the drug might be excreted mainly in a metabolized form. The complete metabolic profile of baicalein in humans is still under investigation. Enrolling female subjects early in the drug-development process is important for the assessment of sex-related differences in PK. The PK of baicalein after administration of a single dose was comparable in female and male subjects. Considering our small
study cohort, however, data from more subjects will be required to confirm the absence of sex differences. Baicalein administration was safe and well tolerated by healthy subjects from 100 mg to 2800 mg, with a low number of AEs being reported. The safety assessments used in our study based on preclinical safety data were, in general, recognized to be reliable and relevant for a Phase I study. The study included routine monitoring of subjects for clinical signs, hematology parameters, liver function tests (alanine transaminase, aspartate aminotransferase, and gamma-glutamyl transferase), kidney function tests (blood, urea, nitrogen; creatine), ECG and urinalyses. No risks were identified according to evaluation of vital signs, ECG, or physical examination and there appeared to be no effect on the QTc interval. Adverse gastrointestinal events such as constipation and abdominal distention were expected due to disruption to intestinal microflora. Dry cough and hemolytic anemia, which occurred in preclinical toxicity tests, were not observed in clinical studies. All reported AEs were transient and mild in intensity. There was no evidence of a dose-related increase in AEs upon baicalein administration.
5. Conclusions Multiple peaks on the plasma concentration–time curves for baicalein and baicalin were found at all doses tested. Dose proportionality was inconclusive in the dose range 100– 2800 mg. Single oral doses of 100–2800 mg of baicalein were safe and well tolerated by healthy subjects. Clinical laboratory assessments showed no signs of toxicity in the liver or kidney. The favorable safety profile and PK properties warrant further clinical studies for baicalein.
Funding sources National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Twelfth Five-year Plan Period (2012ZX09303-008-002, http://www.nmp.gov.cn/).
Acknowledgment This study was financially supported by Kanion pharmaceutical Limited Corporation (CTR20132944) (Jiangsu, P.R. China). We are grateful for the following persons for their assistance in the study: Jiamin Chi, Huili Liu, Jianhua Yang, Huihong Qi, Panpan Xie, Taifeng Li, Yi Jin, Chunxiao Li, Wenjing Hou, and Kongcai Zhu.
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References Chang, W.H., Chen, C.H., Lu, F.J., 2002. Different effects of baicalein, baicalin and wogonin on mitochondrial function, glutathione content and cell cycle progression in human hepatoma cell lines. Planta Medica 68, 128–132. Cheng, Y., He, G., Mu, X., Zhang, T., Li, X., Hu, J., Xu, B., Du, G., 2008. Neuroprotective effect of baicalein against MPTP neurotoxicity: behavioral, biochemical and immunohistochemical profile. Neuroscience Letters 441, 16–20. Dauer, W., Przedborski, S., 2003. Parkinson's disease: mechanisms and models. Neuron 39, 889–909. Du, G.H., Lv, Y., Chang, Y., Cheng, Y.X., He, G.R., Pei, L.X. Two crystal substances of baicalin, and preparations, pharmaceutical composition and uses thereof. Chinese Patent no. CN101434593 B; 2013. Food and Drug Administration, 2005. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research, Rockville, MD. Gao, Z., Huang, K., Yang, X., Xu, H., 1999. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochimica et Biophysica Acta 1472, 643–650. Gough, K., Hutchison, M., Keene, O., Byrom, B., Ellis, S., Lacey, L., McKellar, J., 1995. Assessment of dose proportionality: report from the statisticians in the pharmaceutical industry/pharmacokinetics UK joint working party. Drug Information Journal 29, 1039–1048. Hart, R.G., Pearce, L.A., Ravina, B.M., Yaltho, T.C., Marler, J.R., 2009. Neuroprotection trials in Parkinson's disease: systematic review. Movement Disorders 24, 647–654. Koller, W.C., Tse, W., 2004. Unmet medical needs in Parkinson's disease. Neurology 62 (1 Suppl 1), S1–S8. Lee, H.J., Noh, Y.H., Lee, D.Y., Kim, Y.S., Kim, K.Y., Chung, Y.H., Lee, W.B., Kim, S.S., 2005. Baicalein attenuates 6-hydroxydopamine-induced neurotoxicity in SHSY5Y cells. European Journal of Cell Biology 84, 897–905. Li, X.X., He, G.R., Mu, X., Xu, B., Tian, S., Yu, X., Meng, F.R., Xuan, Z.H., Du, G.H., 2012. Protective effects of baicalein against rotenone-induced neurotoxicity in PC12 cells and isolated rat brain mitochondria. European Journal of Pharmacology 674, 227–233. Lin, C.C., Shieh, D.E., 1996. The anti-inßammatory activity of Scutellaria rivularis extracts and its active components, baicalin, baicalein and wogonin. American Journal of Chinese Medicine 24, 31–36. Mu, X., He, G., Cheng, Y., Li, X., Xu, B., Du, G., 2009. Baicalein exerts neuroprotective effects in 6-hydroxydopamine-induced experimental parkinsonism in vivo and in vitro. Pharmacology Biochemistry and Behavior 92, 642–648.
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Mu, X., He, G.R., Yuan, X., Li, X.X., Du, G.H., 2011. Baicalein protects the brain against neuron impairments induced by MPTP in C57BL/6 mice. Pharmacology Biochemistry and Behavior 98, 286–291. Niranjan, R., 2014. The role of inflammatory and oxidative stress mechanisms in the pathogenesis of Parkinson's disease: focus on astrocytes. Molecular Neurobiology 49, 28–38. Nutt, J.G., Wooten, G.F., 2005. Clinical practice. Diagnosis and initial management of Parkinson's disease. The New England Journal of Medicine 353, 1021–1027. Olanow, C.W., Jankovic, J., 2005. Neuroprotective therapy in Parkinson's disease and motor complications: a search for a pathogenesistargeted, disease-modifying strategy. Movement Disorders 20, S3–S10. Pedrosa, D.J., Timmermann, L., 2013. Management of Parkinson's disease. Neuropsychiatric Disease and Treatment 9, 321–340. Shao, Z.H., Vanden Hoek, T.L., Qin, Y., Becker, L.B., Schumacker, P.T., Li, C.Q., Dey, L., Barth, E., Halpern, H., Rosen, G.M., Yuan, C.S., 2002. Baicalein attenuates oxidant stress in cardiomyocytes. American Journal of Physiology 282, H999–H1006. Shen, Y.C., Chiou, W.F., Chou, Y.C., Chen, C.F., 2003. Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes. European Journal of Pharmacology 465, 171–181. Shieh, D.E., Liu, L.T., Lin, C.C., 2000. Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Research 20, 2861–2865. Smith, B.P., Vandenhende, F.R., DeSante, K.A., Farid, N.A., Welch, P.A., Callaghan, J.T., Forgue, S.T., 2000. Confidence interval criteria for assessment of dose proportionality. Pharmaceutical Research 17, 1278–1283. Srinivas, N.R., 2010. Baicalin, an emerging multi-therapeutic agent: pharmacodynamics, pharmacokinetics, and considerations from drug development perspectives. Xenobiotica 40, 357–367. Taiming, L., Xueha, J., 2006. Investigation of the absorption mechanisms of baicalin and baicalein in rats. Journal of Pharmaceutical Sciences 95, 1326–1333. Tian, S., He, G., Song, J., Wang, S., Xin, W., Zhang, D., Du, G., 2012. Pharmacokinetic study of baicalein after oral administration in monkeys. Fitoterapia 83, 532–540. Tsai, T.H., Shum, A.Y.C., Chen, C.F., 2000. Enterohepatic circulation of chloramphenicol and its glucuronide in the rat by microdialysis using a hepato-duodenal shunt. Life Sciences 66, 363–370. Von Bohlen und Halbach, O., Schober, A., Krieglstein, K., 2004. Genes, proteins, and neurotoxins involved in Parkinson's disease. Progress in Neurobiology 73, 151–177. Xing, J., Chen, X., Zhong, D., 2005. Absorption and enterohepatic circulation of baicalin in rats. Life Sciences 78, 140–146. Yu, X., He, G., Du, G., 2012. Neuroprotective effect of baicalein in patients with Parkinson's disease. Zhongguo Zhong Yao Za Zhi 37, 421–425 ([Article in Chinese]).
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Safety, tolerability, and pharmacokinetics of a single ascending dose of baicalein chewable tablets in healthy subjects Min Li a, Aixin Shi a,n, Hongxian Pang a, Wei Xue a, Yang Li a, Guoying Cao a, Bei Yan a, Fan Dong a, Kexin Li a, Wei Xiao b, Guorong He c, Guanhua Du c, Xin Hu a a Beijing Key Laboratory of Drug Clinical Risk and Personalized Medication Evaluation, Department of Clinical pharmacology, Beijing Hospital of the Ministry of Health, Beijing 100730, China b StateKey Laboratory of New-tech for Chinese Medicine Pharmaceutical Process, JiangsuKanion Parmaceutical CO. LTD., Lianyungang, Jiangsu 222001, China c Beijing Key Laboratory of Drug Targets Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China
art ic l e i nf o
a b s t r a c t
Article history: Received 22 May 2014 Received in revised form 11 August 2014 Accepted 24 August 2014 Available online 8 September 2014
Ethnopharmacological relevance: The root of Scutellaria baicalensis Georgi has been used extensively in traditional Chinese medicine for the treatment of inflammation, fever, cough, dysentery, and hypertension. Baicalein is a flavonoid isolated from the root of Scutellaria baicalensis Georgi and is a novel neuroprotective agent under development for the treatment of Parkinson's disease. We aimed to investigate the pharmacokinetic (PK) properties of baicalein and its main metabolite, bacalin, after single-dose administration in healthy Chinese subjects. The safety and tolerability of baicalein were also assessed. Materials and methods: This was a Phase I, randomized, double-blind, single-dose trial of baicalein (100– 2800 mg) in 72 healthy adults. Samples of blood, urine and feces were collected at regular intervals up to 48 h after administration of the study drug. Baicalein and baicalin were then analyzed using liquid chromatography-tandem mass spectrometry (LC/MS/MS). The maximum concentration that the drug achieved after dosing (Cmax), time to Cmax (Tmax), terminal half-life (t1/2), area under the curve from time zero to time of last quantifiable concentration (AUC(0, t)), area under the curve from time zero to infinity (AUC(0, 1)), apparent total plasma clearance (CL/F), and apparent total volume of distribution (V/F) were determined using non-compartmental models. Dose proportion was tested using a method combining the equivalence criterion and power model. Physical examinations, vital signs, ECG findings, hematology, and urinalysis were monitored before and at regular intervals after administration of the study drug. Results: The PK profile of baicelein and baicelin was characterized by a median Tmax of 0.75–3.5 h and 0.5–3 h, respectively, followed by a multiphasic profile with a t1/2 of 1.90–15.01 h and 4.22–10.80 h, respectively. The estimates of the proportionality coefficient (90% CI) for Cmax, AUC0–t and AUC0–1 were 0.83 (0.70–0.96), 0.91 (0.81–1.00) and 0.92 (0.82–1.02), respectively. All values overlapped within the pre-specified range of (0.89–1.11), (0.93–1.07), and (0.93–1.07), respectively. Dose proportionality was inconclusive for a baicalein dose range of 100–2800 mg. The total urinary clearance of baicalein and baicalin was o 1%. Approximately 27% of baicalein was eliminated as unchanged drug in feces. Baicalein was well tolerated. Eleven treatment-related adverse events were observed, and all were rated as “mild” and resolved without further treatment. No serious adverse events occurred. Conclusions: Single oral doses of 100–2800 mg of baicalein were safe and well tolerated by healthy subjects. Clinical laboratory assessments showed no signs of toxicity in the liver or kidney. The favorable safety profile and PK properties warrant further clinical studies for baicalein. & 2014 Elsevier Ireland Ltd. All rights reserved.
Chemical compounds studied in this article: Baicalein (PubChem CID: 5281605) Baicalin (PubChem CID:64982) Keywords: Baicalein Baicalin Parkinson's Disease Safety Tolerability Pharmacokinetics
1. Introduction
n
Correspondance to: Beijing key laboratory of drug clinical risk and personalized medication evaluation, Department of clinical pharmacology, Beijing Hospital of the Ministry of Health, Beijing, 100730, China Tel:þ 86 10 85133632. E-mail address: [email protected] (A. Shi). http://dx.doi.org/10.1016/j.jep.2014.08.031 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.
Parkinson's disease (PD) is an age-related neurodegenerative disorder caused by the disturbance of the function of the extrapyramidal system. The prevalence of PD in people aged 460 years is E1% (Nutt and Wooten, 2005). The precise pathogenesis of PD is not known, but environmental factors, genetic factors,
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2. Methods 2.1. Study design
Fig. 1. Chemical structures of baicalein (A) and baicalin (B).
inflammatory responses, oxidative stress, and mitochondrial dysfunction might be involved in the development of PD (Dauer and Przedborski, 2003; Von Bohlen und Halbach et al., 2004; Niranjan, 2014). Several drugs have been developed for the treatment of PD, but one that can retard the progression of PD has not been identified (Koller and Tse, 2004; Pedrosa and Timmermann, 2013). Moreover, the long-term utility of these drugs (e.g., levodopa) are often limited by their unavoidable side effects. New drugs with novel mechanisms and fewer side effects are needed urgently. Recently, the concept of “neuroprotective therapy” has emerged as a potential treatment of PD (Olanow and Jankovic, 2005; Hart et al., 2009). “Skullcap”, the root of Scutellaria baicalensis Georgi, has been used extensively in traditional Chinese medicine for the treatment of inflammation, fever, cough, dysentery, and hypertension (Lin and Shieh, 1996). More than 50 compounds have been isolated and identified from skullcap. Flavonoids are the principal components contributing to its bioactivity (Gao et al., 1999). Baicalein (5,6,7-trihydroxyflavone) is a flavonoid originally isolated from skullcap (Fig. 1). Among its multiple biological activities, anti-oxidant, anti-inflammatory and neuroprotective activities have been demonstrated in various in vitro and in vivo models (Shao et al., 2002; Shen et al., 2003). Studies have demonstrated that baicalein exerts neuroprotective effects in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and 6hydroxydopamine-induced experimental models of PD (Lee et al., 2005; Cheng et al., 2008; Mu et al., 2009). Studies on molecular mechanisms have suggested that baicalein exerts its neuroprotective effects by regulating the function of the dopaminergic system and anti-oxidative damage. The anti-inflammatory effects and improved mitochondrial dysfunction of baicalein have also been observed (Mu et al., 2011; Li et al., 2012). Those results suggest that baicalein could have multiple mechanisms of actions on Parkinson's disease (Yu et al., 2012). An application for a clinical trial using baicalein to treat PD has been approved by the China Food and Drug Administration. The primary objective of our study was to investigate the pharmacokinetic (PK) properties of baicalein and its primary metabolite baicalin after administration of single escalating oral doses. The secondary objective was to characterize the safety and tolerability of baicalein in healthy male and female Chinese volunteers.
This trial was a single-center, randomized, double-blind, placebo-controlled, single dose-escalation Phase I study with healthy males and females. Research was conducted in accordance with Good Clinical Practice procedures and the principles of the Helsinki Declaration. The study protocol was approved by the Ethics Committee of the Beijing Hospital of the Ministry of Health (Beijing, China). Written informed consent was obtained from each subject before commencement of any protocol-specific procedures and after full explanation of the study design. The dose started at 100 mg, then escalated to 200, 400, 800, 1200, 1600, 2200, and 2800 mg. Six participants received 100 mg of β-form baicalein in the preliminary study. Then, treatment allocation was undertaken in sequential groups of 8–10 participants per dose group. Among the 8–10 subjects within each group, 2 subjects were assigned randomly to receive the placebo and 6–8 assigned randomly to receive the β-form active drug. Evaluations (safety, PK) were conducted before escalation to the next dose level. Chewable doses were administered with 200 mL of water after an overnight fast of Z10 h and standardized meals were started 4 h after dosing.
2.2. Subjects The study population comprised healthy male and female subjects (18–40 years; body mass index (BMI), 19–25 kg/m 2). Exclusion criteria were clinically significant abnormal medical history or physical examination; excessive alcohol intake or smoking; participation in any other clinical trial o 4 weeks before the first dose or consumption of any prescription/over-the-counter drug o2 weeks before administration; aiming to becoming pregnant in o6 months. Laboratory tests, vital signs and cardiac parameters (electrocardiography (ECG)) had to be within normal parameters. Subjects had to refrain from consumption of alcohol, cigarettes, brassicaceous vegetables, tomato, soya, apples, oranges or grapefruits for 3 days before dosing and during hospital admission.
2.3. Investigational products Baicalein chewable tablets and placebo were manufactured in accordance with applicable GMP and complying with applicable regulatory requirements. Batch sample analyses and characteristics records were maintained. The active ingredient was β-form baicalein. The inactive ingredients included mannitol, microcrystalline cellulose, aspartame, citric acid, sodium hydrogen sulfite, sodium calcium edentate, and other ingredients.
2.4. Safety evaluation Safety assessments comprised recording of all adverse events (AEs) and serious adverse events (SAEs) along with their severities and their relationship to the study drug. Safety assessments also included regular monitoring by: hematology; analyses of serum chemistry; coagulation assays; urinalysis; determination of vital signs; 12-lead ECG; physical examinations. The medications that the subjects were taking concomitantly were noted. Safety and tolerability results were analyzed descriptively.
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2.5. Sampling of plasma, urine and feces
2.7. Statistical analyses
2.5.1. Plasma collection A series of time points was chosen at which to collect the blood samples of subjects over a period of 48 h after dosing. Samples were obtained at time zero (pre-dosing) 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, and 48 h for the preliminary study (100 mg); and at time zero (pre-dosing), 10, 20, 30 min, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, 12, 24, 36, and 48 h for the remainder of the groups (200–2800 mg). At each time for each subject, 3 mL of blood was collected. The blood sample was centrifuged at 1811g for 10 min at 4 1C within 30 min of collection. Plasma was divided into 3 parts and transferred to centrifuge tubes containing Vitamin C (10 mg mL 1) as a stabilizer. These tubes were labeled and stored at 70 1C for subsequent analyses.
The PK parameters were the maximum concentration that the drug achieved after dosing (Cmax); time to Cmax (Tmax); terminal half-life (t1/2); area under the curve from time zero to time of last quantifiable concentration (AUC(0–t)); area under the curve from time zero to infinity (AUC(0–1)); apparent total plasma clearance (CL/F); and the apparent total volume of distribution (V/F). These parameters were derived by non-compartmental analysis using WinNonlins v6.1 (Pharsight, St Louis, MO, USA) and summarized as the mean 7SD. Dose proportionality was assessed using a method combining the equivalence criterion and power model (Gough et al., 1995; Smith et al., 2000).
3. Results 2.5.2. Urine collection Blank urine samples were collected at –2 h to 0 h (pre-dosing). Subsequently, urine samples were collected over six intervals (0–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h) after administration of the study drug. Urine samples were collected at each time point, mixed well with the stabilizer Vc (10 mg mL 1) and volumes measured accurately. 10 mL of each sample was retained in a test tube, divided into three parts, sealed immediately, and stored at – 70 1C for subsequent analyses. 2.5.3. Feces collection Blank feces samples were collected at –12 h to 0 h (pre-dosing). Subsequently, feces samples (if any) were collected over a period of 48 h after dosing. Feces samples were weighed, homogenized with an aqueous solution of 0.1% formic acid, and the volume measured accurately. 10 mL of each sample was retained in a test tube, divided into three parts, mixed with the stabilizer Vc (10 mg mL 1), sealed immediately, and stored at –70 1C for subsequent analyses. 2.6. Bioanalytical methods Samples of plasma, urine and feces were analyzed for concentrations of baicalein and baicalin. Briefly, liquid–liquid extraction was employed for pretreatment of plasma and urine samples, and a protein precipitation method was used for pretreatment of feces samples. Chromatographic separation was undertaken using a C12 column with gradient elution. That is, A: acetonitrile–methanol (50:50, v/v) solution containing 0.5% formic acid; and B: aqueous solution of 3 mM ammonium acetate containing 0.5% formic acid. The gradient flow rate was 0.30 mL min 1 and the entire analysis lasted for 12 min. Concentrations of baicalein and baicalin were measured using a triple quadrupole tandem mass spectrometer in multiple re-action monitoring (MRM) mode via a positive electrospray ionization source. The m/z ratios of monitored parent ions and daughter ions of baicalein were 271.1 einf . Also, 447.1-271.0 were monitored for baicalin in plasma and feces, whereas 447.1lasma were monitored for baicalin in urine. The calibration range in plasma samples was 1–400 ng mL 1 for baicalein and baicalin. The range in urine samples was 1–400 ng mL–1 for baicalein and 2–800 ng mL 1 for baicalin. The range in feces samples was 100–20,000 ng mL 1 for baicalein and 10–4000 ng mL 1 for baicalin. The precision (expressed as the coefficient of variation) of the assay was shown to be 3.4–6.9% for plasma baicalein and ranged between 4.4% and 9.6% for the metabolite. Accuracy was 96–109% for plasma baicalein and 92–103% for baicalin. Similar precision and accuracy were demonstrated for the analysis of samples of urine and feces.
3.1. Subjects Seventy-two healthy male (n ¼46) and female (n ¼26) volunteers (mean age, 29.2 (range, 21–39) years; mean weight, 62.2 (range, 50–80) kg) were enrolled. Two subjects discontinued before drug administration: one because of high blood pressure and the other because of an ECG abnormality. Another subject withdrew 24 h after drug administration because of fear of blood sampling. All 70 subjects who were administered with baicalein were included in the pharmacokinetic analysis. 3.2. Safety There were 34 AEs in 24 subjects after administration of the study drug or placebo. All AEs were rated as “mild” and resolved without further treatment. Of the 34 reported AEs, 11 were determined by the study investigators to be probably or possibly related to the study drug (Table 1). The overall prevalence of AEs was the highest in the 1600-mg group (6/10 subjects, 60%) and ranged from 0% to 50% in the other groups. There was no evidence of a dose-related increase in AEs with baicalein administration. There were no SAEs in this study. None of the subjects withdrew from the study or were discontinued by the investigators because of AEs. There were no clinically relevant changes in blood pressure or ECG in individuals during the study. 3.3. PK The mean plasma concentration–time curves of baicalein and its metabolite baicalin after drug administration are shown in Fig. 2. The PK parameters derived by non-compartmental analysis of baicalein and baicalin are shown in Tables 2 and 3. The mean plasma concentration–time profiles of baicalein administration were characterized by a median tmax that varied from 0.75 h to 3.5 h across the dose groups, with mean Cmax values
Table 1 Summary of treatment-related adverse events. Adverse event
No. (%) of subjects
Hyperactive bowel sounds Abdominal distention Constipation Dizziness Somnolence Blurred vision Plasma fibrinogen decreased Blood leukocyte decreased
1 2 1 1 1 2 2 1
(1.8) (3.6) (1.8) (1.8) (1.8) (3.6) (3.6) (1.8)
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ranging from 5.82 ng mL–1 (100 mg) to 108.17 ng mL–1 (2800 mg). The mean t1/2 of baicalein varied from 1.9 h to 15.01 h. The shape of the plasma concentration–time profiles for baicalin resembled that of the parent compound: it had multiple peaks. The median tmax varied from 0.5 h to 3.0 h across the dose groups. The mean t1/2 varied from 4.22 h to 9.65 h. However, exposure to the metabolite baicalin was much higher compared with bacalein, with mean Cmax values ranging from 75.47 ng mL–1 L (100 mg) to 2264.50 ng mL–1 (2200 mg). The dose proportionality constant, β for baicalein was 0.83 (90% confidence interval (90% Cl), 0.70–0.96), 0.91 (0.81–1.00), and 0.92 (0.82–1.02), for Cmax, AUC(0–t), and AUC(0–1), respectively. These values overlapped with the acceptance interval (0.89–1.11), (0.93– 1.07), and (0.93–1.07), for Cmax, AUC(0–t), and AUC(0–1), respectively, suggesting that dose proportionality of baicalein for Cmax, AUC(0–t), and AUC(0–1) was inconclusive. The dose proportionality constant, β for baicalin was 0.82 (90% confidence interval (90% Cl), 0.69–0.95), 0.89 (0.78–1.00) and 0.88 (0.78–0.99) for Cmax, AUC(0–t) and AUC(0–1), respectively. These values overlapped within the pre-specified range, suggesting that
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dose proportionality of baicalin for Cmax, AUC(0–t), and AUC(0–1) was also inconclusive. The amount of drug excreted in urine from 0 h to 48 h after dosing was 0.20–4.51 mg of baicalein at a dose range of 100– 2800 mg, which corresponded to 0.10–0.27% of the administered dose, respectively. The amount of baicalein excreted in feces from 0 h to 48 h after dosing was 7.72–898.13 mg at a dose range of 100–2800 mg, which corresponded to 7.72% and 40.82% of the administered dose, respectively (Table 2). The amount excreted in urine from 0 h to 48 h after dosing was 1.32 mg to 22.76 mg of baicalin at a dose range of 100 mg to 2800 mg, respectively. The amount of baicalin excreted in feces from 0 h to 48 h after dosing ranged between 0.01 mg and 36.43 mg at a dose range of 100 mg to 2800 mg, respectively (Table 3).
4. Discussion The present first-in-human study was designed to investigate the PK, safety and tolerability of baicalein after oral administration in healthy subjects of both sexes. Previous studies have demonstrated that baicalein has two crystal forms, α-form and β-form. The crystalline pattern of naturally occurred baicalein was α-form with very low bioavailability, which was described in many studies. Therefore, therapeutic applications of naturally occurred baicalein were limited by its low bioavailability. On the other hand, the bioavailability of the newfound β-form was much higher than the α-form, which could achieve therapeutic concentrations in various animal models. For example, the absolute bioavailability of β-form in monkeys ranged from 13% to 23% at 50–500 mg/kg dose level (Tian et al., 2012). The application for the patent of two crystal substances of baicalin, and preparations, pharmaceutical composition and uses thereof has been granted (CN101434593 B, Du et al., 2013). The β-form baicalein used in the present study was expected to achieve higher body exposure in man compared with naturally occurred α-form. The single-ascending-dose design permitted the safety and PK data at a lower dose to be evaluated before proceeding to higher doses. Preclinical toxicology data fully supported the safety of the starting dose. Using the standardized algorithm recommended by FDA (Food and Drug Administration., 2005), 1200-mg was the upper limit of starting doses (12-fold for the 100-mg starting dose we used). Eight single dose levels were investigated from 100 mg to 2800 mg, resulting in a maximum 28-fold increase in dose. The plasma concentration–time profiles of baicalein showed that maximum plasma concentrations were achieved E0.75 h to E3 h after administration and, thereafter, baicalein concentrations decreased in a multiphasic way. The multiple peaks of baicalein in the plasma drug concentration–time profile observed in the present study and the predominant amount of baicalein compared
Fig. 2. Mean plasma concentration vs. time profiles of baicalein (A) and its metabolites baicalin (B) after a single dose (n¼ 6–8 per group) of 100–2800 mg baicalein.
Table 2 Main pharmacokinetic parameters of baicalein in healthy Chinese subjects after administration of a single dose of 100–2800 mg of baicalein. Values are the mean 7 SD for all parameters with the exception of tmax, which is the median (range). Parameter
100 mg
200 mg
400 mg
800 mg
1200 mg
1600 mg
2200 mg
2800 mg
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0-t ( ng/mL h) AUC0-1 (ng/mL h) Vd/F (L) CLz/F (L/h) U_Recovered (%) F_Recovered (%)
1.90 3.5 5.820 21.90 24.95 15,926 4796 0.20 10.30
8.03 3 11.54 69.15 74.94 32,723 2779 0.25 32.06
4.82 3 20.47 98.25 101.72 28,519 5072 0. 19 31.04
8.23 1.5 35.38 210.57 218.02 48,631 4078 0.15 32.18
10.16 2 51.62 307.91 323.48 62,778 4438 0.13 28.03
8.71 1.5 43.06 239.18 255.23 88,281 6892 0.10 26.81
7.29 1.25 131.36 478.45 497.45 59,743 5398 0.16 40.82
15.01 0.75 108.17 540.93 678.23 80,950 4929 0.16 18.15
(1.17) (0.5–5) (3.33) (10.98) (10.78) (18,345) (2491) (0.09) (13.70)
(3.07) (0.5–5) (4.10) (18.74) (17.87) (13,955) (536) (0.05) (22.04)
(2.95) (0.17–5) (11.81) (59.44) (60.90) (15,465) (2384) (0.10) (16.84)
(1.84) (0.17–4) (19.17) (77.41) (78.50) (18,968) (1393.46) (0.06) (28.65)
(4.07) (0.17–5) (46.50) (164.21) (164.63) (25,882) (1824) (0.04) (20.20)
(2.00) (0.5–4) (22.67) (71.22) (80.64) (50,158) (2397) (0.03) (18.97)
(4.16) (0.17–5) (140.25) (263.47) (260.39) (45,724) (2396) (0.10) (36.97)
(14.97) (0.33–4) (83.16) (175.71) (304.95) (47,692) (2153) (0.04) (17.76)
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Table 3 Main pharmacokinetic parameters of baicalin in healthy Chinese subjects after administration of a single dose of 100–2800 mg of baicalein. Values are the mean 7 SD for all parameters with the exception of tmax, which is the median (range). Parameter
100 mg
t1/2 (h) Tmax (h) Cmax (ng/mL) AUC0-t ( ng/mL h) AUC0-1 (ng/mL h) Vd/F (L) CLz/F (L/h) U_Recovered (mg) F_Recovered (mg)
8.58 1.00 75.47 556.44
200 mg
400 mg
800 mg
(5.06) 4.22 (0.42) 4.56 (0.5–4) 3 (0.33–5) 2.75 (26.43) 182.00 (90.53) 234.82 (136.54) 979.65 (369.72) 1314.66
(1.71) 4.84 (0.33–5) 1.5 (140.48) 428.58 (760.59) 3853.07
1200 mg (1.62) 10.80 (0.33–4) 0.5 (156.88) 770.00 (1756.89) 4887.99
580.24 (135.69) 982.69 (369.70) 1322.10 (766.82) 3877.79 (1777.29)
1600 mg
(4.97) 8.64 (0.33–2) 1.5 (749.68) 635.85 (2581.04) 3838.56
5177.42 (2586.50)
2200 mg
2800 mg
(2.59) 5.87 (2.85) 9.65 (0.5–4) 0.75 (0.5–5) 1.25 (230.74) 2264.50 (3022.58) 1847.21 (1186.55) 7391.79 (5118.18) 10,349.15
3917.97 (1181.40) 7500.35 (5106.65)
(5.78) (0.5–4) (1269.03) (3319.71)
11,961.53 (6016.22)
3692 (2588) 299 (74) 1.32 (0.58)
2298 (855) 377 (134) 2.37 (0.68)
4234 (2879) 661 (348) 3.37 (1.33)
2733 (1088) 423 (228) 8.08 (3.88)
6841 (3249) 447 (160) 11.58 (7.00)
8831 (3004) 718 (169) 8.81 (1.78)
7279 (7445) 725 (468) 15.09 (6.85)
5247 (515) 465 (202) 22.76 (7.56)
0.04 (0.04)
0.86 (0.97)
5.61 (2.97)
4.44 (3.83)
1.44 (3.00)
6.48 (6.07)
36.43 (58.38)
7.21 (8.14)
with its metabolite in feces are in accordance with the hypothesis that baicalein undergoes enterohepatic recirculation (Xing et al., 2005). This phenomenon has been reported to complicate absorption and recycling, and affect the PK of certain drugs by influencing t1/2, the area under the plasma concentration–time curve, and bioavailability (Tsai et al., 2000). Studies have indicated that baicalein is well absorbed from the stomach and small intestine with limited absorption from the colon, whereas baicalin is moderately absorbed in the stomach but poorly absorbed from the small intestine and colon. These two moieties maintain a balance of systemic levels in the body through dynamic reconversion. However, it was concluded that baicalein was the preferred species for oral absorption because of a more rapid and complete absorption of baicalein and restoration of baicalin in the systemic circulation by the hydrolysis of baicalein. Circulating levels of baicalin would be expected to re-enter the gastrointestinal tract via biliary excretion (Taiming and Xueha, 2006). In contrast to the relative low systemic levels of the parent compound baicalein, its most abundant metabolite conjugate, baicalin, was present in plasma at a Cmax concentration range of 0.075–1.8 μmol L–1. The neuroprotective effect in rodent models is thought to be mediated primarily via the parent compound. The extensive glucuronidation and sulfation of baicalein with consequent poor bioavailability of the parent compound, as described here and previously, calls into question the role of the metabolite baicalin in the mediation of efficacy. The pharmacologic properties of most baicalein conjugates are not known, but baicalin (the 7βglucuronidase-catalyzed conjugate) has been suggested to contribute to the pharmacologic activity of the parent molecule (Srinivas, 2010). In hepatoma cell lines, baicalin can induce apoptosis or inhibit proliferation (Chang et al., 2002). Baicalin also seems to retain (at least in part) the antioxidant properties of the parent molecule (Shieh et al., 2000). Therefore, the exposure of both baicalein and baicalin in the human body was studied. Preclinical data showed that baicalein is metabolized predominatly by glucuronidation via uridine 50 -diphospho-glucuronosyltransferase systems. The urinary excretion of unchanged baicalein was extremely small. Of all the administered oral doses, E0.7% baicalein was excreted unchanged in urine and 27.1% excreted unchanged in feces within 48 h. These findings suggested that the drug might be excreted mainly in a metabolized form. The complete metabolic profile of baicalein in humans is still under investigation. Enrolling female subjects early in the drug-development process is important for the assessment of sex-related differences in PK. The PK of baicalein after administration of a single dose was comparable in female and male subjects. Considering our small
study cohort, however, data from more subjects will be required to confirm the absence of sex differences. Baicalein administration was safe and well tolerated by healthy subjects from 100 mg to 2800 mg, with a low number of AEs being reported. The safety assessments used in our study based on preclinical safety data were, in general, recognized to be reliable and relevant for a Phase I study. The study included routine monitoring of subjects for clinical signs, hematology parameters, liver function tests (alanine transaminase, aspartate aminotransferase, and gamma-glutamyl transferase), kidney function tests (blood, urea, nitrogen; creatine), ECG and urinalyses. No risks were identified according to evaluation of vital signs, ECG, or physical examination and there appeared to be no effect on the QTc interval. Adverse gastrointestinal events such as constipation and abdominal distention were expected due to disruption to intestinal microflora. Dry cough and hemolytic anemia, which occurred in preclinical toxicity tests, were not observed in clinical studies. All reported AEs were transient and mild in intensity. There was no evidence of a dose-related increase in AEs upon baicalein administration.
5. Conclusions Multiple peaks on the plasma concentration–time curves for baicalein and baicalin were found at all doses tested. Dose proportionality was inconclusive in the dose range 100– 2800 mg. Single oral doses of 100–2800 mg of baicalein were safe and well tolerated by healthy subjects. Clinical laboratory assessments showed no signs of toxicity in the liver or kidney. The favorable safety profile and PK properties warrant further clinical studies for baicalein.
Funding sources National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Twelfth Five-year Plan Period (2012ZX09303-008-002, http://www.nmp.gov.cn/).
Acknowledgment This study was financially supported by Kanion pharmaceutical Limited Corporation (CTR20132944) (Jiangsu, P.R. China). We are grateful for the following persons for their assistance in the study: Jiamin Chi, Huili Liu, Jianhua Yang, Huihong Qi, Panpan Xie, Taifeng Li, Yi Jin, Chunxiao Li, Wenjing Hou, and Kongcai Zhu.
M. Li et al. / Journal of Ethnopharmacology 156 (2014) 210–215
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