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Pharmacokinetics, safety, and tolerability of single and multiple-doses of pinocembrin injection administered intravenously in healthy subjects
Journal of Ethnopharmacology 168 (2015) 31–36
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Pharmacokinetics, safety, and tolerability of single and multiple-doses of pinocembrin injection administered intravenously in healthy subjects Guoying Cao a, Pengyue Ying c, Bei Yan a, Wei Xue a, Kexin Li a, Aixin Shi a, Taohua Sun b, Jiling Yan c, Xin Hu a,n a b c
Department of Clinical Pharmacology, Beijing Hospital, Beijing 100730, China School of Pharmaceutical Sciences, Shandong University, Jinan 250100, Shandong, China Zhongqi Pharmaceutical Technology (Shijiazhuang) Co. Ltd., Shijiazhuang 050051, China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 December 2014 Received in revised form 9 March 2015 Accepted 13 March 2015 Available online 23 March 2015
Ethnopharmacological relevance: Pinocembrin is the most abundant flavonoid in propolis. Preclinical studies have suggested that pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia–reperfusion both in vivo and in vitro. To investigate the safety, tolerability and pharmacokinetics of a new neuroprotective agent, pinocembrin. Materials and method: A double-blind, placebo-controlled, randomized study was carried out in 58 healthy subjects. Single ascending doses of pinocembrin (20–150 mg) were evaluated in 5 cohorts. Multidose was studied at pinocembrin 60 mg. Results: Pinocembrin was well tolerated. No serious adverse events occurred. No subjects were discontinued because of a treatment emergent AE. Treatment related adverse event was acute urticaria. Two subjects in 150 mg cohort developed grade II urticaria during the study. One subject discontinued after 3 days at 60 mg bid because of diarrhea. In the single-dose study, the mean peak plasma pinocembrin concentration was obtained at the end of the 30-min infusion. The Cmax ranged from 0.28 μg mL 1 to 2.46 μg mL 1. AUC (0,1) ranged from 10.34 μg mL 1 min to 89.34 μg mL 1 min. The T1/2 was similar across 5 dose groups, ranging from 40 to 55 min. Both urinary and feces excretion levels of pinocembrin were extremely low and similar among each dose groups, with mean values ranging from 0.07% to 0.17% and 0.94% to 1.94% of the administered dose, respectively. Linear increases in Cmax and AUC(0,1) were observed. The pharmacokinetics of pinocembrin in multiple-dose was similar to those observed in the single-dose study, with no evidence of accumulation. Both urinary and feces excretion levels of pinocembrin were extremely low. Conclusions: Pinocembrin displayed linear plasma pharmacokinetics over the dose range, 20–150 mg and was well tolerated up to 120 mg day 1 when administered intravenously to healthy adults. No major safety concerns were identified that would preclude further clinical development of pinocembrin injection. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pinocembrin Pharmacokinetics Phase I Safety Tolerability
1. Introduction Pinocembrin is the most abundant flavonoid in propolis. It was reported to have multiple actions including anti-platelet aggregant (Jantan et al., 2008), anti-angiogenic activities (Ahn et al., 2009), anti-inflammatory (Soromou et al., 2012), anti-oxidant (Kapoor, 2013; Santos et al., 1998; Yu et al., 2009), and antimicrobial (Paintz and Metzner, 1979). Recently, preclinical studies have suggested pinocembrin protects rat brain against oxidation and apoptosis
n
Corresponding author.
http://dx.doi.org/10.1016/j.jep.2015.03.041 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
induced by ischemia–reperfusion both in vivo and in vitro(Gao et al., 2008; Liu et al., 2008; Rasul et al., 2013; Shi et al., 2011; Soromou et al., 2012; Wang et al., 2013; Wu et al., 2013). Using a permanent focal cerebral ischemia rat model, pinocembrin improved regional cerebral blood flow and reduced the postischemic damage to the neurovascular unit at the dose level of 3, 10, and 30 mg kg 1. Pharmacokinetics, tissue distribution and vitro metabolism have been evaluated for pinocembrin in rat and dog (Yang et al., 2009). The major findings are summarized as follows, single injection doses (67.5, 22.5 and 7.5 mg kg 1) of pinocembrin in rats, displayed a linear dose-exposure profile at three doses. The half-life of the parent drug
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G. Cao et al. / Journal of Ethnopharmacology 168 (2015) 31–36
was 13.9, 14.6 and 7.3 min. Following a single injection of 22.5 mg kg 1 pinocembrin to rats, 0.1%, 2.1% and 26.3% of intravenous dose were recovered in bile, urine and feces respectively, indicated metabolism clearance was the primary route of excretion. After multiple injection doses (80 mg kg 1) to dogs, there was a dose under-proportional increase in exposure and accumulation. Based on our vitro metabolism study, pinocembrin showed a potent inhibition of CYP1A2 with IC50 values of 0.82 μM. Moderate inhibitions of CYP2C9 and CYP2C19 with IC50 values were 13.1 and 22.3 μM, respectively. Very little or no inhibition of CYP 2D6 and 3A4/5 was observed. No evidence of enzyme induction was detected in vitro. The metabolism of pinocembrin involves many enzymes, including CYP and UGT systems. However, overall UGT enzymes appear to play a major role in pinocembrin metabolism. The metabolism involves many enzymes, including CYP1A2, 2C9, CYP2E1 and CYP3A4 systems (not published). This report describes the pharmacokinetic, safety, and tolerability results from a phase I, randomized, double blind, and placebo-controlled study conducted with healthy human subjects to explore escalating once-daily single and multiple-dose administration of pinocembrin injection. 2. Methods 2.1. Study population This study was conducted according to the ethical principles of the Declaration of Helsinki, the International Conference on Harmonization of Good Clinical Practice Guideline and the Guideline for Good Clinical Practice recommended by the State Food and Drug Administration (SFDA) of China. The study protocol and the protocol amendments, as well as the informed consent form, the subject recruitment materials, and the investigator's brochure were reviewed and approved by the ethics committee of Beijing Hospital (Ethical approval record number is “2012L02508”). Healthy subjects were recruited by the phase I clinical trial site at Beijing Hospital (Beijing, China) through the clinical site database. After signing informed consent forms, subjects underwent clinical examinations. Electrocardiogram and clinical chemistry were performed 2 to 7 days before admitting to the Clinical Research Center. Criteria for eligibility included healthy male and female adults, aged 18–40 years, who were nonsmokers, and had a body mass index (BMI) between 19 and 25. All female subjects had negative pregnancy test results at screening. Male subjects had to use a two acceptable methods of contraception for the entire duration of the study, up to the study completion visit. Other exclusion criteria were as follows: any disease or condition that might interfere with the absorption, distribution, metabolism, or excretion of the study drug, a history of drug or alcohol abuse, blood donation ( more than 400 mL) within the past 8 weeks, consumption of other prescribed or over the counterdrugs (vitamins or calcium supplements allowed) within 4 weeks before the study, participation in a similar study within the past 4 weeks, and a history of immunodeficiency disease, including a positive HIV test result, or a positive hepatitis B surface antigen or hepatitis C antibody. These criteria were confirmed by blood testing and patient reports. Routine clinical chemistry tests consisted of hemoglobin, hematocrit, total white blood cell count, blood glucose, triglycerides, total cholesterol, albumin, direct and indirect bilirubin, creatinine, AST, alkaline phosphates, lactic dehydrogenase, potassium, and urinalysis 2.2. Drugs Pinocembrin, manufactured by Central Institute of Pharmaceutical Research Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. in 10 mg powder, with matching placebo.
2.3. Study design This was a phase I, randomized, double-blind, placebo-controlled, parallel-group, single-dose escalation and multiple-dose clinical study to evaluate and compare the safety and PK of pinocembrin compared to placebo in healthy subjects. The single-dose study was designed to enroll 46 subjects into 5 cohorts at dose levels of 20, 40, 80, 120, or 150 mg. The multiple-dose study involved one group of subjects receiving either study drug or placebo from day 0 for 5 consecutive days. In the single-dose study, doses of 20, 40, 80 mg were administered in 10 subjects, while doses of 120 and 150 mg were administered in 8 subjects. Subjects were randomly assigned to receive either pinocembrin injection (n ¼8 subjects in 20, 40, 80 mg and n ¼6 in 120, 150 mg) or placebo (0.9% normal saline in 100 ml; n¼ 2 subjects in each group) administered intravenously (IV) over a 30-min infusion and were monitored for 48 h after the infusion. The principal investigator reviewed all safety data to determine if dose limiting toxicity had occurred. Once the safety of the prior cohort had been determined, the study drug was administered to subjects in the next dose level cohort. Each subject participated in up to a 7-day screening period, 48 h treatment periods, and a study completion evaluation. In the multiple-dose study, 12 subjects were randomly assigned to receive either pinocembrin injection, 60 mg bid (n ¼10 subjects) or placebo (normal saline, n ¼ 2 subjects) on day 2, day 3, day 4 and a single morning dose on day 1 and day 5 for all cohorts. Eligible subjects were admitted to the Clinical Research Center the day before dosing. No other food and drink other than that specified in the protocol was consumed at any time during the study. On the morning of dosing day, subjects were served a breakfast at 7:00. At 8:30 subjects were administered the study medication by a 30-min infusion. Series pharmacokinetics blood samples, urine and feces samples were completed during the 48 h period for the single-dose and up to 5 days for the multiple-doses. 2.4. Safety monitoring Safety was assessed in both studies by vital signs, routine clinical chemistry, urinalysis, serum β2-microglobulin (β2-MG) and spontaneous reporting of adverse events (AEs) by the subjects. Heart rate and rhythm were monitored by dynamic electrocardioscanners prior to study drug administration, during infusion, and for 4 h postinfusion. AEs were recorded for the entire study duration. Investigators assessed all adverse events for severity, duration, outcome, and possible relationship to the study medication. Renal function was assessed from BUN, serum creatinine measurements, and serum β2-MG. AEs were graded according to the DAIDS [http://www.niaid.nih.gov/LabsAndResources/resources/ DAIDSClinRsrch/Documents/gclp.pdf]. 2.5. Pharmacokinetic evaluation In the single-dose study, blood samples were obtained for measurement of pinocembrin plasma levels as follows: pre-dose, 10 min, 20 min, 30 min during infusion time and 10 min, 20 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h after infusion. Urine samples were obtained at the following time intervals: predose and 0–2, 2–4, 4–8, 8–12, 12–24 and 24–48 h after infusion. In multiply dose study, blood samples were obtained from all subjects on day 1 and day 5, a full PK time course was obtained which included the following time points: pre-dose, 10 min, 20 min, 30 min during infusion time and 10 min, 20 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h and 8 h after the last administration intravenous infusion. Urine and feces from the different subjects
G. Cao et al. / Journal of Ethnopharmacology 168 (2015) 31–36
were collected and mixed from for the determination of urine and feces concentrations of pinocembrin. In each of the subjects, two i.v. lines were inserted (one in each forearm), one for the drug injection and the other for blood sampling. Pinocembrin or placebo was infused over the course of 30 min, using an i.v. infusion pump. 1 mL of 0.9% sodium chloride was used to flush the i.v. line after the infusion. Plasma was separated by centrifugation at 3000 g for 10 min at room temperature and stored at 80 1C until quantification. Urine was stored at 4 1C during the collection interval. Urine was weighed and a 10 mL aliquot was stored in a fresh container at 80 1C until analyzed. The remaining urine was discarded. Feces were collected quantitatively for pinocembrin concentration determination after study drug administration. Specimens were weighed and processed to form homogenates by adding an equal weight of Milli-Q water. Blender HR2094 (Phillip, Ltd., Netherlands ) was used to homogenize these samples, which were then weighed and stored at 80 1C until analysis. Pharmacokinetic profiles were evaluated during the study. These included maximum observed pinocembrin plasma concentration (Cmax), time to Cmax (tmax), terminal-phase half life (t1/2), area under the plasma concentration time curve from time 0 to time of last measurable concentration (AUC (0,8)) and area under the plasma concentration time curve from 0 time extrapolated to infinity (AUC (0,1)). All the concentrations below quantitative limit were substituted by a value of zero. 2.6. Analytic methods The quantitation was analyzed with electrospray mass spectrometry (Qtrap 5500 (AB SCIEX, USA) chromatograph equipped with a SIL-20ACXR autosampler (SHIMADZU, Japan). The analytical column was a Luna C8 (150 2.00 mm i.d., 3 mm, Phenomenex, USA) coupled with a C8 guard column (4 3 mm, Phenomenexs). The mobile phase, a mixture of acetonitrile/0.3 mM ammonium acetate solution (pH 2.5) (65:35, v/v), and the flow rate was 0.25 mL min 1. The column oven temperature was set at 40 1C. The entire acquisition time was 6 min. Solid-phase extraction (SPE) of pinocembrin from human plasma was performed using Waters Oasiss HLB cartridges. 500 μL of plasma were placed in a 1.5 mL polypropylene tube and 10 μL of the IS was added. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), all of the supernatant was transferred to a conditioned SPE cartridge. After the cartridge was washed with 1 ml methanol twice and 1 mL ultrapure water twice, the pinocembrin was eluted with 1.0 mL of methanol and 1 mL of 40% methanol–water (v/v). 5 μL of elusion was injected into the HPLC–MS–MS system for analysis. 200 μL of urine samples was mixed with 10 μL of the IS. The tubes were briefly vortex-mixed. They were extracted with 800 μL of ether. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), 600 μL of supernatant was evaporated at 40C1 and then reconstituted in 200 μL of methanol. 5 μL of solution was injected into the HPLC–MS–MS system for analysis. 200 μL of feces homogenates was placed in a 1.5 mL polypropylene tube and 10 μL of the IS was added. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), 15 μL of the supernatant, with 65% 785 μL of acetonitrile, were transferred to a 1.5 mL tube. After vortexmixing and centrifugation (10,000 g for 5 min), 5 μL of supernatant was injected into the HPLC–MS–MS system for analysis. The mass spectrometer was operated in electrospray negative ionization using multiple reaction monitoring (MRM) for the transitions of m/z 255.0-213.1 and m/z 314.0-278.1, for pinocembrin and the IS, respectively. The optimized parameters were as follows: ionspray voltage: 4500 V; curtain gas: 30 psi; ion source gas 1 (nebulizer gas): 55 psi; ion source gas 2 (heater gas): 60 psi; declustering potential: 100 V for pinocembrin and 80 V for IS; collision cell potential: 28 V for
33
pinocembrin and 38 V for IS; collision energy: 28 V for pinocembrin and 22 V for IS. Data acquisition and analysis were performed using the analyst software Analyst version 1.5.1., (Applied Biosystemss, USA). For plasma, the validated range of the assay for quantitation was 1–400 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 5.5% and intrabatch percent CV was within 4.5%. The interassay accuracy ranged between 98.2% and 99.1% and the intraassay accuracy ranged between 99.8% and 100.5%. For urine, the validated range of the assay for pinocembrin was 3–200 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 6.5% and intrabatch percent CV was within 5.0%. The inter-batch accuracy ranged between 98.1% and 100.5% and the intra-batch accuracy ranged between 98.2% and 99.8%. For feces, the validated range of the assay for pinocembrin was 30–4000 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 6.2% and intrabatch percent CV was within 5.1%. The interbatch accuracy ranged between 101.4% and 102.4% and the intrabatch accuracy ranged between 98.6% and 102.2%. 2.7. Pharmacokinetic data analysis PK parameter estimates for pinocembrin were calculated using standard noncompartmental methods of analysis (DAS version 2.0, The Center of Drug Evaluation, Anhui Province, P.R.C). Thus, maximum plasma drug concentration (Cmax) and time to Cmax (tmax) were taken directly from the observed plasma concentration data. The terminal-phase disposition rate constant (λz) was estimated using a log-linear regression of the concentration data in the terminal disposition phase, and terminal phase elimination. Halflife (t1/2) was estimated as ln(2)/λz. AUC(0,8), and AUC(0,1). AUC(0,1) was calculated as AUC(0,8) þ Ct/λz. Pinocembrin concentrations below quantitation limit were substituted by a value of zero. 2.8. Statistical analysis Descriptive statistics were used to summarize all baseline characteristics, treatment administration, and safety variables. The safety population included all subjects who were randomized and received any amount of study drug. The PK population included subjects who received pinocembrin administration and for whom at least one plasma PK variable was available. Dose dependency of Cmax and AUC(0,1) was assessed by the power model (point estimate and 95% confidence interval [95% CI] of the slope (b) of the regression formula (log (Y) ¼a þb log (X)). Dose proportionality was confirmed if the 95% CI of b included 1 and fell within the range of 0.7, 1.43.
3. Result 3.1. Study population The baseline demographics of the study participants are summarized in Table 1. Forty-six subjects were randomized to the single-dose study and all completed the study. There were no spastic differences in baseline characteristics between treatment groups. No subjects were excluded from the safety or the pharmacokinetic analysis populations. 3.2. Safety analysis Compliance was good in all volunteers. No severe or lifethreatening AE occurred. For single-dose study, pinocembrin was well tolerated at 5 treatment groups. No subject was discontinued because
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Table 1 Baseline demographic characteristics of the study population. Dose (mg)
Gender
20
2 (placebo) 8 (pinocembrin) 2 (placebo) 8 (pinocembrin) 2 (placebo) 8 (pinocembrin) 2 (placebo) 6 (pinocembrin) 2 (placebo) 6 (pinocembrin) 2 (placebo) 10 (pinocembrin)
40 80 120 150 60a
a
Male
Female
1 3 0 6 2 5 0 5 1 5 2 6
1 5 2 2 0 3 2 1 1 1 0 4
Mean age (yr range)
Mean weight (kg range)
Mean BMI (range)
30.0 30.2 29.0 26.1 27.5 28.2 29.5 29.8 23.5 28 24.5 26
61.5 58.9 50.2 61.1 66.0 59.7 53.3 61.6 54.5 60.2 62.4 60.8
22.6 23.1 20.4 21.8 22.8 21.7 22–8 21.6 21.5 21.6 23.1 21.5
(26–34) (22–36) (24–34) (20–35) (23–32) (19–36) (29–30) (22–35) (21–26) (22–40) (28–21) (21–36)
(51–72) (52–66) (57–46) (55–69) (64–72) (47–64) (51.7–54.6) (55–68) (52.5–56.4) (54.6–64.8) (61.5–63.2) (52.0–71.0)
(19.8–23.8) (22.4–23.8) (20.4–20.4) (19.4–23.6) (22.1–23.5) (19.9–23.4) (20.3–23.3) (19.8–23.8) (19.8–23.2) (19.6–23.9) (23.9–22.3) (19.5–24.0)
Multiple-dose.
Table 2 Summary of adverse events after intravenous administration of pinocembrin to healthy subject at doses of 20–150 mg once daily and 60 mg twice daily. Adverse events
No. of subjects (%)
Any events β2-MG elevation Urine microprotein Acute urticaria Diarrhea a
Placebo (%)
20 mg (%)
2 (16.7) 1 (8.3) 1 (8.3)
2 (25 ) 1 (12.5) 1 (12.5)
40 mg (%)
80 mg (%)
120 mg (%)
150 mg (%)
60 mga (%)
2 (25) 1 (12.5) 1 (12.5)
1 (16.7) 1 (16.7)
2 (33.3)
1 (10)
2 (33.3) 1 (10)
Multiple-dose.
Table 3 Pharmacokinetics of pinocembrin at dose levels of 20–150 mg after intravenous infusion. Parameters 1
AUC(0,8) (μg mL min) AUC(0,1) (μg mL 1 min) MRT(0,8) (min) MRT(0,1) (min) T1/2 (min) Tmax (min) Vz (L) CLz (L min 1) Cmax (μg mL 1)
20 mg
40 mg
80 mg
120 mg
150 mg
10.247 1.5 10.34 7 1.5 40.0 7 4.8 42.7 7 5.7 47.4 7 14.0 28.0 7 4.0 136.6 7 52.8 1.98 7 0.3 0.28 7 0.1
19.8 7 2.5 20.0 7 2.5 37.8 7 4.4 39.4 7 4.5 40.6 7 8.1 28.0 7 5.0 119.9 7 32.6 2.03 7 0.3 0.6 7 0.1
43.86 7 10.3 44.0 7 10.6 38.5 7 5.7 39.6 7 6.0 55.7 7 24.6 28.0 7 7.0 149.57 0.1 1.93 7 0.6 1.22 7 0.3
59.1 7 12.9 59.3 7 12.9 38.0 7 5.6 38.9 7 6.2 55.8 7 19.0 27.0 7 5.0 174.6 7 90.3 2.1 7 0.5 1.63 7 0.4
89.217 13.5 89.34 7 13.5 38.3 7 4.9 39.0 7 5.0 54.5 7 13.7 22.0 7 8.0 136.6 7 45.5 1.727 0.3 2.46 7 0.4
of a treatment emergent AE. All 46 subjects received at least one dose of the study drug and were included in the safety analysis. AEs were reported by 11 of 46 subjects, including 2 (20%) who received control infusions and 9 of 36 (25%) who received pinocembrin. The most commonly observed AE were acute urticaria, β2-MG elevation, urine microprotein (mAlb), β2-MG elevation and urine mAlb occurred in both pinocembrin and placebo-treated subjects. Skin acute urticaria occurred only in pinocembrin injection subjects. Urinalysis for renal damage was via BUN, serum creatinine, serum β2-MG and urine mAlb. In the present studies, BUN and serum creatinine remained within the normal limits throughout the treatment period. However, three subjects (two pinocembrin subjects and one placebo subject) had a mild elevation of serum β2-MG. Three subjects (two pinocembrin subjects and one placebo subject) had urine mAlb. PI thinks they were attributed to laboratory testing variability. Two subjects enrolled in 150 mg cohort developed grade II skin acute urticaria during the study. Urticaria was found in chest, eyes. It occurred 1.5–2.0 h after dosing and resolved within 3 h without any specific intervention. This event was not considered related to the study drug. The AE was deemed mild by the investigator and suspected to be related to study medication.
The PI and the sponsor think these AEs raise the safety concern and agreed to discontinue of the study. The preceding dose level, 120 mg, is defined as the maximum tolerated dose. In the multiple-dose study, pinocembrin was similarly welltolerated. One AE occurred in pinocembrin group. The subject was discontinued from the study because of grade II diarrhea, which occurred on the third day of the multiple-dose and recovered within 24 h. Diarrhea was deemed not suspected to be related to study medication and resolved without any treatment. Table 2 summarizes all AEs by body system, both in pinocembrin and placebo-treated groups. All the AEs were mild or moderate in intensity, none was characterized as serious or potentially life-threatening AEs occurred, and most resolved spontaneously without intervention. 3.3. Pharmacokinetic evaluation Data for all 36 subjects who received pinocembrin injection are included in the single-dose PK analyses. PK parameters are presented in Table 3. The mean peak plasma concentration was obtained at the end of the 30-min infusion (Fig. 1). The Cmax ranged from 0.28 μg mL 1
3000.00
Concentration (ng ml-1)
2500.00 2000.00 20 mg 1500.00
40 mg 80 mg
1000.00
120 mg 150 mg
500.00 0.00
0
100
200
300
400
0.30 0.25 20mg
0.20
40mg
0.15
80mg
0.10
120mg
0.05
150mg
0.00
0
5
10
15
20
25
30
35
40
45
50
Time (minute)
Fig. 1. Graph of mean d pinocembrin concentration versus time plots at the 20–150 mg dose levels in the single dose study.
Fig. 3. The mean pinocembrin urine concentrations at 20–150 mg dose levels after a single 30-min intravenous infusion.
Table 4 Pharmacokinetics of pinocembrin on day 1 and at the end of dosing period (day 5) at dose level of 60 mg twice daily.
120.00 100.00 AUCinf (μg mL-1 min)
35
0.35
500
Time (minute)
80.00 60.00 40.00 20.00 0.00
Cumulative fraction of pinocembrin
G. Cao et al. / Journal of Ethnopharmacology 168 (2015) 31–36
0
50
100
150
200
Parameters
Day 1
Day 5
AUC(0,8) (μg mL 1 min) AUC(0,1) (μg mL 1 min) MRT(0,8) (min) MRT(0,1) (min) T1/2 (min) Tmax (min) Vz (L) CLz (L min 1) Cmax (μg mL 1)
32.117 2.6 32.217 2.5 38.7 7 2.5 39.3 7 2.5 39.7 7 6.9 26.0 7 7.0 106.57 14.3 1.87 7 0.14 0.86 7 0.09
30.737 6.06 30.82 7 6.07 39.17 7.2 40.2 7 7.4 50.3 7 12.3 28.0 7 4.0 145.6 7 44.1 2.02 7 0.41 0.82 7 0.13
Dose (mg)
Pinocembrin plasma pharmacokinetics parameters in multipledose study are presented in Table 4. There were no significant changes in observed parameters on day 5 versus day 1. On average, 0.11% and 1.86% of the administered dose was recovered in urine and feces as unchanged pinocembrin during the 7 study period. Both urinary and feces excretion levels of pinocembrin were extremely low and similar to those observed in the singledose study.
3.50
Cmax (ng ml -1 )
3.00 2.50 2.00 1.50 1.00 0.50 0.00
4. Discussion 0
50
100
150
200
Dose (mg) Fig. 2. Pinocembrin AUC (0,1) (y ¼0.577x 2.691; R2 ¼ 0.896) (A) and Cmax (y¼ 0.015x 0.078; R2 ¼ 0.889) (B) versus dose (mg) following a once-daily 30-min IV infusion of pinocembrin injection in 36 healthy subjects.
in the 20 mg group to 2.46 μg mL 1 in the 150 mg group. AUC(0,1) ranged from 10.34 μg mL 1 min to 89.34 μg mL 1 min. The elimination half-lives were similar across five dose groups, ranging from 40 to 60 min. Clearances were also similar across all dose groups, ranging from 1.72 to 2.10 L min 1. Dose dependency of Cmax and AUC(0,1) (20–150 mg) was assessed. Both Cmax and AUC(0,1) met the dose proportionality criteria (Fig. 2). The mean pinocembrin urine concentrations after a single 30-min intravenous infusion reached the highest in the first two post-dose collection intervals (0–2 and 2–4 h, Fig. 3). Both urinary and feces excretion levels of pinocembrin were extremely low and similar among each dose groups, with mean values ranging from 0.07% to 0.17% and 0.94% to 1.94% of the administered dose, respectively.
This report describes the first-in-human studies of pinocembrin following single and multiple intravenous dose administrations. Pinocembrin was well tolerated by the healthy volunteers; all adverse events were mild to moderate and reversible. No clinically relevant changes in renal and hepatic indices from baseline were observed. In addition to assessing safety, our study represented the first evaluation of the pharmacokinetics of pinocembrin in healthy male and female subjects. When pinocembrin was intravenously administered to healthy subjects at doses of 20–150 mg, the Cmax and AUC(0,1) were proportional to dose, with a rapid Tmax and a relatively short plasma T1/2 following administered pinocembrin injection. Across all dose groups, the single-dose and the multipledose PK look similar, with no evidence of accumulation. No evidence of dose-dependent clearance is apparent within the pinocembrin injection doses tested. Clearance is most likely to occur predominantly through metabolism. Urinary excretion of the unchanged compound was o1%. In the metabolism study of pinocembrin on average, more than 80% of the administered doses were metabolized by hepatic phase II, and the metabolites were not found in feces (which is done in our lab and not published).
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The present study characterized urinary and fecal elimination in humans based on the administration of a nonradiolabeled dose. Given the low proportion of drug recovered in urine and feces, an extensive study period with radio labeled drug would have been required to better characterize pharmacokinetics of pinocembrin in humans. However, such a study would not be approved by IRB because of ethics concerns in China. Despite this limitation, the present results together with that from preclinical studies in rats following the administration of 14C-labeled doses can help to understand the distribution, metabolism, and elimination of pinocembrin in humans. 5. Conclusion The results indicate that pinocembrin injection given in singledose up to 120 mg, and 60 mg bid, were generally well tolerated. No major safety concerns were identified that would preclude further clinical development of pinocembrin injection. Acknowledgment We thank Zhongqi Pharmaceutical Technology Corporation Ltd. for kindly providing the pinocembrin formulations. Staff at the clinical site declares that they have no financial relationship with Zhongqi Pharmaceutical Technology Corporation Ltd. The authors have indicated they have no conflicts of interests regarding the contents of this paper. References Ahn, M.R., Kunimasa, K., Kumazawa, S., Nakayama, T., Kaji, K., Uto, Y., Hori, H., Nagasawa, H., Ohta, T., 2009. Correlation between antiangiogenic activity and antioxidant activity of various components from propolis. Mol. Nutr. Food Res. 53, 643–651. Gao, M., Liu, R., Zhu, S.Y., Du, G.H., 2008. Acute neurovascular unit protective action of pinocembrin against permanent cerebral ischemia in rats. J. Asian Nat. Prod. Res. 10, 551–558.
Jantan, I., Raweh, S.M., Sirat, H.M., Jamil, S., Mohd Yasin, Y.H., Jalil, J., Jamal, J.A., 2008. Inhibitory effect of compounds from Zingiberaceae species on human platelet aggregation. Phytomedicine 15, 306–309. Kapoor, S., 2013. Comment on isolation and identification of compounds from Penthorum chinense Pursh with antioxidant and antihepatocarcinoma properties: pinocembrin and its rapidly emerging neuroprotective effects. J. Agric. Food. Chem. 61, 1416. Liu, R., Gao, M., Yang, Z.H., Du, G.H., 2008. Pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia–reperfusion both in vivo and in vitro. Brain Res. 1216, 104–115. Paintz, M., Metzner, J., 1979. On the local anaesthetic action of propolis and some of its constituents. Pharmazie 34, 839–841. Rasul, A., Millimouno, F.M., Ali Eltayb, W., Ali, M., Li, J., Li, X., 2013. Pinocembrin: a novel natural compound with versatile pharmacological and biological activities. BioMed Res. Int. 2013, 379850. Santos, A.C., Uyemura, S.A., Lopes, J.L., Bazon, J.N., Mingatto, F.E., Curti, C., 1998. Effect of naturally occurring flavonoids on lipid peroxidation and membrane permeability transition in mitochondria. Free Radic. Biol. Med. 24, 1455–1461. Shi, L.L., Qiang, G.F., Gao, M., Zhang, H.A., Chen, B.N., Yu, X.Y., Xuan, Z.H., Wang, Q.Y., Du, G.H., 2011. Effect of pinocembrin on brain mitochondrial respiratory function. Acta Pharm. Sin. 46, 642–649. Soromou, L.W., Chu, X., Jiang, L., Wei, M., Huo, M., Chen, N., Guan, S., Yang, X., Chen, C., Feng, H., Deng, X., 2012. In vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses. Int. Immunopharmacol. 14, 66–74. Wang, S.B., Pang, X.B., Gao, M., Fang, L.H., Du, G.H., 2013. Pinocembrin protects rats against cerebral ischemic damage through soluble epoxide hydrolase and epoxyeicosatrienoic acids. Chin. J. Nat. Med. 11, 207–213. Wu, C.X., Liu, R., Gao, M., Zhao, G., Wu, S., Wu, C.F., Du, G.H., 2013. Pinocembrin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress induced apoptosis. Neurosci. Lett. 546, 57–62. Yang, Z., Liu, R., Li, X., Tian, S., Liu, Q., Du, G., 2009. Development and validation of a high-performance liquid chromatographic method for determination of pinocembrin in rat plasma: application to pharmacokinetic study. J. Pharm. Biomed. Anal. 49, 1277–1281. Yu, Y.S., Hsu, C.L., Yen, G.C., 2009. Anti-inflammatory effects of the roots of Alpinia pricei Hayata and its phenolic compounds. J. Agric. Food Chem. 57, 7673–7680. 〈http://www.niaid.nih.gov/LabsAndResources/resources/DAIDSClinRsrch/Docu ments/gclp.pdf〉.
Contents lists available at ScienceDirect
Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Pharmacokinetics, safety, and tolerability of single and multiple-doses of pinocembrin injection administered intravenously in healthy subjects Guoying Cao a, Pengyue Ying c, Bei Yan a, Wei Xue a, Kexin Li a, Aixin Shi a, Taohua Sun b, Jiling Yan c, Xin Hu a,n a b c
Department of Clinical Pharmacology, Beijing Hospital, Beijing 100730, China School of Pharmaceutical Sciences, Shandong University, Jinan 250100, Shandong, China Zhongqi Pharmaceutical Technology (Shijiazhuang) Co. Ltd., Shijiazhuang 050051, China
art ic l e i nf o
a b s t r a c t
Article history: Received 20 December 2014 Received in revised form 9 March 2015 Accepted 13 March 2015 Available online 23 March 2015
Ethnopharmacological relevance: Pinocembrin is the most abundant flavonoid in propolis. Preclinical studies have suggested that pinocembrin protects rat brain against oxidation and apoptosis induced by ischemia–reperfusion both in vivo and in vitro. To investigate the safety, tolerability and pharmacokinetics of a new neuroprotective agent, pinocembrin. Materials and method: A double-blind, placebo-controlled, randomized study was carried out in 58 healthy subjects. Single ascending doses of pinocembrin (20–150 mg) were evaluated in 5 cohorts. Multidose was studied at pinocembrin 60 mg. Results: Pinocembrin was well tolerated. No serious adverse events occurred. No subjects were discontinued because of a treatment emergent AE. Treatment related adverse event was acute urticaria. Two subjects in 150 mg cohort developed grade II urticaria during the study. One subject discontinued after 3 days at 60 mg bid because of diarrhea. In the single-dose study, the mean peak plasma pinocembrin concentration was obtained at the end of the 30-min infusion. The Cmax ranged from 0.28 μg mL 1 to 2.46 μg mL 1. AUC (0,1) ranged from 10.34 μg mL 1 min to 89.34 μg mL 1 min. The T1/2 was similar across 5 dose groups, ranging from 40 to 55 min. Both urinary and feces excretion levels of pinocembrin were extremely low and similar among each dose groups, with mean values ranging from 0.07% to 0.17% and 0.94% to 1.94% of the administered dose, respectively. Linear increases in Cmax and AUC(0,1) were observed. The pharmacokinetics of pinocembrin in multiple-dose was similar to those observed in the single-dose study, with no evidence of accumulation. Both urinary and feces excretion levels of pinocembrin were extremely low. Conclusions: Pinocembrin displayed linear plasma pharmacokinetics over the dose range, 20–150 mg and was well tolerated up to 120 mg day 1 when administered intravenously to healthy adults. No major safety concerns were identified that would preclude further clinical development of pinocembrin injection. & 2015 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pinocembrin Pharmacokinetics Phase I Safety Tolerability
1. Introduction Pinocembrin is the most abundant flavonoid in propolis. It was reported to have multiple actions including anti-platelet aggregant (Jantan et al., 2008), anti-angiogenic activities (Ahn et al., 2009), anti-inflammatory (Soromou et al., 2012), anti-oxidant (Kapoor, 2013; Santos et al., 1998; Yu et al., 2009), and antimicrobial (Paintz and Metzner, 1979). Recently, preclinical studies have suggested pinocembrin protects rat brain against oxidation and apoptosis
n
Corresponding author.
http://dx.doi.org/10.1016/j.jep.2015.03.041 0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
induced by ischemia–reperfusion both in vivo and in vitro(Gao et al., 2008; Liu et al., 2008; Rasul et al., 2013; Shi et al., 2011; Soromou et al., 2012; Wang et al., 2013; Wu et al., 2013). Using a permanent focal cerebral ischemia rat model, pinocembrin improved regional cerebral blood flow and reduced the postischemic damage to the neurovascular unit at the dose level of 3, 10, and 30 mg kg 1. Pharmacokinetics, tissue distribution and vitro metabolism have been evaluated for pinocembrin in rat and dog (Yang et al., 2009). The major findings are summarized as follows, single injection doses (67.5, 22.5 and 7.5 mg kg 1) of pinocembrin in rats, displayed a linear dose-exposure profile at three doses. The half-life of the parent drug
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was 13.9, 14.6 and 7.3 min. Following a single injection of 22.5 mg kg 1 pinocembrin to rats, 0.1%, 2.1% and 26.3% of intravenous dose were recovered in bile, urine and feces respectively, indicated metabolism clearance was the primary route of excretion. After multiple injection doses (80 mg kg 1) to dogs, there was a dose under-proportional increase in exposure and accumulation. Based on our vitro metabolism study, pinocembrin showed a potent inhibition of CYP1A2 with IC50 values of 0.82 μM. Moderate inhibitions of CYP2C9 and CYP2C19 with IC50 values were 13.1 and 22.3 μM, respectively. Very little or no inhibition of CYP 2D6 and 3A4/5 was observed. No evidence of enzyme induction was detected in vitro. The metabolism of pinocembrin involves many enzymes, including CYP and UGT systems. However, overall UGT enzymes appear to play a major role in pinocembrin metabolism. The metabolism involves many enzymes, including CYP1A2, 2C9, CYP2E1 and CYP3A4 systems (not published). This report describes the pharmacokinetic, safety, and tolerability results from a phase I, randomized, double blind, and placebo-controlled study conducted with healthy human subjects to explore escalating once-daily single and multiple-dose administration of pinocembrin injection. 2. Methods 2.1. Study population This study was conducted according to the ethical principles of the Declaration of Helsinki, the International Conference on Harmonization of Good Clinical Practice Guideline and the Guideline for Good Clinical Practice recommended by the State Food and Drug Administration (SFDA) of China. The study protocol and the protocol amendments, as well as the informed consent form, the subject recruitment materials, and the investigator's brochure were reviewed and approved by the ethics committee of Beijing Hospital (Ethical approval record number is “2012L02508”). Healthy subjects were recruited by the phase I clinical trial site at Beijing Hospital (Beijing, China) through the clinical site database. After signing informed consent forms, subjects underwent clinical examinations. Electrocardiogram and clinical chemistry were performed 2 to 7 days before admitting to the Clinical Research Center. Criteria for eligibility included healthy male and female adults, aged 18–40 years, who were nonsmokers, and had a body mass index (BMI) between 19 and 25. All female subjects had negative pregnancy test results at screening. Male subjects had to use a two acceptable methods of contraception for the entire duration of the study, up to the study completion visit. Other exclusion criteria were as follows: any disease or condition that might interfere with the absorption, distribution, metabolism, or excretion of the study drug, a history of drug or alcohol abuse, blood donation ( more than 400 mL) within the past 8 weeks, consumption of other prescribed or over the counterdrugs (vitamins or calcium supplements allowed) within 4 weeks before the study, participation in a similar study within the past 4 weeks, and a history of immunodeficiency disease, including a positive HIV test result, or a positive hepatitis B surface antigen or hepatitis C antibody. These criteria were confirmed by blood testing and patient reports. Routine clinical chemistry tests consisted of hemoglobin, hematocrit, total white blood cell count, blood glucose, triglycerides, total cholesterol, albumin, direct and indirect bilirubin, creatinine, AST, alkaline phosphates, lactic dehydrogenase, potassium, and urinalysis 2.2. Drugs Pinocembrin, manufactured by Central Institute of Pharmaceutical Research Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. in 10 mg powder, with matching placebo.
2.3. Study design This was a phase I, randomized, double-blind, placebo-controlled, parallel-group, single-dose escalation and multiple-dose clinical study to evaluate and compare the safety and PK of pinocembrin compared to placebo in healthy subjects. The single-dose study was designed to enroll 46 subjects into 5 cohorts at dose levels of 20, 40, 80, 120, or 150 mg. The multiple-dose study involved one group of subjects receiving either study drug or placebo from day 0 for 5 consecutive days. In the single-dose study, doses of 20, 40, 80 mg were administered in 10 subjects, while doses of 120 and 150 mg were administered in 8 subjects. Subjects were randomly assigned to receive either pinocembrin injection (n ¼8 subjects in 20, 40, 80 mg and n ¼6 in 120, 150 mg) or placebo (0.9% normal saline in 100 ml; n¼ 2 subjects in each group) administered intravenously (IV) over a 30-min infusion and were monitored for 48 h after the infusion. The principal investigator reviewed all safety data to determine if dose limiting toxicity had occurred. Once the safety of the prior cohort had been determined, the study drug was administered to subjects in the next dose level cohort. Each subject participated in up to a 7-day screening period, 48 h treatment periods, and a study completion evaluation. In the multiple-dose study, 12 subjects were randomly assigned to receive either pinocembrin injection, 60 mg bid (n ¼10 subjects) or placebo (normal saline, n ¼ 2 subjects) on day 2, day 3, day 4 and a single morning dose on day 1 and day 5 for all cohorts. Eligible subjects were admitted to the Clinical Research Center the day before dosing. No other food and drink other than that specified in the protocol was consumed at any time during the study. On the morning of dosing day, subjects were served a breakfast at 7:00. At 8:30 subjects were administered the study medication by a 30-min infusion. Series pharmacokinetics blood samples, urine and feces samples were completed during the 48 h period for the single-dose and up to 5 days for the multiple-doses. 2.4. Safety monitoring Safety was assessed in both studies by vital signs, routine clinical chemistry, urinalysis, serum β2-microglobulin (β2-MG) and spontaneous reporting of adverse events (AEs) by the subjects. Heart rate and rhythm were monitored by dynamic electrocardioscanners prior to study drug administration, during infusion, and for 4 h postinfusion. AEs were recorded for the entire study duration. Investigators assessed all adverse events for severity, duration, outcome, and possible relationship to the study medication. Renal function was assessed from BUN, serum creatinine measurements, and serum β2-MG. AEs were graded according to the DAIDS [http://www.niaid.nih.gov/LabsAndResources/resources/ DAIDSClinRsrch/Documents/gclp.pdf]. 2.5. Pharmacokinetic evaluation In the single-dose study, blood samples were obtained for measurement of pinocembrin plasma levels as follows: pre-dose, 10 min, 20 min, 30 min during infusion time and 10 min, 20 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h after infusion. Urine samples were obtained at the following time intervals: predose and 0–2, 2–4, 4–8, 8–12, 12–24 and 24–48 h after infusion. In multiply dose study, blood samples were obtained from all subjects on day 1 and day 5, a full PK time course was obtained which included the following time points: pre-dose, 10 min, 20 min, 30 min during infusion time and 10 min, 20 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h and 8 h after the last administration intravenous infusion. Urine and feces from the different subjects
G. Cao et al. / Journal of Ethnopharmacology 168 (2015) 31–36
were collected and mixed from for the determination of urine and feces concentrations of pinocembrin. In each of the subjects, two i.v. lines were inserted (one in each forearm), one for the drug injection and the other for blood sampling. Pinocembrin or placebo was infused over the course of 30 min, using an i.v. infusion pump. 1 mL of 0.9% sodium chloride was used to flush the i.v. line after the infusion. Plasma was separated by centrifugation at 3000 g for 10 min at room temperature and stored at 80 1C until quantification. Urine was stored at 4 1C during the collection interval. Urine was weighed and a 10 mL aliquot was stored in a fresh container at 80 1C until analyzed. The remaining urine was discarded. Feces were collected quantitatively for pinocembrin concentration determination after study drug administration. Specimens were weighed and processed to form homogenates by adding an equal weight of Milli-Q water. Blender HR2094 (Phillip, Ltd., Netherlands ) was used to homogenize these samples, which were then weighed and stored at 80 1C until analysis. Pharmacokinetic profiles were evaluated during the study. These included maximum observed pinocembrin plasma concentration (Cmax), time to Cmax (tmax), terminal-phase half life (t1/2), area under the plasma concentration time curve from time 0 to time of last measurable concentration (AUC (0,8)) and area under the plasma concentration time curve from 0 time extrapolated to infinity (AUC (0,1)). All the concentrations below quantitative limit were substituted by a value of zero. 2.6. Analytic methods The quantitation was analyzed with electrospray mass spectrometry (Qtrap 5500 (AB SCIEX, USA) chromatograph equipped with a SIL-20ACXR autosampler (SHIMADZU, Japan). The analytical column was a Luna C8 (150 2.00 mm i.d., 3 mm, Phenomenex, USA) coupled with a C8 guard column (4 3 mm, Phenomenexs). The mobile phase, a mixture of acetonitrile/0.3 mM ammonium acetate solution (pH 2.5) (65:35, v/v), and the flow rate was 0.25 mL min 1. The column oven temperature was set at 40 1C. The entire acquisition time was 6 min. Solid-phase extraction (SPE) of pinocembrin from human plasma was performed using Waters Oasiss HLB cartridges. 500 μL of plasma were placed in a 1.5 mL polypropylene tube and 10 μL of the IS was added. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), all of the supernatant was transferred to a conditioned SPE cartridge. After the cartridge was washed with 1 ml methanol twice and 1 mL ultrapure water twice, the pinocembrin was eluted with 1.0 mL of methanol and 1 mL of 40% methanol–water (v/v). 5 μL of elusion was injected into the HPLC–MS–MS system for analysis. 200 μL of urine samples was mixed with 10 μL of the IS. The tubes were briefly vortex-mixed. They were extracted with 800 μL of ether. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), 600 μL of supernatant was evaporated at 40C1 and then reconstituted in 200 μL of methanol. 5 μL of solution was injected into the HPLC–MS–MS system for analysis. 200 μL of feces homogenates was placed in a 1.5 mL polypropylene tube and 10 μL of the IS was added. After vortex-mixing for 30 s and centrifugation (10,000 g for 5 min), 15 μL of the supernatant, with 65% 785 μL of acetonitrile, were transferred to a 1.5 mL tube. After vortexmixing and centrifugation (10,000 g for 5 min), 5 μL of supernatant was injected into the HPLC–MS–MS system for analysis. The mass spectrometer was operated in electrospray negative ionization using multiple reaction monitoring (MRM) for the transitions of m/z 255.0-213.1 and m/z 314.0-278.1, for pinocembrin and the IS, respectively. The optimized parameters were as follows: ionspray voltage: 4500 V; curtain gas: 30 psi; ion source gas 1 (nebulizer gas): 55 psi; ion source gas 2 (heater gas): 60 psi; declustering potential: 100 V for pinocembrin and 80 V for IS; collision cell potential: 28 V for
33
pinocembrin and 38 V for IS; collision energy: 28 V for pinocembrin and 22 V for IS. Data acquisition and analysis were performed using the analyst software Analyst version 1.5.1., (Applied Biosystemss, USA). For plasma, the validated range of the assay for quantitation was 1–400 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 5.5% and intrabatch percent CV was within 4.5%. The interassay accuracy ranged between 98.2% and 99.1% and the intraassay accuracy ranged between 99.8% and 100.5%. For urine, the validated range of the assay for pinocembrin was 3–200 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 6.5% and intrabatch percent CV was within 5.0%. The inter-batch accuracy ranged between 98.1% and 100.5% and the intra-batch accuracy ranged between 98.2% and 99.8%. For feces, the validated range of the assay for pinocembrin was 30–4000 ng mL 1. The interbatch percent coefficient of variation (CV) for quality control (QC) levels was within 6.2% and intrabatch percent CV was within 5.1%. The interbatch accuracy ranged between 101.4% and 102.4% and the intrabatch accuracy ranged between 98.6% and 102.2%. 2.7. Pharmacokinetic data analysis PK parameter estimates for pinocembrin were calculated using standard noncompartmental methods of analysis (DAS version 2.0, The Center of Drug Evaluation, Anhui Province, P.R.C). Thus, maximum plasma drug concentration (Cmax) and time to Cmax (tmax) were taken directly from the observed plasma concentration data. The terminal-phase disposition rate constant (λz) was estimated using a log-linear regression of the concentration data in the terminal disposition phase, and terminal phase elimination. Halflife (t1/2) was estimated as ln(2)/λz. AUC(0,8), and AUC(0,1). AUC(0,1) was calculated as AUC(0,8) þ Ct/λz. Pinocembrin concentrations below quantitation limit were substituted by a value of zero. 2.8. Statistical analysis Descriptive statistics were used to summarize all baseline characteristics, treatment administration, and safety variables. The safety population included all subjects who were randomized and received any amount of study drug. The PK population included subjects who received pinocembrin administration and for whom at least one plasma PK variable was available. Dose dependency of Cmax and AUC(0,1) was assessed by the power model (point estimate and 95% confidence interval [95% CI] of the slope (b) of the regression formula (log (Y) ¼a þb log (X)). Dose proportionality was confirmed if the 95% CI of b included 1 and fell within the range of 0.7, 1.43.
3. Result 3.1. Study population The baseline demographics of the study participants are summarized in Table 1. Forty-six subjects were randomized to the single-dose study and all completed the study. There were no spastic differences in baseline characteristics between treatment groups. No subjects were excluded from the safety or the pharmacokinetic analysis populations. 3.2. Safety analysis Compliance was good in all volunteers. No severe or lifethreatening AE occurred. For single-dose study, pinocembrin was well tolerated at 5 treatment groups. No subject was discontinued because
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Table 1 Baseline demographic characteristics of the study population. Dose (mg)
Gender
20
2 (placebo) 8 (pinocembrin) 2 (placebo) 8 (pinocembrin) 2 (placebo) 8 (pinocembrin) 2 (placebo) 6 (pinocembrin) 2 (placebo) 6 (pinocembrin) 2 (placebo) 10 (pinocembrin)
40 80 120 150 60a
a
Male
Female
1 3 0 6 2 5 0 5 1 5 2 6
1 5 2 2 0 3 2 1 1 1 0 4
Mean age (yr range)
Mean weight (kg range)
Mean BMI (range)
30.0 30.2 29.0 26.1 27.5 28.2 29.5 29.8 23.5 28 24.5 26
61.5 58.9 50.2 61.1 66.0 59.7 53.3 61.6 54.5 60.2 62.4 60.8
22.6 23.1 20.4 21.8 22.8 21.7 22–8 21.6 21.5 21.6 23.1 21.5
(26–34) (22–36) (24–34) (20–35) (23–32) (19–36) (29–30) (22–35) (21–26) (22–40) (28–21) (21–36)
(51–72) (52–66) (57–46) (55–69) (64–72) (47–64) (51.7–54.6) (55–68) (52.5–56.4) (54.6–64.8) (61.5–63.2) (52.0–71.0)
(19.8–23.8) (22.4–23.8) (20.4–20.4) (19.4–23.6) (22.1–23.5) (19.9–23.4) (20.3–23.3) (19.8–23.8) (19.8–23.2) (19.6–23.9) (23.9–22.3) (19.5–24.0)
Multiple-dose.
Table 2 Summary of adverse events after intravenous administration of pinocembrin to healthy subject at doses of 20–150 mg once daily and 60 mg twice daily. Adverse events
No. of subjects (%)
Any events β2-MG elevation Urine microprotein Acute urticaria Diarrhea a
Placebo (%)
20 mg (%)
2 (16.7) 1 (8.3) 1 (8.3)
2 (25 ) 1 (12.5) 1 (12.5)
40 mg (%)
80 mg (%)
120 mg (%)
150 mg (%)
60 mga (%)
2 (25) 1 (12.5) 1 (12.5)
1 (16.7) 1 (16.7)
2 (33.3)
1 (10)
2 (33.3) 1 (10)
Multiple-dose.
Table 3 Pharmacokinetics of pinocembrin at dose levels of 20–150 mg after intravenous infusion. Parameters 1
AUC(0,8) (μg mL min) AUC(0,1) (μg mL 1 min) MRT(0,8) (min) MRT(0,1) (min) T1/2 (min) Tmax (min) Vz (L) CLz (L min 1) Cmax (μg mL 1)
20 mg
40 mg
80 mg
120 mg
150 mg
10.247 1.5 10.34 7 1.5 40.0 7 4.8 42.7 7 5.7 47.4 7 14.0 28.0 7 4.0 136.6 7 52.8 1.98 7 0.3 0.28 7 0.1
19.8 7 2.5 20.0 7 2.5 37.8 7 4.4 39.4 7 4.5 40.6 7 8.1 28.0 7 5.0 119.9 7 32.6 2.03 7 0.3 0.6 7 0.1
43.86 7 10.3 44.0 7 10.6 38.5 7 5.7 39.6 7 6.0 55.7 7 24.6 28.0 7 7.0 149.57 0.1 1.93 7 0.6 1.22 7 0.3
59.1 7 12.9 59.3 7 12.9 38.0 7 5.6 38.9 7 6.2 55.8 7 19.0 27.0 7 5.0 174.6 7 90.3 2.1 7 0.5 1.63 7 0.4
89.217 13.5 89.34 7 13.5 38.3 7 4.9 39.0 7 5.0 54.5 7 13.7 22.0 7 8.0 136.6 7 45.5 1.727 0.3 2.46 7 0.4
of a treatment emergent AE. All 46 subjects received at least one dose of the study drug and were included in the safety analysis. AEs were reported by 11 of 46 subjects, including 2 (20%) who received control infusions and 9 of 36 (25%) who received pinocembrin. The most commonly observed AE were acute urticaria, β2-MG elevation, urine microprotein (mAlb), β2-MG elevation and urine mAlb occurred in both pinocembrin and placebo-treated subjects. Skin acute urticaria occurred only in pinocembrin injection subjects. Urinalysis for renal damage was via BUN, serum creatinine, serum β2-MG and urine mAlb. In the present studies, BUN and serum creatinine remained within the normal limits throughout the treatment period. However, three subjects (two pinocembrin subjects and one placebo subject) had a mild elevation of serum β2-MG. Three subjects (two pinocembrin subjects and one placebo subject) had urine mAlb. PI thinks they were attributed to laboratory testing variability. Two subjects enrolled in 150 mg cohort developed grade II skin acute urticaria during the study. Urticaria was found in chest, eyes. It occurred 1.5–2.0 h after dosing and resolved within 3 h without any specific intervention. This event was not considered related to the study drug. The AE was deemed mild by the investigator and suspected to be related to study medication.
The PI and the sponsor think these AEs raise the safety concern and agreed to discontinue of the study. The preceding dose level, 120 mg, is defined as the maximum tolerated dose. In the multiple-dose study, pinocembrin was similarly welltolerated. One AE occurred in pinocembrin group. The subject was discontinued from the study because of grade II diarrhea, which occurred on the third day of the multiple-dose and recovered within 24 h. Diarrhea was deemed not suspected to be related to study medication and resolved without any treatment. Table 2 summarizes all AEs by body system, both in pinocembrin and placebo-treated groups. All the AEs were mild or moderate in intensity, none was characterized as serious or potentially life-threatening AEs occurred, and most resolved spontaneously without intervention. 3.3. Pharmacokinetic evaluation Data for all 36 subjects who received pinocembrin injection are included in the single-dose PK analyses. PK parameters are presented in Table 3. The mean peak plasma concentration was obtained at the end of the 30-min infusion (Fig. 1). The Cmax ranged from 0.28 μg mL 1
3000.00
Concentration (ng ml-1)
2500.00 2000.00 20 mg 1500.00
40 mg 80 mg
1000.00
120 mg 150 mg
500.00 0.00
0
100
200
300
400
0.30 0.25 20mg
0.20
40mg
0.15
80mg
0.10
120mg
0.05
150mg
0.00
0
5
10
15
20
25
30
35
40
45
50
Time (minute)
Fig. 1. Graph of mean d pinocembrin concentration versus time plots at the 20–150 mg dose levels in the single dose study.
Fig. 3. The mean pinocembrin urine concentrations at 20–150 mg dose levels after a single 30-min intravenous infusion.
Table 4 Pharmacokinetics of pinocembrin on day 1 and at the end of dosing period (day 5) at dose level of 60 mg twice daily.
120.00 100.00 AUCinf (μg mL-1 min)
35
0.35
500
Time (minute)
80.00 60.00 40.00 20.00 0.00
Cumulative fraction of pinocembrin
G. Cao et al. / Journal of Ethnopharmacology 168 (2015) 31–36
0
50
100
150
200
Parameters
Day 1
Day 5
AUC(0,8) (μg mL 1 min) AUC(0,1) (μg mL 1 min) MRT(0,8) (min) MRT(0,1) (min) T1/2 (min) Tmax (min) Vz (L) CLz (L min 1) Cmax (μg mL 1)
32.117 2.6 32.217 2.5 38.7 7 2.5 39.3 7 2.5 39.7 7 6.9 26.0 7 7.0 106.57 14.3 1.87 7 0.14 0.86 7 0.09
30.737 6.06 30.82 7 6.07 39.17 7.2 40.2 7 7.4 50.3 7 12.3 28.0 7 4.0 145.6 7 44.1 2.02 7 0.41 0.82 7 0.13
Dose (mg)
Pinocembrin plasma pharmacokinetics parameters in multipledose study are presented in Table 4. There were no significant changes in observed parameters on day 5 versus day 1. On average, 0.11% and 1.86% of the administered dose was recovered in urine and feces as unchanged pinocembrin during the 7 study period. Both urinary and feces excretion levels of pinocembrin were extremely low and similar to those observed in the singledose study.
3.50
Cmax (ng ml -1 )
3.00 2.50 2.00 1.50 1.00 0.50 0.00
4. Discussion 0
50
100
150
200
Dose (mg) Fig. 2. Pinocembrin AUC (0,1) (y ¼0.577x 2.691; R2 ¼ 0.896) (A) and Cmax (y¼ 0.015x 0.078; R2 ¼ 0.889) (B) versus dose (mg) following a once-daily 30-min IV infusion of pinocembrin injection in 36 healthy subjects.
in the 20 mg group to 2.46 μg mL 1 in the 150 mg group. AUC(0,1) ranged from 10.34 μg mL 1 min to 89.34 μg mL 1 min. The elimination half-lives were similar across five dose groups, ranging from 40 to 60 min. Clearances were also similar across all dose groups, ranging from 1.72 to 2.10 L min 1. Dose dependency of Cmax and AUC(0,1) (20–150 mg) was assessed. Both Cmax and AUC(0,1) met the dose proportionality criteria (Fig. 2). The mean pinocembrin urine concentrations after a single 30-min intravenous infusion reached the highest in the first two post-dose collection intervals (0–2 and 2–4 h, Fig. 3). Both urinary and feces excretion levels of pinocembrin were extremely low and similar among each dose groups, with mean values ranging from 0.07% to 0.17% and 0.94% to 1.94% of the administered dose, respectively.
This report describes the first-in-human studies of pinocembrin following single and multiple intravenous dose administrations. Pinocembrin was well tolerated by the healthy volunteers; all adverse events were mild to moderate and reversible. No clinically relevant changes in renal and hepatic indices from baseline were observed. In addition to assessing safety, our study represented the first evaluation of the pharmacokinetics of pinocembrin in healthy male and female subjects. When pinocembrin was intravenously administered to healthy subjects at doses of 20–150 mg, the Cmax and AUC(0,1) were proportional to dose, with a rapid Tmax and a relatively short plasma T1/2 following administered pinocembrin injection. Across all dose groups, the single-dose and the multipledose PK look similar, with no evidence of accumulation. No evidence of dose-dependent clearance is apparent within the pinocembrin injection doses tested. Clearance is most likely to occur predominantly through metabolism. Urinary excretion of the unchanged compound was o1%. In the metabolism study of pinocembrin on average, more than 80% of the administered doses were metabolized by hepatic phase II, and the metabolites were not found in feces (which is done in our lab and not published).
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