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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 1 pp. 299ñ307, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
PRECLINICAL PHARMACOKINETIC ANALYSIS OF
(E)-METHYL-4-ARYL-4-OXABUT-2-ENOATE, A NOVEL SER/THR
PROTEIN KINASE B INHIBITOR, IN RATS
QIAN-QIAN ZHAI1,2, JING PANG1, GUO-QING LI1, CONG-RAN LI1, YU-CHENG WANG1,
LI-YAN YU1, JIAN LI2 and XUE-FU YOU1,*
Beijing Key Laboratory of Anti-infective Agents, Institute of Medicinal Biotechnology,
Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
2
Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture,
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
1
Abstract: (E)-Methyl-4-aryl-4-oxabut-2-enoate, designated YH-8, is a novel Ser/Thr protein kinase B (PknB)
inhibitor, which is designed for the treatment of tuberculosis. The aim of this study was to investigate the pharmacokinetics, bioavailability, tissue distribution and excretion characteristics of YH-8 in rats and study its plasma protein binding in vitro. The pharmacokinetic properties were examined after intravenously injected YH-8
at 10 and 20 mg/kg and oral administrated YH-8 at 50, 100 and 200 mg/kg to rats. The concentrations of YH8 in plasma were determined with LC-MS/MS, with a liquidñliquid extraction. The tissue distribution and urinary, fecal and biliary excretion patterns of YH-8 were investigated following a single oral dosing of 100
mg/kg. The plasma protein binding rates of YH-8 were determined using ultra-filtration method. After intravenous and oral administration, YH-8 showed dose-independent pharmacokinetic characteristics, with T1/2 of
approximately 5.5 h and 7.1 h, respectively. The oral absolute bioavailability of YH-8 was relatively low (about
12%). YH-8 was widely distributed in various tissues and showed substantial deposition in intestine, stomach,
liver, lung and kidney. The drug was mainly eliminated via fecal excretion and its binding rate with plasma protein was concentration-dependent. In conclusion, this study as first provided the full pharmacokinetic characteristics of YH-8, which would be helpful for its further development and clinical application.
Keywords: (E)-methyl-4-aryl-4-oxabut-2-enoate, pharmacokinetics, bioavailability, distribution, excretion,
protein binding
(8-11). PknB is necessary for the survival of M.
tuberculosis, and also plays an important role in the
growth of M. tuberculosis (12-14). Phosphorylation
or the changes of expression index and activity of
PknB can lead to the alteration of growth rate and
morphology of M. tuberculosis, due to the defects in
cell wall synthesis and cell division (13, 15-17).
Because of the significant differences between M.
tuberculosis PknB and the human protein kinases,
PknB is widely accepted as a drug target for antiTB. To date, several PknB inhibitors have been
reported, and some of them have shown certain
degree of anti-TB capability. Most of these compounds are aminopyrimidines, aminoguanidines and
anthraquinones (18-23).
(E)-Methyl-4-aryl-4-oxabut-2-enoate (designated YH-8; molecular formula: C12H12O4; molecular weight: 220 (Fig. 1), is a novel small-molecule
Tuberculosis (TB) is one of the oldest human
diseases and also one of the major diseases that
endangers humanís health. With the drug-resistant
TB especially multidrug-resistant TB (MDR-TB)
and extensively drug-resistant TB (XDR-TB) dissemination, the treatment of TB faced up more difficulties (1). Although human have raced with the
Mycobacterium tuberculosis (M. tuberculosis) drug
resistance since the discovery of the anti-TB drug
rifampin (2), there was only bedaquiline being
approved for clinical treatment to TB in recent 40
years (3-6). Therefore, it is essential and urgent to
develop new anti-TB drugs for the prevention and
treatment of drug-resistant TB.
M. tuberculosis Ser/Thr protein kinase PknB is
one of the most important Ser/Thr protein kinases
for M. tuberculosis (7), which is able to autophosphorylate and combine with ATP and its analogues
* Corresponding author: e-mail: e-mail: [email protected]; phone: + 86 10 67061033, fax. +86 10 67017302
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QIAN-QIAN ZHAI et al.
Figure 1. The chemical structure of YH-8
anti-TB candidate of unsaturated butene acid derivatives with good inhibitory activity on PknB (24).
YH-8 inhibits the activity of PknB by acting as a
substrate analogue binding to the active site of
PknB. In vitro anti-TB studies demonstrated that the
good anti-TB activities of YH-8 had no cross-resistance with the first-line anti-tuberculosis drugs with
the minimum inhibitory concentration (MIC) value
of 0.125 µg/mL against M. tuberculosis including
MDR-TB (24). The anti-TB activity of YH-8 is significantly higher than those of the reported PknB
inhibitors of aminopyrimidines, aminoguanidines,
and anthraquinones classes (18-24). In addition, the
results of acute toxicity test in mice have indicated
YH-8 also has good safety with the oral LD50 higher
than 500 mg/kg (unpublished data).
Pharmacokinetic studies play a very important
role in drug discovery and development, not only to
support toxicological and clinical studies but also to
optimize drug candidates (25). Therefore, the objective of the current study was to examine the plasma
pharmacokinetics, tissue distribution, excretion
characteristics of YH-8 in rats and to study its protein binding ability in vitro.
MATERIALS AND METHODS
Chemicals and reagents
(E)-Methyl-4-aryl-4-oxabut-2-enoate (YH-8,
purity > 98%) was synthesized by Institute of
Medicinal Biotechnology, Chinese Academy of
Medical Sciences (Beijing, China). Polysorbate
Tween-80 and dimethyl sulfoxide (DMSO) were
purchased from SigmañAldrich Corporation
(Steinheim, Germany). HPLC-grade methanol was
purchased from Fisher Scientific (New Jersey,
USA). Ultrapure water was obtained from a
Millipore system (Bedford, MA, USA). All other
reagents were of analytical grade. Nitrogen gas was
provided by the Gas Company of Beijing (China).
Animals
Sprague-Dawley (SD) rats (Certificate No.
SCXK-(Military) 2007-004), 6-8 weeks old and
weighed 180-220 g, were supplied by Academy of
Military Medical Sciences (Beijing, China). The rats
were acclimated in the laboratory for at least seven
days prior to the experiments, and were housed
under controlled environmental conditions (temperature, 23 ± 3OC; humidity, 55-75%) with commercial food diet and water freely available. All animal
experiments were carried out under the permission
and supervision of the Ethics Committee of Institute
of Medicinal Biotechnology, Chinese Academy of
Medical Sciences and Peking Union Medical
College, Beijing, China.
Preparation of solutions
YH-8 solutions for oral administration were
prepared by grinding the required amount of YH-8
in appropriate volume of water supplemented with
1.0% sodium carboxymethyl cellulose. YH-8 solution for intravenous administration was prepared by
dissolving the powder of the compound in saline
with 1.0% DMSO and 0.5% Tween-80.
Pharmacokinetic studies in rats
Oral administration of drug
Eighteen male SD rats were randomly divided
into three groups (six per group). The animals were
fasted for 12 h with free access to water prior to the
start of the experiment. Each rat in the three groups
was given a single oral administration of YH-8 at a
dose of 50, 100 and 200 mg/kg, respectively. The volume of YH-8 solution administrated orally to each rat
was 1 mL/100 g. Approximately 0.3 mL blood samples were collected from the post-orbital venous
plexus into heparinized tubes at 0.17, 0.33, 0.5, 0.75,
1, 1.5, 2, 4, 6, 8, 10, 12, 24, 36, 48 and 60 h after oral
administration of the drug. The blood samples were
immediately centrifuged at 2292 ◊ g for 15 min at
4OC. Then, the plasma (100 µL) was transferred to
clean tubes and stored at -70OC until analysis.
Intravenous administration of drugs
Twelve male SD rats were randomly divided
into two groups (six per group). YH-8 at doses of 10
and 20 mg/kg was intravenously injected to the two
groups of overnight fasted rats, respectively. The
volume of YH-8 solution injected intravenously to
each rat was 1 mL/100 g. Blood samples of 0.3 mL
were taken via the post-orbital venous plexus vein at
0.083, 0.167, 0.333, 0.667, 1, 1.5, 3, 6, 12, 18, 24
and 36 h after intravenous injection of the drug. The
blood samples were immediately centrifuged at
2292 ◊ g for 15 min at 4OC. Then, the plasma (100
µL) was transferred to clean tubes and stored at
-70OC until analysis.
Preclinical pharmacokinetic analysis of...
Tissue distribution study
Twenty-four rats were divided into four groups
(six per group and three per sex) randomly, and were
orally administrated with 100 mg/kg YH-8. After
administration, the rats were sacrificed at 10 min, 1
h, 8 h, and 30 h for each group, respectively. Tissue
samples were collected from heart, liver, spleen,
lung, kidney, intestine, muscle, adipose, stomach,
brain, ovary and testicle. The blood samples were
collected at the same time. All the tissues were
rinsed in saline, wiped dry with absorbent paper,
accurately weighed and grinded together with three
times (v/w) of ultrapure water. Then, a subpackage
of 100 µL tissue homogenate was placed into an
electro-polishing (EP) tube, which was kept at -70OC
until analysis.
Excretion studies
Urinary and fecal excretion
Six rats (three male and three female rats) were
used in this experiment. The rats were individually
placed in a stainless-steel metabolic cage that
allowed for the separate collection of urine and
feces. They were provided with standard food and
water ad libitum throughout the experiment. Urine
and feces were collected on the day before drug dosing for the background count. Then, each rat
received a single oral dose of YH-8 at 100 mg/kg,
and the excreted urine and excreted feces samples
were collected from each rat at 0-5, 5-12, 12-24, 2436, 36-48, 48-60 and 60-72 h after drug administration. The volume of each collected urine sample was
recorded separately. Fecal samples were air dried,
individually weighed, and homogenized with ultrapure water to permit reasonably accurate pipetting.
Samples were stored at -70OC until analysis.
Biliary excretion
Six rats (three per sex) were anesthetized by
intraperitoneal injection with 2% sodium pentobarbital and bile duct cannulation was performed. Bile
was then collected before the drug was administered. Following that, YH-8 at a dose of 100 mg/kg
was orally administrated to rats. The bile samples
containing YH-8 were collected at 0ñ3, 3ñ6, 6ñ9,
9ñ12, 12ñ15, 15ñ18, 18ñ21 and 21ñ24 h and the volume of each collected sample was recorded separately. Samples were stored at -70OC until analysis.
Plasma protein binding
In order to determine the plasma protein binding rates, the ultrafiltration method was used. A volume of 8 µL YH-8 was added to 392 µL freshly
obtained blank plasma to give final concentrations
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of 5, 50 and 500 ng/mL. The compound was incubated with plasma at 37OC for 4 h before centrifugation. A 100 µL aliquot of the incubated mixture was
removed for the analysis of the total drug concentration in each sample using LC-MS/MS.
Ultrafiltration was performed in the remaining volume using filters. Centrifugation was carried out at
12000 ◊ g for 120 min. A 100 µL aliquot of the centrifuged plasma water was analyzed for the free drug
concentration using the procedure described for the
total concentration. A 300 µL phosphoric acid buffer
solution of YH-8 at 5, 50 and 500 ng/mL was analyzed using the same apparatus to assess the membrane binding extent of YH-8. The difference
between the plasma binding and phosphoric acid
buffer solution binding was assumed to be due to
YH-8-protein binding.
Sample preparation procedure
Before analysis, plasma samples were thawed
at room temperature together with calibration standards and QC samples. A 100 µL volume of the
plasma sample was extracted with 400 µL ethyl
acetate by vortex for 1 min. The sample was then
centrifuged at 5867 ◊ g for 15 min and the supernatant was transferred to a new tube. The residue
was extracted one more time with 400 µL ethyl
acetate and the supernatant was combined with the
first round supernatant. These sample extracts were
evaporated to dryness at room temperature under a
stream of nitrogen, reconstituted with 200 µL of
mobile phase, vortexed for 1 min, filtered through a
0.22 µm pore size filter (Millipore), and then 5 µL
of the solution was injected into the LC-MS/MS system. The process of analysis was similar for samples
taken from feces, urine and bile extraction as well as
protein binding and tissue homogenate samples.
LC-MS/MS analysis
The plasma, tissue, bile, urine, feces and plasma protein binding samples were analyzed using the
developed and validated LC-MS/MS method (26).
The quantification of YH-8 was performed on a
Thermo Fisher (San Jose, USA) triple quadrupole
LCñMS/MS spectrometer, consisting of a
Finniganô SurveyorÆ MS pump, a Finniganô
SurveyorÆ autosampler and a Finniganô TSQ
Quantum Discovery mass detector. The chromatographic separation was achieved using a C18 column
(50 ◊ 2.1 mm, 3.5 µm, Waters XTerraÆ, Ireland) at
35OC. A mobile phase consisting of ultrapure water
(A) and methanol (B) was delivered for separation
of analytes using a gradient elution program at a
flow rate of 0.3 mL/min. The 16 min gradient pro-
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gram was conducted as follows: 0-5.0 min, 10% B;
5.0ñ6.0 min, 10-50% B; 6.0ñ14.0 min, 50% B;
14.0ñ14.01 min, 50ñ10% B; 14.01ñ16.0 min, 10%
B. Solvent eluted from chromatography column during the first 4 min was switched to waste before it
entered the ion source. Autosampler was set at 4OC
and the injection volume was 5 µL. Quantification
of YH-8 was performed in positive selected reaction
monitoring mode with (m/z)+ 221→161. The MS
operating conditions were optimized and set as follows: sheath gas pressure, 30 arbitrary units; auxiliary gas pressure, 10 arbitrary units; capillary temperature, 350OC; spray voltage, 3800 V and source
collision-induces dissociation (CID), 15 V. The collision gas was argon and the collision energy was 17
eV. The calibration curves were linear over the concentration range of 1-500 ng/mL with determination
coefficients greater than 0.99 and the weighting
index of 1/x2. The low limit of quantification
(LLOQ) was 1 ng/mL. The precisions, both intraand inter-day, were less than 7%, and the accuracies
were in the range of 100.69ñ106.18%. The mean
extraction recoveries of YH-8 from spiked rat plasma under the liquid extraction conditions were
above 85% and no relevant cross-talk and matrix
effect were observed at the concentrations of 5, 50
and 500 ng/mL. The mean stability values of YH-8
during sample handling (freeze-thaw, short-term
temperature, long-term, post-preparative) were all
above 92%.
Pharmacokinetic and statistical analysis
Pharmacokinetic parameter calculations were
performed with WinNonlinÆ (Version 6.1, Pharsight
Corp., Mountain View, CA, USA) using non-compartmental model. The maximum concentration
(Cmax) was read directly from the observed concentration and the time to maximum concentration (Tmax)
was determined as the blood sampling time corresponding to Cmax. AUC0-t (area under the plasma concentration vs. time curve from time 0, Cp0, to the
time of the last measurable plasma concentration,
Cplast) was calculated by using the linear trapezoidal
rule, followed by extrapolation to infinity (AUC0-∞)
using the ratio Cplast/λz. The elimination rate constant
(λz) was estimated from the terminal linear segment
of the log plasma concentration vs. time data. The
elimination half-life (T1/2) was calculated from ln2
divided by the λz. The mean residence time to last
measurable concentration (MRT0-t) was calculated by
AUMC0-t/AUC0-t and extended to infinity (MRT0-∞)
by AUMC0-∞/AUC0-∞. The clearance (CL) was calculated by dose/AUC0-∞. The apparent volume of distribution (Vz) was calculated as CL/λz.
The statistical analysis was conducted using
SPSS software (version 17.0, IBM Co., Armonk,
NY). Levels of statistical significance were assessed
using the Duncanís multiple range tests among the
three or more means for unpaired data, or the
unpaired t-test between the two means following an
ANOVA analysis. Values of p < 0.05 were considered to be significantly different. All the data were
expressed as the mean ± standard deviation.
RESULTS
Pharmacokinetic study
Pharmacokinetic study after intravenous administration
The plasma concentrationñtime curves and the
pharmacokinetic parameters of YH-8 after its intravenous administration (10 and 20 mg/kg) are pre-
Table 1. Pharmacokinetic parameters of YH-8 in Sprague-Dawley rats after intravenous administration at two
different doses (n = 6, Mean ± SD).
Parameters
10 mg/kg
20 mg/kg
λz(1/h)
0.125 ± 0.027
0.124 ± 0.031
T1/2 (h)
5.52 ± 0.35
5.54 ± 0.23
C0 (ng/mL)
563.66 ± 26.34
1116.16 ± 50.27
AUC0-t (ng∑h/mL)
898.27 ± 41.66
1778.74 ± 93.51
AUC0-∞ (ng∑h/mL)
902.12 ± 41.60
1786.38 ± 91.14
Vz (L/kg)
88.54 ± 10.73
89.42 ± 11.94
CL (L/h/kg)
11.09 ± 1.37
11.20 ± 1.55
MRT (h)
3.27 ± 0.39
3.22 ± 0.87
C0: initial concentration; λz: elimination rate constant; AUC0ñt: area under curve from time zero to the last sampling time; AUC0-∞: area under curve from time zero to infinity; T1/2: the plasma elimination half-life; MRT:
mean residence time; CL: Clearance; Vz: volume of distribution. Data are expressed as the mean ± SD.
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Preclinical pharmacokinetic analysis of...
Figure 2. Mean plasma concentration-time profile of YH-8 in rats after (A) intravenous administration of 10 and 20 mg/kg and (B) oral
administration of 50, 100 and 200 mg/kg. Each point and bar represents the mean ± SD (n = 6)
Table 2. Pharmacokinetic parameters of YH-8 in Sprague-Dawley rats after oral administration at three different doses (n = 6, Mean ± SD).
Parameters
50 mg/kg
100 mg/kg(26)
200 mg/kg(26)
λzz(1/h)
0.097 ± 0.008
0.098 ± 0.008
0.097 ± 0.007
T1/2 (h)
7.16 ± 0.62
7.17 ± 0.56
7.21 ± 0.57
Tmax (h)
0.83 ± 0.59
0.63 ± 0.49
0.58 ± 0.46
Cmax (ng/mL)
172.73 ± 21.38
289.72 ± 37.54
457.08 ± 49.77
AUC0-t (ng∑h/mL)
473.70 ± 40.63
1137.41 ± 167.62
2101.66 ± 270.92
AUC0-∞ (ng∑h/mL)
478.86 ± 41.42
1148.46 ± 167.47
2119.45 ± 269.42
Vz/F (L/kg)
1053.48 ± 150.13
1028.07 ± 155.71
996.43 ± 163.73
CL/F (L/h/kg)
99.77 ± 16.58
98.11 ± 16.03
95.66 ± 12.30
MRT (h)
11.68 ± 1.91
11.73 ± 2.74
13.11 ± 3.18
Cmax: the maximum concentration; Tmax: the time to maximum concentration; λzz: elimination rate constant; AUC0ñt: area under curve from
time zero to the last sampling time; AUC0-∞: area under curve from time zero to infinity; T1/2: the plasma elimination half-life; MRT: mean
residence time; CL: Clearance; Vz: volume of distribution; F: bioavailability. Data are expressed as the mean ± SD
sented in Fig. 2(A) and Table 1, respectively. After
its intravenous administration, YH-8 was moderately eliminated from the plasma with T1/2 of approximately 5.5 h. The AUC0ñt and the initial concentration (C0) values were proportional to the intravenous
doses, with no statistical differences of the dose-normalized AUC0ñt (AUC0ñt/D) and C0 (C0/D) between
the two doses (p > 0.05). However, other pharmacokinetic parameters such as T1/2, CL, and Vz were
independent of the intravenous doses.
Pharmacokinetic study after oral administration
As shown in Fig. 2(B) and Table 2, after oral
administration of YH-8 at doses of 50, 100 and 200
mg/kg, Tmax and T1/2 were both dose independent and
were approximately 0.7 h and 7.1 h, respectively.
AUC0ñt and dose, as well as Cmax and dose, showed
good linear relationships, with correlation coefficients (R2) both above 0.99. In addition, the dosenormalized AUC0ñt (AUC0ñt/D) and Cmax (Cmax/D)
were not significantly different among the three
doses analyzed by ANOVA (p > 0.05).
Oral absolute bioavailability of YH-8
The absolute bioavailability F(%) of YH-8 was
estimated with the following equation: F(%) =
[(AUCoral ◊ Doseiv)/(AUCiv ◊ Doseoral)] ◊ 100%,
where AUCoral and AUCiv are the areas under the
concentrationñtime curves, Doseiv and Doseoral represent the doses intravenously and orally administered, respectively. The oral absolute bioavailability
of YH-8 administered at 50, 100, and 200 mg/kg
was 10.69 ± 0.12%, 12.88 ± 0.15% and 11.84 ±
0.03%, respectively, and bioavailability was thus
dose independent (p > 0.05). The final average oral
absolute bioavailability was 11.82 ± 0.04%.
Tissue distribution in rats
The tissue distribution of YH-8 was investigated following a single oral dose of 100 mg/kg. The
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QIAN-QIAN ZHAI et al.
concentrations of YH-8 in the various tissues were
measured at 10 min, 1, 8 and 30 h after administration (Fig. 3). YH-8 was identified in all the tissues
tested 10 min after administration, suggesting its
rapid and wide distribution. In most tissues, the concentration of YH-8 reached a peak at 1 h and was
still high at 8 h after drug administration. The abundant YH-8 distributions were detected in the intestine, stomach, liver, lung and kidney. The drug concentration in the liver was higher than that in the
kidney. By 30 h, less than 5% of the maximum YH8 remained in most tissues.
50 and 500 ng/mL) of YH-8, the transmissivity (%)
of the filter membrane were determined to be 57.65
± 1.43, 56.78 ± 1.21 and 57.54 ± 1.31, respectively.
This factor was taken into consideration for the protein binding rates analysis. As a result, the binding
rates (%) of different concentrations (5, 50 and 500
ng/mL) of YH-8 to human plasma protein were
determined to be 41.12 ± 1.21, 48.24 ± 1.13, and
58.31 ± 1.49, respectively. The binding rates (%) to
rat plasma protein of 5, 50 and 500 ng/mL YH-8
were 40.71 ± 1.23, 48.54 ± 1.32 and 57.71 ± 1.26
respectively.
Excretion
The excretion data after a single oral administration of 100 mg/kg YH-8 to intact and bile duct
cannulated rats are illustrated in Figure 4. In intact
rats, feces excretion was the dominant route of elimination after oral administration, as 37.44 ± 3.20%
of administered YH-8 were recovered in feces and
2.64 ± 1.21% were recovered in urine within 72 h,
respectively. When YH-8 was administered to bile
duct cannulated rats, 2.11 ± 1.10% of the dosed drug
was excreted into bile up to 24 h post dosing.
DISCUSSION
Protein binding ability of YH-8
The plasma protein binding rates of YH-8 were
determined over the plasma drug concentrations as
observed in animals. At different concentrations (5,
In the light of developing a new promising
anti-TB drug, we evaluated the pharmacokinetics,
bioavailability, tissue distribution, excretion properties and protein binding ability of YH-8. The results
of this study should provide a meaningful basis for
the use of YH-8 in prevention and treatment of
tuberculosis.
The results of this study showed that YH-8 was
eliminated moderately from the plasma after intravenous and oral administration. The plasma concentrations of YH-8 could be detectable up to 36 and 60
h in rats according to intravenous and oral administration using the analytical method described above.
After its intravenous administration, the pharmacoki-
Figure 3. Mean concentrations of YH-8 in various tissues and plasma in rats at 10 min, 1, 8, and 30 h after oral administration at the dose
of 100 mg/kg (n = 6), Data represent the mean ± SD
Preclinical pharmacokinetic analysis of...
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Fgure 4. The excretion of YH-8 in bile fluid, urine and feces after the one-off oral administration of 100 mg/kg to rats. (A). The average
excretion of YH-8 into the bile fluid, urine and feces after a single oral administration of 100 mg/kg to rats. (B). The accumulation of excretion of YH-8 in bile fluid, urine and feces after the one-off oral administration of 100 mg/kg to rats. Data represent the mean ± SD (n = 6)
netic parameters of YH-8 such as AUC0ñt and C0 were
proportional to the intravenous doses with no statistical differences of the dose-normalized AUC0ñt
(AUC0ñt/D) and C0 (C0/D) between the two doses, but
the T1/2, CL, and Vz values were dose independent.
After the oral administration, both AUC0ñt and Cmax of
YH-8 showed perfect linear relationships with the oral
doses, and there were no significant differences of the
dose-normalized AUC0ñt (AUC0ñt/D) and Cmax (Cmax/D)
among the three doses (50, 100 and 200 mg/kg),
whereas other pharmacokinetic parameters such as
T1/2, CL, and Vd were dose independent. These findings suggested that YH-8 has linear pharmacokinetic
profiles in rats within the dose range studied. The values of CL/F were much larger than the hepatic blood
flow rate (about 55.2 mL/min/kg) (27), suggesting a
rapid clearance of YH-8 from the rat body. The values
of the apparent volume of distribution (Vd/F) were
much larger than the total blood volume (about 54.0
mL/kg) and the total body water in rat (about 671.0
mL/kg) after oral dosing (27), demonstrating the distribution of YH-8 into the extravascular systems was
wide and the affinity of YH-8 to rat tissues was considerable. In addition, after oral administration of YH8 at dosages of 50, 100 and 200 mg/kg, the drug concentrations in plasma were maintained above the
MICs against M. tuberculosis (0.125 µg/mL) for
approximately 2, 3 and 4.5 h, respectively, indicating
good prospect of YH-8 for clinical treatment of tuberculosis. The low oral bioavailability of YH-8 in rats
could be due to a poor absorption in the gastrointestinal tract and/or small intestine or hepatic first-pass
metabolism when administered orally. During the
experiment, YH-8 presented poor water solubility.
The low water solubility and the dissolution process in
the gastrointestinal fluid could limit the drug absorption. The bioavailability of YH-8 can be improved by
modifying it as prodrug or changing the pharmaceutical dosage forms.
The tissue distribution of a drug is vital when
investigating its major target sites and interpreting
its disposition in vivo (28). In this study, YH-8 presented a wide distribution profile for the drug was
detected in all the tissues examined, which were
consistent with the results of Vd/F in the pharmacokinetic study. After its oral administration, the concentrations of YH-8 peaked at 1 h in most tissues
and declined significantly by 30 h, indicating that
there was no apparent accumulation of YH-8 in tissues. YH-8 showed substantial deposition in intestine, stomach, liver, lung, kidney, heart and spleen
during the experiment. Among the tested
tissues/organs, the intestines and stomach had the
highest concentration distribution of YH-8 due to
the mode of administration being oral. For the clearance organs, liver initially absorbed more YH-8 than
kidney, and both of them decreased rapidly. After 30
h, the drug concentrations in the liver and kidney
were almost the same. This implied that the liver
played a more important role than the kidneys did in
the elimination of YH-8. Therefore, monitoring of
the hepatic function becomes necessary and a dose
adjustment is needed in patients who have impaired
hepatic metabolic functions. YH-8 was also
detectable in the skeletal muscle in 10 min, but the
amounts were found to be low and approximately
equal to that of the level in adipose. Notably, the
reproductive tissues also absorbed a little of the YH8. YH-8 could be detected in the brain. It indicated
that the lipid solubility enabled YH-8 to cross the
blood-brain barrier and distribute in the brain. In
addition, since the lung is regarded as the major target organ and pathological organ in tuberculosis, the
relatively high concentration distribution of YH-8 in
lung would be a positive message for the prevention
and treatment of tuberculosis.
In our excretion experiments, we examined the
unchanged fraction of YH-8 in the urine, feces, and
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bile. The maximum excretion route of YH-8 was via
the feces (37.44% of the dose) followed by a very
low urinary cumulative excretion (2.64%) and low
biliairy cumulative dose (2.11%), indicating that
YH-8 underwent important intestinal excretion. In
addition, when the excretion rate was subanalyzed,
the highest rates of urinary, fecal and biliary excretion were between 0ñ5 h, 0ñ12 h and 0ñ3 h, respectively. The extensive fecal excretion of YH-8 was
mainly caused by its low gastrointestinal absorption,
because in bile duct of cannulated rats the biliary
excretion of YH-8 was non-significant. Furthermore,
three demethylation metabolites of YH-8 by phase I
metabolic pathway were found in urine. These evidences indicated YH-8 could be metabolized by
phase I metabolic enzymes of liver or intestine.
The pharmacokinetic and pharmacodynamic
properties of drugs are largely due to the reversible
binding between drugs and serum or plasma proteins. The protein binding ability of a drug in blood
plasma is a very important factor influencing its free
concentration in blood as well as its tissue distribution. Generally, only the unbound drug is available
for diffusion or transport across cell membranes,
and for the interaction with the pharmacological target. As a result, the extent of plasma protein binding
to a drug influences the action of the drug in vivo.
The binding rate of YH-8 with plasma protein was
concentration-dependent. In the inspection concentration scope, the binding forces of YH-8 with rat
plasma protein and human plasma protein were
increased with the increase of drug concentrations,
suggesting that the association of YH-8 with other
drugs requires dose adjustment to prevent the emergence of toxicity.
CONCLUSION
We evaluated the pharmacokinetics, tissue distribution and excretion characteristics of YH-8 in
rats and studied its protein binding ability in vitro.
Following its oral administration, YH-8 can be rapidly absorbed. The average bioavailability of YH-8
was not very high. Tissue distribution studies
showed that YH-8 could be rapidly and widely distributed into various tissues. The feces excretion is
the predominant excretion route of YH-8 in rats,
while the contribution of urine and bile made for the
elimination of YH-8 was insignificant. The binding
ability of YH-8 with plasma protein was found to be
concentration dependent, which should be taken into
consideration when designing dose escalation protocols as well as to prevent possible toxicity induced
by YH-8. These results provided reliable scientific
data, which helps the further development of the
drug. To best of our knowledge, this is the first
report to evaluate the pharmacokinetics, tissue distribution, plasma protein binding and excretion of
YH-8 in rats.
Acknowledgments
This work was supported by the National
Mega-project for Innovative Drugs (No.
2014ZX09201001-011, 2012ZX09301-002-001020), and the National Natural Science Foundation
of China (No. 81273427).
Conflicts of interest
The authors declare that they have no conflict
of interest.
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Received: 30. 01. 2016