Download Polysaccharide peptides from COV-1 strain of Coriolus versicolor

Food and Chemical Toxicology 44 (2006) 1414–1423
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Polysaccharide peptides from COV-1 strain of Coriolus versicolor
inhibit tolbutamide 4-hydroxylation in the rat in vitro and in vivo
John H.K. Yeung *, Siu-Lung Chan, Penelope M.Y. Or
Department of Pharmacology, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, NT, Hong Kong, China
Received 18 October 2005; accepted 18 March 2006
Abstract
Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. In this study, the effects of whole PSP extract and water extract of PSP on 4-hydroxylation of tolbutamide were investigated in rat liver microsomes in vitro and in vivo in the rat. Both the whole PSP extract and the water soluble fraction (0.5–20 lM)
decreased the metabolism of tolbutamide to 4-hydroxytolbutamide in vitro. Enzyme kinetics studies showed that PSP inhibited tolbutamide 4-hydroxylase activity in a competitive, concentration-dependent manner. The whole PSP extract had a Ki value of 12.6 lM
and IC50 at 18.4 lM, while the water extract had a Ki value of 6.9 lM and IC50 at 9.8 lM. Sulphaphenazole, a specific human CYP2C9
inhibitor, showed a Ki value of 30.8 lM and IC50 at 44.0 lM in the test system. In the pharmacokinetic studies in vivo, acute PSP
(4 lmol/kg, i.p.) treatment did not produce significant changes in tolbutamide clearance, but produced a decrease in the Cinitial
(7.4%) and an increase in the Vd (7.4%). Sub-chronic pre-treatment of PSP (1–2 lmol/kg/day, i.p.) for three days did not affect the clearance and AUC of tolbutamide, but the Cinitial was decreased, together with increases in the T1/2, and Vd. The formation of 4-hydroxytolbutamide in vivo was decreased in both acute and sub-chronic studies. Taken together, this study demonstrated the PSP can inhibit
tolbutamide 4-hydroxylation both in vitro and in vivo. Despite the fact that CYP isoforms that metabolise tolbutamide are different
between rat and human liver due to different catalytic characteristics, and rat studies may not be directly extrapolatable to man, the concomitant use of PSP with other CYP2C substrates should be carefully monitored.
2006 Elsevier Ltd. All rights reserved.
Keywords: Polysaccharide peptide (PSP); Coriolus versicolor; Tolbutamide metabolism; 4-Hydroxytolbutamide
1. Introduction
The biotransformation of drugs is an important determinant of its efficacy, duration of action and toxicity.
The drug-metabolizing enzymes, cytochrome P450s, are
responsible for the metabolism, and subsequent physiological clearance of these compounds from the body. Cytochrome P450 (CYP) represents an important group of
Phase I drug-metabolizing enzymes for the biotransformation of xenobiotics (Zhou et al., 2003). The relative abundance and significance of the individual CYP enzyme in
human drug metabolism varies, with the more important
*
Corresponding author. Tel.: +852 26096886; fax: +852 26035139.
E-mail address: [email protected] (J.H.K. Yeung).
0278-6915/$ - see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2006.03.007
CYP isoform families including CYP3A, CYP2D and
CYP2C. Individual variation in the expression of CYP in
man is affected by both genetic and environmental factors.
The inducibility of the cytochrome P450 enzymes after
exposure to different drugs and chemicals is directly linked
to a number of drug-induced toxicity and drug interactions
leading to treatment failure. These drug interactions are
now more common as a result of polypharmacy (and/or
exposure to environmental pollutants) and the increasing
use of alternative medicine, including herbs and natural
products. With the increasing consumption of medicinal
herbs, often in combination with conventional therapeutic
drugs, it is important to find out if constituents in
herbal preparations or natural products may be substrates,
inhibitors or inducers of CYPs and thus affect the
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
pharmacokinetics of the co-administered drugs which rely
on CYP for their metabolism and clearance.
The fungus Coriolus versicolor is found almost worldwide, but its biological activity varies depending on the
strain and the habitat in which it grows (Monro, 2003).
Polysaccharide peptide (PSP) is a protein-bound polysaccharide isolated from Chinese fungus (Yun Chi), strain
COV-1 of C. versicolor, which has been shown to possess
immunomodulatory effect in human tumour cell lines
in vitro (Wang et al., 1999) and in animals (Yang et al.,
1993). PSP has been suggested to be useful as an adjunct
to chemotherapy and radiotherapy in phase three clinical
trials completed at 14 hospitals throughout China since
1997. Results from these clinical trials suggested that PSP
improved the quality of life by decreasing cancer treatment-related symptoms such as fatigue, loss of appetite,
nausea and vomiting, and pain in patients (Liu et al.,
1999). The objective response rate of the phase III study
was 85.8% compared to control groups (41.9%). When
combined with 5-fluorouracil, PSP improved symptoms
like dryness of throat and mouth, night sweating, and vomiting in patients with stomach cancer (Zhang et al., 1999).
In another study, PSP restored cyclophosphamide-induced
immuno-suppression by increasing the functions of natural
killer cells in patients (Qian et al., 1999), although the
mechanisms remain uncertain. In Hong Kong, PSP is
commonly used as a health product to enhance the ‘‘immunomodulatory functions’’ such that PSP and similar
products are freely available over-the-counter. A similar
compound to PSP, Polysaccharide Krestin (PSK), isolated
from CM-101 strain of C. versicolor, has also been shown
to significantly extend the five-year survival rates of
patients with cancer in the stomach (Kodama et al.,
1982), colon-rectum (Mitomi et al., 1992), esophagus (Ogoshi et al., 1995), nasopharynx (Go and Chung, 1989), and
lung (Hayakawa et al., 1993) in Japan. Both PSK and
PSP are now commonly used as health supplements in
the Southeast Asia regions.
Despite the potential use of PSP and other similar mushroom glucans and proteoglycans as adjuvants to cancer
treatments (Kidd, 2000), the metabolism of PSP and other
similar compounds has not been elucidated, although one
would predict that PSP would be metabolized to polysaccharides and other smaller peptides in vivo. Previous
in vivo study in the rat showed that the phase II metabolites of paracetamol (glucuronide and sulphate) were significantly increased by PSP, which may reflect either phase II
enzyme induction or a decrease rate of metabolism which
utilized the phase I pathways (Yeung et al., 1995). However, an earlier study that showed PSP dose-dependently
decreased the covalent binding of paracetamol to microsomal proteins in vitro suggested that the P450-mediated
generation of the chemically reactive metabolite of paracetamol was decreased in the presence of PSP (Yeung et al.,
1994). More recently, PSP has been shown to affect the
clearance of cyclophosphamide in the rat in vivo, possibly
through metabolic mechanisms involving cytochrome P450
1415
(Chan and Yeung, 2006a). Cyclophosphamide is a prodrug that requires activation by CYP-mediated 4-hydroxylation to form the active cytotoxic nitrogen mustards
(Zhang et al., 2005). Cyclophosphamide is metabolized to
a number of metabolites via P450-mediated oxidative pathways involving human CYP2B6, CYP2C9 and CYP3A4
(Chang et al., 1993) and rat CYP2B1 and CYP2C6/2C11
(Sladek, 1972; Yu and Waxman, 1996). In addition, further
in vivo studies using the model substrate antipyrine confirmed that PSP (1–3 lmol/kg, i.p., 3 days) decreased the
clearance (30–35%) and increased the plasma half-life
(55%) of antipyrine (Chan and Yeung, 2006b). Since both
antipyrine and cyclophosphamide are metabolised through
a variety of CYP isoforms in the rat and in man, the
involvement of individual CYP isoforms could not be
established only by in vivo studies. In the rat, the CYP2C
isoforms are the more abundantly expressed P450 whereas
CYP3A4 is the most abundant in man. In this study, the
effects of PSP on the metabolism of tolbutamide to 4hydroxytolbutamide, mediated by CYP2C in both human
(CYP2C9) and in rat (CYP2C11), was studied to determine
the potential of PSP in affecting CYP2C mediated phase I
metabolism in the rat, notwithstanding that PSP may also
influence the CYP2B- and CYP3A-mediated metabolism.
The acute and sub-chronic effects of PSP on tolbutamide
pharmacokinetics were also investigated in vivo to complement the in vitro studies.
2. Materials and methods
2.1. Animals
Male Sprague–Dawley rats (250–300 g) were supplied by the Laboratory Animal Service Center, The Chinese University of Hong Kong
(CUHK). The animals were kept in animal holding room under standard
conditions with 12-h light–dark cycle, with free access to rodent cubes
(Glen Forrest Stockfeeders, Australia) and tap water. All the experimental
procedures had been approved by the Animal Experimentation Ethics
Committee (CUHK) in accordance to the Department of Health
(HKSAR) guidelines in Care and Use of Animals.
2.2. Materials
Polysaccharide peptide (PSP), isolated from deep-layer cultivated
mycelia of C. versicolor, was a gift from Winsor Health Products Ltd.
(Hong Kong). Tolbutamide, 4-hydroxytolbutamide, chlorpropamide,
b-nicotinamide adenine dinucleotide phosphate (NADP), D-glucose 6phosphate, glucose 6-phosphate dehydrogenase, heparin sodium, urethane, and phenacetin were from Sigma Chemical Co. (St. Louis, MO,
USA). Sulphaphenazole was from UFC Limited (Manchester, UK).
Acetonitrile (HPLC Grade) was purchased from Labscan Analytical Sciences (Bangkok, Thailand). Methanol (HPLC Grade) was from BDH
Laboratory Supplies (Poole, UK), ethyl acetate (HPLC grade) was from
Fisher Chemicals (Leicester, UK). Acetic acid, glacial, (HPLC grade) was
from Scharlau Chemie (Barcelona, Spain). Phenobarbitone sodium was
obtained from Universal Pharmaceutical Lab. (Hong Kong). Carbon
monoxide was supplied by Hong Kong Special Gas Co.
2.3. Preparation of PSP extracts
The PSP sample was supplied by Professor Q.Y. Yang (Shanghai
Teachers’ University, China) through Winsor Health Products Ltd. (Hong
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J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
Kong) where an initial screening test has been carried out that the batch of
PSP was free from endotoxins and other contaminants. The mycelia of C.
versicolor (strain COV-1) was extracted in boiling water for 3 h, followed
by extraction with ethanol (1 part extract : 4 parts ethanol). The extracts
were concentrated in vacuo and dried to produce the PSP sample in
powder form. The PSP sample used in this study has a molecular weight of
100 kDaltons, analysed by gel electrophoresis, according to the method by
Zhou and Yang (1999) and was free from lipopolysaccharide (LPS). The
PSP sample, composed of 90% polysaccharides (74.6% glucose, 4.8%
xylose, 2.7% galactose, 1.5 % mannose, 2.4% arabinose and 4.8% rhamnose) and 10% peptides (18 amino acids are present in the protein portion:
aspartic acid, 4%; threonine 2.3%; serine 3.2%; glutamic acid, 5.8%; praline 1%; glycine, 2.6%; alanine 2.6%; cysteine, 0.9%; valine, 1.8%; methionine, 0.4%; isoleucine, 2.2%; leucine 2.4%; tyrosine, 1.5%; phenylalanine,
1.5%; tryptophan, 1.7%; lysine, 2.3; histidine, 0.7% and arginine, 1.8%),
was authenticated by Professor Q.Y. Yang (Shanghai Teachers’ University, China). Fig. 1 shows the typical partial structures of polysaccharide
portions of the polysaccharide peptide (PSP) of COV-1 strain of C. versicolor (Zhou and Yang, 1999). The water soluble fraction of PSP was
obtained by boiling in distilled water the powder form of the whole PSP
extract, with the insoluble material removed by centrifugation and filtration of the supernatant using a 0.22-lm filter. Spectral analysis (wavelength 200–450 nm) did not show obvious changes between the whole PSP
extract and the water extract, except for a slightly lower absorbance for
the latter.
2.4. Preparation of rat liver microsomes
Sprague–Dawley Rats (male, 250–300 g) were killed by exsanguinations. The liver was excised, rinsed with ice-cold 0.9% NaCl solution,
weighed and homogenised in a 0.1 mM phosphate buffer (pH 7.4) containing 0.25 M sucrose. The homogenate was centrifuged at 10,000g at 4
for 30 min. the supernatant was then centrifuged at 105,000g at 4 for
60 min. The pellet was reconstituted with 0.1 mM phosphate buffer (pH
7.4), and the protein concentration of the liver microsomes was determined by a protein assay (Lowry et al., 1951).
incubated with 0.05 M Tris/KCl buffer (pH 7.4) with NADPH-regenerating system (10 mM NADP, 5 mM G6P, 2 units/ml G6PDH, and 5 mM
magnesium chloride). The incubation mixtures were incubated in an
Eppendorf Thermomixer at 800 r.p.m. at 37. The reaction was initiated
by addition of the rat liver microsomes. Termination of the enzyme
reaction was by addition of 500 ll of ice-cold acetonitrile. The incubation
tubes were centrifuged and the supernatant extracted with 500 ll ethyl
acetate. The organic layer was dried under a gentle stream of nitrogen,
resuspended in 120 ll methanol, with 50 ll used in HPLC analysis.
Chlorpropamide (50 lg/ml, 10 ll) was used as the internal standard for
extraction and HPLC analysis.
2.6. Inhibition kinetics studies
PSP (1–10 lM) and tolbutamide (5–300 lM) were used for inhibition
kinetics studies. Sulphaphenazole (25 lM), a selective human CYP2C9
inhibitor, was used as a positive control for enzyme inhibition.
2.7. HPLC analysis of tolbutamide and 4-hydroxytolbutamide
Analysis of tolbutamide and 4-hydroxytolbutamide was by a well
established method, with modifications (Veronese et al., 1990). The HPLC
system consisted of a Hewlett Packard (HP) 1050 series pumping system
and a multiple wavelength detector (seat at 313 nM). The eluate was
analysed by passing through a guard column (Eclipse XDB-C18) and an
Alltech Allsphere ODS-2 column (250 mm · 4.6 mm, 5 lm). The mobile
phase, containing 40% acetonitrile in 10 mM sodium acetate at pH 4.3,
was eluted by gradient mode at a flow rate of 0.7 ml/min. Detection was
by u.v. absorbance at 230 nm. Standard curves of tolbutamide and 4hydroxytolbutamide were linear between 0.1 and 5 lg/ml. Precision tests
gave good reproducibility of the HPLC analysis, the relative standard
deviation was 3.7% at 50 lg/ml and 2.3% at 100 lg/ml. The accuracy of
the standard curve is 96%, 98% and 99% at 25, 50 and 100 lg/ml,
respectively.
2.8. Effects of PSP treatments on the pharmacokinetics of
tolbutamide
2.5. Analysis of (S)-warfarin hydroxylase enzyme activities
Cytochrome P450 content of the rat liver microsomes was quantified
and activity of P450 determined as described (Schenkman and Jansson,
1998). The interaction between PSP and tolbutamide towards (S)-warfarin
hydroxylase (CYP2C11) activity in rat liver microsomes in vitro was
evaluated by adding these substances at various concentrations (PSP 0.5–
20 lM, tolbutamide 5 lM). Control incubations for tolbutamide
4-hydroxylation contained no PSP. Liver microsome (1 mg/ml) was
For the acute treatments, 8 animals per group were treated with PSP
(4 lmol/kg, i.p.), or saline (control). Lower doses (1–2 lmol/kg) of PSP
were not selected because they did not affect the clearance of cyclophosphamide (Chan and Yeung, 2006a). After the pre-treatments, the rats were
anaesthetized with urethane (20% w/v, 6 ml/kg, i.p.). The carotid artery
was cannulated for collecting blood sample, jugular vein for injection of
tolbutamide and replacing saline, and trachea for removing mucous. A
single dose of tolbutamide (10 mg/kg, i.v.) was given via jugular vein
CH2OH
O
OH
HO
1
3
β
O
OH
CH2OH
CH2OH
O
OH
1
HO
O
O
β
HO
OH
1
3
β
1
3
O
β
HO
OH
CH2OH
O
O
O
HO
OH
CH2OH
CH2
1
3
O
O
β
HO
OH
CH2OH
1
3
O
O
β
HO
OH
HOH
3
OH
m
n
Fig. 1. A diagram showing the typical partial structures of polysaccharide portions of the polysaccharide peptide (PSP) of COV-1 strain of Coriolus
versicolor (adopted from Zhou and Yang, 1999).
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
100
IC 50 = 18.4 μM
*
% formation of 4-hydroxytolbutamide
30 min after the treatment. Serial blood samples (0.3 ml) were collected via
the carotid artery at 0, 10, 20, 40, 60, 90, 120, 180, 240, 300 and 360 min
after tolbutamide administration. Heparinized saline (100 units/ml) was
given to prevent blood coagulation. Saline was replaced via jugular vein
each time after a blood sample collection. Plasma was separated and
stored at 20 C prior to analysis by High Performance Liquid Chromatography (HPLC). Cimetidine (60 mg/kg, i.p.) was used as positive
controls for enzyme inhibition studies.
For the sub-chronic treatments, 8 animals per group were pre-treated
with PSP (1–2 lmol/kg/day, i.p.) or saline (control, i.p.) for 3 days. The
dosages of PSP used were based on reports previously when PSP was welltolerated and showed no sign of toxicity to the laboratory animals (Yeung
et al., 1994, 1995) and similar studies with cyclophosphamide (Chan and
Yeung, 2006a). During the pre-treatment period, the rats were kept in a
12-h light/dark cycle animal room with controlled temperature and
humidity. Free access to laboratory rodent diet and tap water was allowed.
One day after the final pre-treatment, the rats were anaesthetized and
experiments were performed as described in previous section. Phenobarbitone (40 mg/kg/day, i.p., 3 days) was used as positive controls for
enzyme induction.
1417
***
75
***
***
50
25
0
-0.50
-0.25
0.00
0.25
0.50
(a)
0.75
1.00
1.25
1.50
log [PSP]
2.9. Data analysis
V ¼
V0
S
1 þ ð1=IC50 Þ
;
where V0 is uninhibited velocity, V is observed velocity, S is slope factor
and I is inhibitor concentration. A Lineweaver–Burk Plot is a double reciprocal plot in which varying substrate concentrations are plotted against
reaction velocities to obtain linear transformation. The enzyme parameter
Michaelis constant (Km) and Vmax values were obtained from Lineweaver–
Burk Plot. The inhibition constant (Ki), the inhibitor concentration at
which the reaction is half of the maximal rate, was obtained by a secondary plot using the slope of the primary Lineweaver–Burk Plot and fitted by
GraphPad Prism 4.
100
% formation of 4-hydroxytolbutamide
Statistical analysis of the data was carried out using ANOVA by a
computer program (Statview 9.0, Abacus Concepts, USA). Enzyme
kinetics data were fitted by non-linear regression analysis using GraphPad
Prism 4 (GraphPad Software, CA, USA). IC50 values (concentration of
inhibitor to cause 50% inhibition of original enzyme activity) were
determined by GraFit where appropriate using the following equation:
3.1. Effect of PSP on (S)-warfarin hydroxylase enzyme
activities in vitro
PSP inhibited the formation of 4-hydroxytolbutamide in
a concentration-dependent manner (Fig. 2(a) and (b)) with
a decrease in the 4-hydroxytolbutamide/tolbutamide ratio
(Fig. 3(a) and (b)). The IC50 of the whole PSP extract
was 18.38 lM and the IC50 for the water extract PSP was
9.87 lM. Enzyme kinetics studies of the rat (S)-warfarin
hydroxylase (CYP2C11) were determined with various tolbutamide concentrations, in the presence or absence of
PSP. As shown in Fig. 4(a), the Lineweaver–Burke linear
transformation of the enzyme velocities versus substrate
concentration showed that PSP act as a competitive inhibitor. Lineweaver–Burke plot of the whole PSP extract
(Fig. 4(a)) and the subsequent secondary plot (Fig. 4(b))
confirmed that the inhibition was competitive, with a Ki
of 12.62 lM. The water extract of PSP showed similar
competitive inhibition (Fig. 5(a)) and from the secondary
(b)
IC50 = 9.8 μM
75
***
***
50
***
25
0
-0. 50
3. Results
*
-0 .25
0.00
0.25
0.50
0.75
1.00
1.25
1.50
log [PSP]
Fig. 2. Inhibition of 4-hydroxytolbutamide formation in rat liver microsomes by (a) whole PSP extract and (b) water extract of PSP. Results were
mean ± SEM of 6 separate assays. Statistical significance was determined
by analysis of variance; ***p < 0.001 and *p < 0.05 when compared with
control.
plot, Ki was calculated as 6.97 lM (Fig. 5(b)). The inhibitory effects of PSP on tolbutamide 4-hydroxylation were
comparable to sulphaphenazole, a specific human CYP2C9
inhibitor.
3.2. Effects of acute PSP treatment on 4-hydroxylation
and pharmacokinetics of tolbutamide
Figs. 6(a) and 6(b) shows the plasma concentration–time
profiles of tolbutamide and 4-hydroxytolbutamide, respectively, after acute PSP (4 lmol/kg, i.p.) treatment. Acute
treatment of PSP did not alter the clearance, T1/2, and
AUC of tolbutamide (Table 1) although the Cinitial was
1418
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
0.25
1/V (1/nmol/mg protein/min)
4-hydroxytolbutamide / tolbutamide ratio
4
3
**
***
2
***
No PSP
1μM PSP
2μM PSP
0.20
5μM PSP
10μM PSP
0.15
0.10
0.05
***
1
***
-0.05
0.05
0.10
0.15
0.20
0.25
1/[tolbutamide] (1/μM)
0
(a)
20
10
5.
0
2.
0
0
1.
0.
5
0
-0.05
Fig. 4(a). Lineweaver–Burke plots of rat CYP2C11 activity versus
tolbutamide concentration in the presence of various concentrations of
whole PSP extract.
PSP (whole) (μM)
1.4
3
*
1.2
Slope of Primary plot
4-hydroxytolbutamide / tolbutamide ratio
4
2
***
***
1
1.0
0.8
y=0.04674x + 0.5897
R 2 = 0.9419
0.6
0.4
***
0.2
Ki = 12.6 μM
(b)
20
10
5.
0
2.
0
1.
0
0.
5
0
0
PSP (soluble extract) (μM)
-14
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
PSP (μM)
Fig. 3. Effects of PSP on 4-hydroxytolbutamide/tolbutamide ratio in rat
liver microsomes studies with whole PSP extract (a) and water extract of
PSP (b). Results were mean ± SEM of 6 rats. Statistical significance was
determined by analysis of variance; ***p < 0.001, **p < 0.01 and *p < 0.05
when compared with control.
decreased by 7.4% and the Vd increased by a similar magnitude (7.4%). The formation of 4-hydroxytolbutamide was
decreased in the first 60 min.
3.3. Effects of sub-chronic PSP treatment on
4-hydroxylation and pharmacokinetics of tolbutamide
The plasma concentration–time profiles of tolbutamide
and 4-hydroxytolbutamide after sub-chronic PSP pre-treatment at different doses were shown in Figs. 7(a), 8(a), 7(b)
and 8(b), respectively. As shown in Table 2, sub-chronic
pre-treatment of PSP (1–2 lmol/kg/day, i.p.) for three days
did not affect the clearance and AUC of tolbutamide. How-
Fig. 4(b). Secondary plot of rat tolbutamide 4-hydroxylase activity using
the slopes of the primary Lineweaver–Burke plots versus concentrations of
whole PSP. The Ki for whole PSP extract was 12.6 lM.
ever, the Cinitial was decreased, together with increases in
the T1/2 and Vd. The increases in Vd after both acute and
sub-chronic PSP pre-treatment indicated PSP caused
changes in the plasma distribution of tolbutamide. The
effects of cimetidine, a P450 inhibitor, and phenobarbitone,
an enzyme inducer were as expected in terms of their ability
to alter tolbutamide 4-hydroxylation (Figs. 9(b) and 10(b))
and caused changes in the pharmacokinetics of tolbutamide (Figs. 9(a) and 10(a)).
4. Discussion
Botanical and other natural products including herbal
products and nutraceuticals are increasingly used in
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
80
No PSP
1μM PSP
2μM PSP
5μM PSP
10μM PSP
0.20
Control
4 μmol/kg PSP
70
60
0.15
0.10
0.05
-0.05
0.05
0.10
0.15
0.20
0.25
1/[tolbutamide] (1/μM)
Tolbuttamide conc. (μg/ml)
1/V (1/nmol/mg protein/min)
0.25
1419
-0.05
50
**
**
40
30
20
Fig. 5(a). Lineweaver–Burke plots of rat CYP2C11 activity versus
tolbutamide concentration in the presence of various concentrations of
water extract of PSP.
10
0
60
0
1.4
120
180
240
300
360
Time (min)
Fig. 6(a). Concentration–time profile of tolbutamide (10 mg/kg, i.v.) after
acute treatment with saline (control) or PSP (4 lmol/kg). Results were
mean ± SEM of 8 rats. **p < 0.01 when compared with control.
Slope of Primary plot
1.2
1.0
y=0.07299x + 0.5084
0.8
3.0
R 2 = 0.9683
Control
4 μmol/kg PSP
0.6
0.4
0.2
Ki = 6.9 μM
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
PSP (μM)
Fig. 5(b). Secondary plot of rat tolbutamide 4-hydroxylase activity using
the slopes of the primary Lineweaver–Burke plots versus concentrations of
water extract of PSP. The Ki for whole PSP extract was 6.9 lM.
4-hydroxytolbutamide conc. (μg/ml)
2.5
2.0
1.5
*
*
1.0
0.5
different countries. These products are commonly known
as dietary supplements in United States of America, natural health products in Canada, phytomedicines in Europe
or traditional medicines in other developing countries (Foster et al., 2005). The potential interaction of these herbal
products with drugs is a major safety concern, especially
for drugs with narrow therapeutic indices or in patients
receiving drug treatment for chronic illness/disease, which
may lead to serious clinical consequences. The importance
of herb–drug interactions and the diversity of such interactions has been recently reviewed (Hu et al., 2005). The
induction or inhibition of hepatic and intestinal drug met-
0.0
0
60
120
180
240
300
360
Time (min)
Fig. 6(b). Concentration–time profile of 4-hydroxytolbutamide (10 mg/
kg, i.v.) after acute treatment with saline (control) or PSP (4 lmol/kg).
Results were mean ± SEM of 8 rats. *p < 0.05 when compared with
control.
abolising enzymes such as cytochrome P450 (CYP) and/or
drug transporters such as P-glycoprotein (P-gp) represent
the major mechanism for the enhanced or reduced
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J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
Table 1
Pharmacokinetics of tolbutamide (10 mg/kg, i.v.) after acute treatment of PSP whole extract (4 lmol/kg, i.p.)
Cinital (lg/ml)
AUC (lg h/ml)
Vd (ml/kg)
CL (ml/min/kg)
T1/2 (min)
Control (saline)
PSP (4 lmol/kg)
Cimetidine (60 mg/kg)
Phenobarbitone
(40 mg/kg/d, 3 days)
55.8 ± 1.0
297.2 ± 10.7
177.7 ± 3.4
0.56 ± 0.0
222.0 ± 7.7
51.7 ± 0.7*
288.5 ± 7.5
190.8 ± 1.5*
0.40 ± 0.0
232.2 ± 4.3
56.9 ± 1.4
431.6 ± 34.6***
174.7 ± 3.7
0.40 ± 0.0**
314.4 ± 22.2***
40.0 ± 1.4***
119.5 ± 1.8***
250.5 ± 8.9***
1.39 ± 0.0***
124.8 ± 5.5***
Results were mean ± SEM of 8 animals.
***
p < 0.001,
**p
< 0.01 and *p < 0.05 when compared with controls.
100
3.0
Control
Control
1 μmol/kg/day PSP
1 μmol/kg/day PSP
2.5
4-hydroxytolbutamide conc. (μg/ml)
Tolbutamide conc. (μg/ml)
80
60
***
***
***
40
***
***
***
***
***
20
***
2.0
1.5
1.0
0.5
0
0
60
120
180
240
300
360
Time (min)
0.0
0
60
120
180
240
300
360
Time (min)
Fig. 7(a). Concentration–time profile of tolbutamide (10 mg/kg, i.v.) after
sub-chronic (3 days) treatment with saline (control), or PSP (1 lmol/kg/
day). Results were mean ± SEM of 8 rats. ***p < 0.001 when compared
with control.
Fig. 7(b). Concentration–time profile of 4-hydroxytolbutamide after subchronic (3 days) treatment with saline (control) or PSP (1 lmol/kg/day).
Results were mean ± SEM of 8 rats.
bioavailability of drugs when herbal/botanical products are
co-administered (Zhou et al., 2003, 2004). Initial evidence
that PSP may affect cytochrome P450-mediated metabolism came from the toxicological study (Yeung et al.,
1994) in which PSP reduced the covalent binding of paracetamol in rat liver microsomes in vitro and protection
against paracetamol toxicity in vivo in the rat. The chemically reactive metabolite of paracetamol was widely
accepted as the intermediate generated via cytochrome
P450-mediated metabolism when paracetamol was present
at a high concentration such as in an overdose situation,
the decrease in the covalent binding of paracetamol by
PSP (Yeung et al., 1994) would be indicative of less of
the cytochrome P450-mediated chemically reactive metabolite being formed. Recently, PSP has been shown to
decrease the clearance of cyclophosphamide in the rat
in vivo (Chan and Yeung, 2006a). The current understanding in the metabolism of cyclophosphamide (Chang et al.,
1993) suggested that CYP2B6, CYP2C9 and CYP3A may
be involved.
CYP2C9 is a major cytochrome P450 enzyme involved
in the metabolism of a wide range of therapeutic agents,
including non-steroidal anti-inflammatory drugs, oral anticoagulants and oral hypoglycaemic agents (Rettie and
Jones, 2005). More than 100 currently used drugs are
known substrates of CYP2C9 which corresponds to 10–
20% of commonly prescribed drugs. In addition, CYP2C9
also significantly contributes to the metabolism of fatty
acids, prostanoids and steroid hormones (Kirchheiner
and Brockmoller, 2005). The rat forms of the human
CYP2C9 equivalent CYP2C isoforms include CYP2C6
and CYP2C11 where CYP2C11 being a homologue of
the human 2C9 with 77% homology. In the rat, the CYP2C
isoforms are more important since they are the more abundantly expressed CYP, in a way equivalent to CYP3A4 in
man. It is clear from the in vitro inhibition kinetic studies
in rat liver microsomes that PSP can act as a competitive
inhibitor to rat (S )-warfarin hydroxylase (CYP2C11) which
is mainly responsible for tolbutamide 4-hydroxylation.
Although tolbutamide hydroxylation has been shown to
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
100
Control
2 μmol/kg/day PSP
Table 2
Pharmacokinetics of tolbutamide (10 mg/kg, i.v.) after sub-chronic
treatment of PSP whole extract (1–2 lmol/kg/day, i.p.) for three days
Control (saline)
PSP
(1 lmol/kg/ day)
PSP
(2 lmol/kg/ day)
62.2 ± 1.1
407.2 ± 10.5
141.9 ± 2.1
0.41 ± 0.0
245.0 ± 6.5
52.2 ± 0.7***
401.9 ± 8.3
185.1 ± 2.4***
0.40 ± 0.0
321.1 ± 9.5***
49.9 ± 1.5***
407.2 ± 10.5
195.3 ± 6.0***
0.43 ± 0.01
319.5 ± 10.3***
Tolbutamide conc. (μg/ml)
80
Cinital (lg/ml)
AUC (lg h/ml)
Vd (ml/kg)
CL (ml/min/kg)
T1/2 (min)
60
***
Results were mean ± SEM of 8 animals. ***p < 0.001 when compared with
control.
***
***
40
1421
***
***
***
***
***
20
***
80
*
Control
Cimetidine
0
60
120
180
240
300
360
Time (min)
Fig. 8(a). Concentration–time profile of tolbutamide (10 mg/kg, i.v.) after
sub-chronic (3 days) treatment with saline (control), or PSP (2 lmol/kg/
day). Results were mean ± SEM of 8 rats. ***p < 0.001 and *p < 0.05 when
compared with control.
3.0
60
Tolbutamide conc. (μg/ml)
0
**
40
*
**
*
20
Control
2 μmol/kg/day PSP
4-hydroxytolbutamide conc. (μg/ml)
2.5
0
0
2.0
60
120
180
240
300
360
Time (min)
Fig. 9(a). Concentration–time profile of tolbutamide (10 mg/kg, i.v.) after
acute treatment with saline (control), or cimetidine (60 mg/kg). Results
were mean ± SEM of 8 rats. ***p < 0.001, **p < 0.01and *p < 0.05 when
compared with control.
1.5
**
****
1.0
**
**
***
**
**
*
**
0.5
0.0
0
60
120
180
240
300
360
Time (min)
Fig. 8(b). Concentration–time profile of 4-hydroxytolbutamide after subchronic (3 days) treatment with saline (control) or PSP (2 lmol/kg/day).
Results were mean ± SEM of 8 rats. ***p < 0.001, **p < 0.01 and *p < 0.05
when compared with control.
be increased following phenobarbital (an inducer of
CYP2B in rat liver) pre-treatment (Veronese et al., 1990),
other studies showed that tolbutamide 4-hydroxylation
was observed in both human and uninduced rat liver
microsomes (Easterbrook et al., 2001). Thus the results
presented here are in agreement with our previous in vivo
studies when PSP has been shown to decrease antipyrine
clearance (Chan and Yeung, 2006b) and decrease the clearance of cyclophosphamide (Chan and Yeung, 2006a), both
drugs utilising CYP2C9 as one of the enzymes for their
metabolism in man. In this study, the Ki (6.9 lM) of PSP
to inhibit tolbutamide 4-hydroxylation in rat liver microsomes was lower than that of sulphaphenazole, a specific
human CYP2C9 inhibitor. However, it should be noted
that sulphaphenazole was much more potent at inhibiting
tolbutamide metabolism in human liver microsomes than
rat liver microsomes (Eagling et al., 1998). It would be of
interest to study how PSP may affect the CYP2C9 activity
in human liver microsomes or other specific individual
human CYP isoforms, especially CYP3A4 which is responsible for the metabolism of about 50% of therapeutic drugs.
Thus, it is possible that PSP may affect CYP3A4, given the
1422
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
4
5
Cimetidine
***
4-hydroxytolbutamide conc. (μg/ml)
4-hydroxytolbutamide conc. (μg/ml)
4
3
2
***
**
1
** ***
***
**
***
***
*
**
0
***
***
**
***
3
2
**
1
0
0
60
120
180
240
300
360
Fig. 9(b). Concentration–time profile of 4-hydroxytolbutamide after acute
treatment with saline (control), or cimetidine (60 mg/kg). Results were
mean ± SEM of 8 rats. ***p < 0.001, **p < 0.01 and *p < 0.05 when
compared with control.
80
Control
Phenobarbitone
60
***
***
40
***
***
***
20
***
***
***
***
***
240
300
360
0
0
60
120
180
0
60
120
180
240
300
360
Time (min)
Time (min)
Tolbutamide conc. (μg/ml)
Control
Phenobarbitone
***
Control
Time (min)
Fig. 10(a). Concentration–time profile of tolbutamide (10 mg/kg, i.v.)
after sub-chronic (3 days) treatment with saline (control), or phenobarbitone (40 mg/kg/day, i.p.). Results were mean ± SEM of 8 rats.
***p < 0.001 and *p < 0.05 when compared with control.
abundance of this CYP isoform in man and its wide-range
of reactions.
The effects of PSP in vivo have also been carried out in
this study, to investigate any enzyme inducing effects on
Fig. 10(b). Concentration–time profile of 4-hydroxytolbutamide after
sub-chronic (3 days) treatment with saline (control), or phenobarbitone
(40 mg/kg/day, i.p.). Results were mean ± SEM of 8 rats. ***p < 0.001,
**p < 0.01 and *p < 0.05 when compared with control.
CYPs as well as to check for any the pharmacokinetic
changes by PSP. The dosage selection of PSP in acute
and sub-chronic studies was based on previous in vitro
and in vivo studies with antipyrine and cyclophosphamide
(Chan and Yeung, 2006a,b). Acute PSP (4 lmol/kg, i.p.)
treatment only led to minimum changes in tolbutamide
pharmacokinetics and tolbutamide 4-hydroxylation. However, sub-chronic PSP treatment at lower doses (1–2 lmol/
kg/day) produced more pronounced changes in the pharmacokinetics of tolbutamide and 4-hydroxylation of tolbutamide. Although the formation of 4-hydroxytolbutamide
was decreased, the major pharmacokinetic parameter
affected was the apparent volume of distribution (Vd)
which suggested that PSP, in addition to inhibiting P450mediated reactions, may also affect the distribution of tolbutamide. The significant changes in the Vd thus led to an
overall increase in plasma half-life of tolbutamide, but with
no effect on the clearance. It was also confirmed in this
study that PSP has no inducing effects on CYPs, which
again complemented with our previous findings when antipyrine was the model compound used.
It is clear from the large amount of literature available
that findings related to the metabolism of xenobiotics in
rodents do not necessary correlate to humans because of
the different specificities in the CYPs. Extrapolation of animal data to humans and the conclusions must be treated
with caution. However, the availability of animal models
to study the potential herb–drug interactions would be a useful initial screening for the increasing number of herbs and
natural products now available, especially with combined
J.H.K. Yeung et al. / Food and Chemical Toxicology 44 (2006) 1414–1423
pharmacodynamic–pharmacokinetic (PD–PK) studies that
are relatively easier to perform in animal models. Taken
together results from previous studies with antipyrine and
the effects of PSP on tolbutamide pharmacokinetics and
inhibition of tolbutamide 4-hydroxylation in vitro and
in vivo in the current study, careful monitoring would be
essential for the concomitant use of PSP with other drugs,
particularly those metabolized by CYP2C9.
Acknowledgements
The authors gratefully acknowledge the gift of PSP from
Winsor Health Products Ltd. (Hong Kong) and Professor
Q.Y. Yang (Shanghai Teachers’ University, China) for
authentication of the PSP sample. Dr. S.L. Chan received
a post-graduate studentship from The Chinese University
of Hong Kong.
References
Chan, S.L., Yeung, J.H.K., 2006a. Effects of polysaccharide peptide (PSP)
from C. versicolor on the pharmacokinetics of cyclophosphamide in
the rat and cytotoxicity in HepG2 cells. Food Chem. Toxicol. 44, 689–
694.
Chan, S.L., Yeung, J.H.K., 2006b. Modulation of antipyrine clearance by
polysaccharide peptide isolated from C. versicolor in the rat Food
Chem. Toxicol., in press, doi:10.1016/j.fct.2006.03.008.
Chang, T.K., Weber, G.F., Crespi, C.L., Waxman, D.J., 1993. Differential
activation of cyclophosphamide and ifosphamide by cytochromes P450
2B and 3A in human liver microsomes. Cancer Res. 53, 5629–5637.
Eagling, V.A., Tjia, J.F., Back, D.J., 1998. Differential selectivity of
cytochrome P450 inhibitors against probe substrates in human and rat
liver microsomes. Br. J. Clin. Pharmacol. 45 (2), 107–114.
Easterbrook, J., Fackett, D., Li, A.P., 2001. A comparison of aroclor
1254-induced and uninduced rat liver microsomes to human liver
microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation,
tolbutamide 4-hydroxylation, S-mephenytoin 4 0 -hydroxylation, chloroxazone 6-hydroxylation and testosterone 6b-hydroxylation. Chem.
Biol. Interact. 134, 243–249.
Foster, B.C., Arnason, J.T., Briggs, C.J., 2005. Natural health products
and drug disposition. Annu. Rev. Pharmacol. Toxicol. 45, 203–226.
Go, P., Chung, C.H., 1989. Adjuvant PSK immunotherapy in patients
with carcinoma of the nasopharynx. J. Int. Med. Res. 17, 141–
149.
Hayakawa, K., Mitsuhashi, N., Saito, Y., Takahashi, M., Katano, S.,
Shiojima, K., Furuta, M., Niibe, H., 1993. Effect of krestin (PSK) as
adjuvant treatment on the prognosis after radical radiotherapy in
patients with non-small cell lung cancer. Anticancer Res. 13, 1815–
1820.
Hu, Z., Yang, X., Ho, P.C.L., Chan, S.Y., Heng, P.W.S., Chan, E., Duan,
W., Koh, H.L., Zhou, S., 2005. Herb–drug interactions: a literature
review. Drugs 65 (9), 1239–1282.
Kidd, P.M., 2000. The use of mushroom glucans and proteoglycans in
cancer treatment. Altern. Med. Rev. 5, 4–27.
Kirchheiner, J., Brockmoller, J., 2005. Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin. Pharmacol. Ther. 77, 1–16.
Kodama, Y., Kano, T., Tamada, R., Kumashiro, R., Okamura, T.,
Inokuchi, K., 1982. Combined effect of prophylactic lymphadenectomy and long term combination chemotherapy for curatively resected
carcinoma of the stomach. Jpn. J. Surg. 12, 244–248.
Liu, J.X., Zhou, J.Y., Liu, T.F., 1999. Phase III clinical trial for the Yun
Zhi polysacchride (PSP) capsules. In: Yang, Q.Y. (Ed.), Advanced
Research in PSP 1999. The Hong Kong Association for Health Care
Limited, Hong Kong, pp. 295–303.
1423
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 243, 1331–
1332.
Mitomi, T., Tsuchiya, S., Iijima, N., Aso, K., Suzuki, K., Nishiyama, K.,
Amano, T., Takahashi, T., Murayama, N., Oka, H., et al., 1992.
Randomized, controlled study on adjuvant immunochemotherapy
with PSK in curatively resected colorectal cancer. The Cooperative
Study Group of Surgical Adjuvant Immunochemotherapy for Cancer
of Colon and Rectum (Kanagawa). Dis. Colon. Rectum. 35, 123–130.
Monro, J.A., 2003. Treatment of cancer with mushroom products. Achiv.
Environ. Health 58 (8), 533–537.
Ogoshi, K., Satou, H., Isono, K., Mitomi, T., Endoh, M., Sugita, M.,
1995. Possible predictive markers of immunotherapy in esophageal
cancer: retrospective analysis of a randomized study. Cancer Invest.
13, 363–369.
Qian, Z.M., Xu, M.F., Tang, P.L., 1999. Polysaccharide peptide (PSP)
restores immunosuppression induced by cyclophosphamide in rats. In:
Yang, Q.Y. (Ed.), Advanced Research in PSP 1999. The Hong Kong
Association for Health Care Limited, Hong Kong, pp. 154–163.
Rettie, A.E., Jones, J.P., 2005. Clinical and toxicological relevance of
CYP2C9: Drug–drug interactions and pharmacogenetics. Annu. Rev.
Pharmacol. Toxicol. 45, 477–494.
Schenkman, J.B., Jansson, I., 1998. Spectral analysis of cytochrome P450.
In: Phillips, I.R., Stephard, E.A. (Eds.), Methods in Molecular
Biology, Cytochrome P450 Protocols, vol. 107. Human Press Inc.,
Totowa, pp. 25–33.
Sladek, N.E., 1972. Therapeutic efficiency of cyclophosphamide as a
function of its metabolism. Cancer Res. 32, 535–542.
Veronese, M.E., Mcmanus, M.E., Laupattarakasem, P., Minors, J.O.,
Birkett, D.J., 1990. Tolbutamide hydroxylation by human, rabbit and
rat liver microsomes by purified forms of cytochrome P450. Drug
Metab. Dispos. 18, 356–361.
Wang, S.Y., Hsu, M.L., Yang, X.T., 1999. Growth inhibition and
apoptosis in human leukemia cells induced by cytokines released from
PSP-activated blood mononuclear cells. In: Yang, Q.Y. (Ed.),
Advanced Research in PSP 1999. The Hong Kong Association for
Health Care Limited, Hong Kong, pp. 128–130.
Yang, Q.Y., Hu, Y.J., Li, X.Y., Yang, J.C., Liu, J.X., Liu, T.F., Xu,
G.M., Liao, M.L., 1993. A new biological response modifier substance
– PSP. In: Yang, Q.Y., Kwok, C.Y. (Eds.), 1993 PSP International
Symposium, Anthology of Theses and Abstracts. Fudan University
Press, Shanghai, pp. 55–72.
Yeung, J.H.K., Chiu, L.C.M., Ooi, V.E., 1994. Effect of polysaccharide
peptide (PSP) on glutathione and protection against paracetamolinduced hepatotoxicity in the rat. Meth. Find Exp. Clin. Pharmacol.
16, 723–729.
Yeung, J.H.K., Chiu, L.C.M., Ooi, V.E., 1995. Effect of polysaccharide
peptide (PSP) on in vivo sulphation and glucuronidation of paracetamol in the rat. Eur. J. Drug Metab. Pharmacokinet. 20, 287–292.
Yu, L., Waxman, D.J., 1996. Role of cytochrome P450 in oxa-phosphorine metabolism: deactivation via N-dechloroethylation and activation
via 4-hydroxylation catalyzed by distinct subsets of rat liver cytochromes P450. Drug. Metab. Dispos. 24, 1254–1262.
Zhang, L.Y., Zhong, Y., Zhou, J., 1999. The observation of PSP decrease
60 cases chemotherapeutical stomach cancer’s side effect. In: Yang,
Q.Y. (Ed.), Advanced Research in PSP 1999. The Hong Kong
Association for Health Care Limited, Hong Kong, p. 328.
Zhang, J., Tian, Q., Chan, Y.S., Li, S.C., Zhou, S., Duan, W., Zhu, Y.Z.,
2005. Metabolism and transport of oxazaphosphorines and the clinical
implications. Drug Metab. Rev. 37 (4), 611–703.
Zhou, Y.L., Yang, Q.Y., 1999. Active principles from Coriolus sp. In:
Yang, Q.Y. (Ed.), Advanced Research in PSP 1999. The Hong Kong
Association for Health Care Limited, Hong Kong, pp. 111–124.
Zhou, S., Gao, Y., Jiang, W., Huang, M., Xu, A., Paxton, J.W., 2003.
Interactions of herbs with Cytochrome P450. Drug Meta. Rev. 35 (1),
35–98.
Zhou, S., Lim, L.Y., Chowbay, B., 2004. Herbal modulation of Pglycoprotein. Drug Meta. Rev. 36 (1), 57–104.