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Introduction
Para-toluene-sulfonamide (p-methylbenzenesulfonamide; PTS) is a novel anticancer reagent for which phase III clinical
trials are being conducted in China. PTS is intended mainly for the treatment of breast cancer and head and neck squamous
cancer. It is delivered by intravenous or intratumoral injection as an adjunct to chemotherapy and radiation therapy. The
reagent has good lipophilic ability, and its clinical pharmacokinetics accord with the one-compartment model.
Because cytochrome P450 enzyme play a prominent role in the metabolism of many pharmaceutical agents and activation
or deactivation of potential carcinogens, it would be useful to know as early in the development process as possible that
which CYP enzymes are likely to process a new chemical entity (NCE), which CYP activities are likely to be altered by an NCE
and the magnitude of the alteration. Moreover, CYP genes have promise in enhancing the sensitivity of tumor cells to cancer
chemotherapeutic drugs[1]. Inhibition screening is very useful not only for investigating the potential influence of new drugs
on liver CYP, but also for future applications of pharmacogenomics and pharmacogenetics to personalize treatment
regimens[2].
In our experiments, we chose five CYP isoforms and their specific substrates (phenacetin for CYP1A2, tolbutamide for
CYP2C7, dextromethorphan for CYP2D1 and CYP3A2, and chlorozoxazone for CYP2E1). Dextromethorphan is reduced to
dextrorphan by the rat CYP2D1 enzyme. In a
parallelpathway, it is N-demethylated to 3-methoxymorphinan, which is
catalyzed by CYP3A2[3]. The known inhibitors selected for CYP1A2, CYP2C7, CYP2D1 and CYP3A2 were, respectively,
a-naphthoflavone, sulfaphenazole, quinidine, and ketoconazole. The present study was designed to: (1) investigate the effect
of PTS pretreatment on rat liver CYP content; (2) examine the kinetic parameters of PTS incubation metabolism toward CYP;
(3) to identify the principal CYP isoforms that metabolize PTS; (4) assess the inhibitory mechanism of known inhibitors on
PTS metabolism in vitro; (5) investigate the modulation effect of PTS on the activities of selected CYP isoforms.
Materials and methods
Chemicals PTS standard was manufactured by Aldrich (Wyoming, IL, USA), phenacetin (Phe), acetaminophen (Ace),
tolbutamide (Tol), 4-hydroxytolbutamide (4-OH-Tol), chlorozoxazone (Chl), 6-hydroxychlorozoxazone (6-OH-Chl),
dextromethorphan hydrobromide monohydrate (Dex), dextrorphan (Dor), 3-methoxymorphinan (3-MM),
a-naphtho- flavone (a-Naph), sulfaphenazole (Sulf), quinidine (Qui), ketoconazole (Ket),
antipyrine, b-nicotinamide adenine dinucleotide
phosphate (b-NADP), glucose-6-phosphate (G-6-P), and glucose-6-phosphate dehydrogenase (G-6-PDH) were obtained from
Sigma-Aldrich (St Louis, MO, USA). Chromatographic
grade acetonitrile was purchased from Fisher Scientific (Fair Lawn,
NJ, USA). All other supplies were of the highest grades available from standard commercial sources.
Solutions Stock solutions of the analytes were prepared separately by dissolving each compound in water with
acetonitrile at concentrations lower than 1%
(v/v)[4]. G-6-PDH was dissolved in 5
mmol/L sodium citrate and was kept at -80
oC until used.
Microsome preparation Male Wistar rats (3 months old, weighing 230-260 g) were purchased from the Department of
Laboratory Animal Sciences, Capital University of Medical Sciences. Animals were divided into 4 groups, with 6 animals in
every group. The groups received the following treatments: blank control, phenobarbital iv injection (40 mg/kg per
d)[5], or PTS intravenous injection via the tail vein
at dosages of 33 and 99 mg/kg perd .
Rats were killed after the animals had been treated for 4 consecutive days. Microsomes were prepared by differential
centrifugation and the preparation was stored at -80
oC until used. All procedures were performed at 0-4
oC[6]. Total CYP content and microsome protein were measured by using the method of
Omura[7] and Lowry[8]. SPSS pharmaceutical software
was used for statistical comparisons. The significance level was set at
P<0.05.
Microsome incubation and sample preparation
The
incubation volume was 0.5 mL, containing 1.0 mg protein, 100 mmol/L potassium phosphate buffer (pH 7.4), and an NADPH-
generating system (0.5 mmol/L b-NADP, 2.0 mmol/L G-6-P,
5 mmol/L MgCl2, and 0.1 mmol/L ethylenediamine tetraacetic
acid). The samples were preincubated for 5 min at 37
oC prior to the addition of 2 U G-6-PDH. Reactions were carried out for
30 min and terminated by placement into icy water and the addition of 100 µL 7% perchloric acid. The samples were
centrifuged at 16 100× g for 10 min to pellet the protein precipitate. The supernatant was transferred for HPLC analysis. All
incubations were carried out in triplicate, and the mean values were used for
analysis[9].
Apparatus and chromatographic conditions
[10] The samples were analyzed on an Agilent 1100 series liquid
chromatograph (Agilent Technologies, Waldbronn, Germany) at room temperature. An aliquot (50 µL) from each sample was injected
onto an Agilent XDB C18 column (4.6 mm×250 mm, 5 µm). Compounds were quantified using their peak areas.
For CYP1A2 phenacetin O-deethylase, the mobile phase consisted of a gradient of acetonitrile/water with 0.1% acetic acid
(pH 3.5) monitored at 230 nm. The flow rate was 1.2
mL/min. The retention times of Ace and Phe were 4.7 min and 9.9 min, respectively.
For CYP2C7 tolbutamide methylhydroxylation, mobile phase A consisted of acetonitrile, and mobile phase B consisted of
water with 0.1% acetic acid (pH 3.5; 40:60), monitored at 230 nm. The flow rate was 1.2 mL/min. The retention times of
4-OH-Tol and Tol were 3.8 min and 11.3 min, respectively.
For CYP2D1 dextro-O-demethylation and CYP3A2
dextro-N-demethylation, the mobile phase consisted of a gradient of
acetonitrile/water with acetic acid and 0.1% triethylamine (pH 4.5) monitored at 277 nm. The flow rate was 1.0 mL/min. The
retention times of Dor, 3MM and Dex were 4.7 min, 10.6 min and 11.1 min,
respectively.
For CYP2E1 chlorzoxazone 6-hydroxylation, mobile phase A consisted of acetonitrile and mobile phase B consisted of
water with 0.1% acetic acid (pH 3.5; 40:60) monitored at 278
nm. The flow rate was 1.2 mL/min. The retention times of
6-OH-Chl and Chl were 3.1 min and 6.2 min, respectively.
For PTS, mobile phase A consisted of acetonitrile and mobile phase B consisted of water with 0.1% acetic acid (pH
3.5; 20:80) monitored at 230 nm. The flow rate was 1.2 mL/min. The retention times of the metabolites, internal standard
(IS) and PTS were 3.0 min, 5.8 min and 9.7 min, respectively.
PTS incubation and kinetics assays To determine the kinetics of PTS metabolism, an incubation mixture contained
PTS at 0, 10, 20, 40, 80, 100, 120, 200, 300, 400, and 600
mmol/L. The apparent Km and
Vmax values were estimated by nonlinear
regression analysis of V (enzyme activity) and
[S] (substrate concentration) using the
Michaelis-Mentenmodel:V=Vmax[S]/(Km+[S])
Inhibition study in rat liver microsomes
The incubation mixture contained microsome protein, the relevant inhibitor,
PTS, and NADPH-generating system. Various concentrations of
a-Naph (1.0-50.0 µmol/L), Sulf (1.0-200.0 µmol/L), Qui
(1.0-100.0 µmol/L), or Ket (0.5-20.0 µmol/L) were co-incubated with 40
µmol/L PTS. Because PTS metabolite standard was not
available, the analyte to internal standard (antipyrine) peak area ratio was used instead of using absolute
quantitation[11]. Both positive (in the presence of known inhibitors and specific substrates) and negative (in the presence of PTS or substrate
and in the absence of the inhibitor) control samples were included in each assay to ensure the integrity of the microsomal
incubation system. The result, expressed as percentage of control activity, was calculated based on a comparison between
the peak area ratio of the sample and that of the negative control
samples[2].
The inhibitory effects of Sulf, Qui, and Ket on PTS metabolism were derived from Lineweaver-Burk plots of PTS
metabolite formation by varying the concentration of the PTS at several fixed concentrations of the
inhibitor[12]. Sulf at concentrations of 0, 50, and 100 µmol/L, Qui at concentrations of 0, 50, and 100
µmol/L, or Ket at concentrations of 0, 5, and 10 µmol/L
were co-incubated with PTS at concentrations ranging from 10.0 to 160.0 µmol/L. To investigate the effect of PTS on CYP
isoform activity, PTS at concentrations of 0-120
µmol/L were added to the incubation mixture with CYP substrates. A single
fixed concentration at approximately the
Km for each substrate (20 µmol/L Phe, 100
µmol/L Tol, 20 µmol/L
Dex, or 40 µmol/L Chl) was used in the
incubation sample. The areas for Ace, 4-OH-Tol, Dor, 3-MM and
6-OH-Chl were used for analysis.
Results
Total CYP and microsome protein content There was no significant difference in total CYP and microsome protein
content between the PTS pretreatment and blank control
groups. PTS pretreatment at the investigated concentrations had
little effect on rat liver CYP content (Table 1).
Validation of PTS assay Validation of the HPLC method developed for PTS assay was performed with 6 calibration
standards ranging from 10.0-320.0 µmol/L, and 3
qualitycontrol (QC) samples (20, 40, and 80 µmol/L). Standard samples were
added to the boiled microsome mixture and prepared as described earlier. The calibration curve was constructed by linear
least-squares regression of standard concentrations against peak
area ratio of PTS and internal standard. The calibration
curve Y=0.065X+0.2265 had excellent linearity, with a correlation coefficient of 0.9996
(n=6). The average recovery for the method was 106.2%±4.5% from different concentrations. The relative standard deviations of intra-day and inter-day
variation in the concentrations determined were less than 9.4%.
Mass spectrometry for PTS and its metabolite
We hoped to gather more information about the metabolites and
metabolism of PTS by in vitro incubation. In HPLC chroma tograms, the retention times of the PTS metabolite and PTS itself
were, respectively, 3.0 min and 9.7 min. There was no interfering peak found at the same retention time for the metabolite and
PTS in the chromatogram for microsomal incubation. We collected the material with a retention time of approximately 3.0 min
during the HPLC separation of the PTS incubation sample. This material was analyzed by
using a Finnigan LCQ Deca XP Max (Sunnyvale, CA, USA) mass spectrometer. The metabolite with
[M+1] + 188>171 in the mass spectrometry could be a hydroxylated derivative(Figure 1).
Kinetics assays of PTS metabolism relative to
CYP The Km and
Vmax values were estimated using the Michaelis-Menten
model. The Kmand
Vmax of the CYP-catalyzed reaction of PTS metabolism were 92.2 µmol/L and 0.0137
nmol/min per mg protein, respectively (Figure
2).
Inhibition screening of PTS metabolism PTS metabolism was inhibited by Sulf, Qui and Ket. The results indicated that
CYP2C7, CYP2D1, and CYP3A2 might be responsible for the CYP-catalyzed metabolism of PTS in liver microsomes of male
Wistar rats (Figure 3).
Mechanisms of inhibitors The three inhibitors (Sulf, Qui and Ket) were assessed with respect to their inhibitory mechanisms.
Lineweaver-Burk plots of PTS metabolite formation in the presence of Sulf, Qui and Ket are shown. As seen in Figure 4,
PTS metabolism was inhibited by both Sulf and Ket through a mixed inhibitory mechanism. Qui had a noncompetitive
mechanism. Noncompetitive inhibition mainly implies that an intermediate substance hinders the release of the substrate
(Figure 4).
Effect of PTS on rat CYP isoform
activity No significant changes were found with respect to the areas of Ace, 4-OH-Tol,
Dor, 3-MM and 6-OH-Chl when the concentration of PTS changed from 0 to 120 µmol/L. This study indicated that PTS had
neither an inhibitive nor an inductive effect on the CYP1A2, CYP2C7, CYP2D1, CYP3A2 and CYP2E1 reactions selected in our
experiments (Figure 5).
Discussion
Acquiring metabolic information and determining the effect of an NCE on CYP are important in developing clinically safe and
efficient medications[13]. Drug co-administration and individual differences in therapeutic effectiveness are common in
cancer therapy. In the present article, we provide a relatively complete description of PTS metabolism involving CYP. This
was a prerequisite for further toxicological risk assessment using this animal model.
In preclinical animal pharmacokinetic research, it was found that PTS could be given to rats at dosages of 33-198 mg/kg
per d, for which the T1/2 was 2.90-3.48 h, and the
Cmax was 29.24-151.23 mg/mL. There was one metabolite detected in the
aqueous and organic phases, respectively, in urine samples. PTS might be used at higher concentrations in the clinical
setting to treat different severities and types of tumors. Further experiments in human microsomes with different PTS
concentrations are expected.
Because of various anti-tumor mechanisms, the anticancer drug degradation process could be very different and could
have no relation with liver CYP enzymes at all. We arrived at a preliminary conclusion that CYP was responsible for PTS
metabolism because of two pieces of evidence. First, inhibition screening studies showed that inhibition of the activity of
CYP2C7, CYP2D1, and CYP3A2 slowed down the metabolism of PTS. Secondly, the result that CYP was responsible for PTS
metabolism had also been verified by PTS metabolism research in a
rat in situ liver perfusion model. In the latter experiment,
there was difference in the PTS metabolism curves between the groups receiving Ket and PB pretreatment and the blank
controls. The inhibitors Sulf, Qui and Ket gave us further evidence that CYP2C7, CYP2D1, and CYP3A2 might contribute to
PTS rat liver metabolism. However, specific CYP isoforms and different effective inhibitors (especially for CYP3A) for
screening research will be needed to verify our
results.
Inhibitors of CYP2C7, CYP2D1, and CYP3A2 could slow down PTS metabolism and potentiate its activity or toxicity. Of
Sulf (up to 100 µmol/L , 56.8% of control activity), Qui (up to 100
µmol/L , 65.1%), and Ket (up to 20
µmol/L , 40.1%), Ket was the most effective inhibitor of PTS metabolism. Agents that modulate CYP3A will always need to be administered with care,
and the same is true for PTS administration.
PTS had no effect on phenacetin O-deethylase, tolbutamide methylhydroxylation, dextro
O-demethylation, dextro N-demethylation, or chlorzoxazone
6-hydroxylationreactions, which respectively represent the activities
ofCYP1A2,CYP2C7, CYP2D1, CYP3A2 and CYP2E1
in vitro. Our data predict that PTS could be used relatively safely with many current drugs,
and that PTS would be suitable for pharmaceutical development. PTS has a simple structure, and it could be manufactured
easily and cheaply. In conclusion, studies of CYP correlations with NCE metabolism provide a firm scientific basis for the safe
and effective use of drugs, personalized medicine, and drug discovery.
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