CBI 7048 No.

of Pages 8, Model 5G
4 June 2014

Chemico-Biological Interactions xxx (2014) xxx–xxx

1

Contents lists available at ScienceDirect

Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

6
7

3 Pretreatment with turmeric modulates the inhibitory influence

4 of cisplatin and paclitaxel on CYP2E1 and CYP3A1/2 in isolated
5 rat hepatic microsomes
8 Q1 Enas M. Ahmed a, Shohda A. EL-Maraghy b,⇑, Zakaria A. Teleb a, Amira A. Shaheen b
9 Q2 a
Biochemistry & Molecular Biology Department, National Organization for Drug Control & Research, Egypt
10 b
Biochemistry Department, Faculty of Pharmacy, Cairo University, Egypt

12
11
13
a r t i c l e i n f o a b s t r a c t
1
2 5
8
16 Article history: Previous animal studies have shown that turmeric can significantly modulate the activity of several drug 29
17 Received 24 March 2014 metabolizing enzymes, this may dramatically affect the bioavailability of several drugs resulting in over 30
18 Received in revised form 7 May 2014 dose or less therapeutic effects. This study was directed to evaluate the inhibitory effects of cisplatin and 31
19 Accepted 15 May 2014
paclitaxel on two CYP450 enzymes namely CYP2E1 and CYP3A1/2 in hepatic microsomes isolated from 32
20 Available online xxxx
normal and turmeric pretreated rats. Cisplatin and paclitaxel were added by different concentrations to 33
hepatic microsomes isolated from untreated and turmeric (100 mg/kg/day) pretreated rats for 15 days after 34
21 Keywords:
receiving pyrazole or dexamethasone for induction of CYP2E1 and CYP3A1/2 respectively. The kinetic 35
22 Turmeric
23 Cisplatin
potency of these drugs as CYP inhibitors was determined by analysis of Lineweaver–Burk plot. Addition 36
24 Paclitaxel of cisplatin or paclitaxel by (10, 50 and 100 lM) to hepatic microsomes from normal or turmeric pretreated 37
25 CYP450 isoenzyme rats caused a concentration dependent inhibition of CYP2E1, with an evidence of less inhibition in turmeric 38
26 Inhibition pretreated microsomes particularly at higher concentration. Both drugs at 100 lM displayed a mixed type 39
27 of inhibition of CYP2E1 in normal or turmeric pretreated microsomes where paclitaxel was the most potent 40
inhibitor. Cisplatin (10, 50 and 100 lM) caused a concentration dependant inhibition of CYP3A1/2 that was 41
enhanced by turmeric pretreatment. The inhibition of CYP3A1/2 by cisplatin (100 lM) was in non- 42
competitive manner with a smaller Ki value in turmeric pretreated microsomes. The inhibitory influence 43
of paclitaxel (10, 50 and 100 lM) on CYP3A1/2 decreased with increasing the drug concentration and this 44
inhibition was augmented by turmeric pretreatment. Interestingly, the inhibition of this enzyme by 45
paclitaxel (10 lM) was switched from mixed type in normal microsomes to competitive manner in 46
turmeric pretreated ones with a marked reduction of Ki values reflecting greater inhibitory influence of 47
Q3 paclitaxel on CYP3A1/2 by turmeric pretreatment. In conclusion, turmeric pretreatment attenuated the 48
inhibitory influence of cisplatin and paclitaxel on CYP2E1 activity and magnified their inhibition on 49
CYP3A1/2, thus the use of turmeric with drugs or other medications should raise concern for drugs–herb 50
interactions. 51
Ó 2014 Elsevier Ireland Ltd. All rights reserved. 52
53

54
55

56 1. Introduction animal studies have shown that curcumin can significantly 63

modulate the activity of several drug metabolizing enzymes by 64
57 Turmeric is a yellow product in the rhizomes of Curcuma longa either induction, down regulation or direct inhibition [42,3] and 65
58 belonging to the family Zingiberaceae and containing curcumin [40]. This may dramatically affect the bioavailability of several drugs 66
59 which is the most active component of turmeric. It is a popular die- resulting in over dose or less therapeutic effects. Repetitive admin- 67
60 tary spice, pigment and widely used in medicine. It’s reported to istration of curcumin to rats resulted in down regulation of intestinal 68
61 have a beneficial activities including anticancer, antioxidant, CYP3A, CYP1A1/2 [42] and esophageal CYP2B1 and CYP2E1 [24]. 69
62 anti-inflammatory [19,4,36] and [26]. Previous in vivo and in vitro Appiah-Opong et al. [3] demonstrated that oral co-administration 70
of curcumin with other drugs-metabolized by these CYPs has the 71
potential to cause clinically significant drug–drug interactions. 72
⇑ Corresponding author. Address: 23 Kasr El-Aini Street, Cairo, Egypt. Tel.: +20 Thus, the oral bioavailability of docetaxel, an important anticancer 73
01223101126. was enhanced by the regular co-administration of curcumin. As 74
E-mail address: Shohda.elmaraghy@pharma.cu.edu.eg (S.A. EL-Maraghy).

http://dx.doi.org/10.1016/j.cbi.2014.05.007
0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: E.M. Ahmed et al., Pretreatment with turmeric modulates the inhibitory influence of cisplatin and paclitaxel on CYP2E1
and CYP3A1/2 in isolated rat hepatic microsomes, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.05.007
 CBI 7048 No. of Pages 8, Model 5G
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2 E.M. Ahmed et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx

75 pretreatment of rat with curcumin has been shown to attenuate the 2. Materials and methods 140
76 activity of CYP3A in liver microsome, an enzyme involved in meta-
77 bolic inactivation of this anticancer drug [40]. 2.1. Drugs and chemicals 141
78 Cisplatin (platinum based drug) [27,38] and Paclitaxel (origi-
79 nally derived from the bark of the Pacific yew tree Taxus brevifolia) Cisplatin was obtained from Korea United Pharm. (KUP) under 142
80 [6,41] are potent antineoplastic agents used for the treatment of a the technical assistance of United Douglas Pharm., USA, imported 143
81 wide range of cancers. Being chemotherapeutic drugs, they are by Egyptian International Medical Center (EIMC) Pharmaceuticals 144
82 often associated with various degrees of interaction with the hepa- Company. Paclitaxel was obtained from filaxis laboratories, Argen- 145
83 tic metabolizing enzymes. The ability of JM216 (a platinum deriv- tine. Dexamethasone, pyrazole, erythromycin, p-nitrophenol and 146
84 ative) to inhibit P450s (CYP2E1, CYP2C8 and CYP3A4) in human all other chemicals used throughout the present work were 147
85 hepatic microsomes was verified by Ando et al. [2] indicating obtained in analytical and purified grade and were purchased from 148
86 strong and non-specific inhibitory effects of the drug. On the other Sigma Chemical Co., St. Louis, USA. 149
87 side, paclitaxel is primarily metabolized by CYP3A1/2 and CYP2C8
88 Q4 into virtually inactive component exerted in bile [30]. Predating to
89 that, Kostrubsky et al. [13] reported that paclitaxel at high concen-
2.2. Preparation of turmeric extract 150
90 trations, higher than 10 ll resulted in inhibition of CYP3A activity
91 in isolated human hepatocytes.
Rhizomes of Curcuma longa were purchased as powder from 151
92 Cytochrome P450 (P450) is a hemoprotein superfamily of isoen-
the folk market, extracted by 70% ethanol according to Lee et al. 152
93 zymes that are responsible for the metabolism of a wide variety of
[18], and the extract was lyophilized, and the lyophilized powder 153
94 foreign chemicals including anticancer drugs [7,1]. P450s system is
was resuspended in saline and used in appropriate dose. The 154
95 mainly localized in the liver more abundant than in any other
extract is standardized by GC mass analysis. Thirty-five com- 155
96 organs such as lung, kidney or intestine [33]. Of various P450 iso-
pounds were present in the ethanol extract; seventeen of them 156
97 enzymes, CYP2E1 is mainly expressed in liver and in small
have a relative percentage about 1.0%. Curcumin and its derivatives 157
98 amounts in kidney, lung and gut [5]; Ingelman-Sundberg et al.
were identified by their mass spectrum as the major components 158
99 [10]. It is a major isoenzyme involved in the bioactivation of chem-
which represent (67.5%). 159
100 icals and drugs to toxic metabolites supporting its role in hepato-
101 toxicity by many drugs [10]. CYP2E1 itself is also an effective
102 enzyme for ROS production, exhibiting enhanced NADPH oxidase
activity, and elevated rates of O 2.3. Experimental animals 160
103 2 and H2O2 production even in
104 the absence of substrate [17,21].
105 Another P450 isoenzymes are CYP3A which comprise the larg- Adult male Wistar albino rats aged 10–12 weeks and weighed 161

106 est portion of the liver and small intestinal CYP protein and they 180 ± 20 g were obtained from the animal house of the National 162

107 are involved in the metabolism of 45–60% of all currently used Organization for Drug Control & Research, Giza, Egypt. Animals 163

108 drugs. The wide substrate spectrum of CYP3A is the reason behind were housed under controlled temperature (25 ± 2 °C) and con- 164

109 their frequent involvement in drug–drug interactions. Induction or stant light/dark cycle (12/12 h). They allowed free access to a stan- 165

110 inhibition of these isoenzymes can lead to clinically significant dard rodent chow diet and water. The investigations complies with 166

111 alterations of the pharmacokinetics and pharmacological activity the guide for care and use of laboratory animals published by US 167

112 of drugs leading to adverse reaction following their co-administra- National institutes of Health (NIH NO.85-23, revised in 1985) and 168

113 tion. Clinically, the most relevant drug interaction is the CYP3A was approved by the Ethis Committee for animal experimental at 169

114 inhibition which may include one of the following mechanisms: Faculty of Pharmacy, Cairo University. 170

115 competitive, non-competitive, uncompetitive and mechanism Rats were divided into 4 groups of 6 animals each. Group 2 and 171

116 based irreversible inhibition [39,8,34]. 4 received at the beginning turmeric extract at an oral dose of 172

117 Inhibition of P450 system by curcumin [3] or by various antiox- 100 mg/kg/day [40] for 15 consecutive days. Group 1 and 2 173

118 idants [28] were found to be effective in protecting the liver received i.p. dose of pyrazole 250 mg/kg/day for two days to induce 174

119 against the toxicity of a wide variety of toxic agents. Inhibition of CYP2E1 [14] which started at the 14th day in group 2. Group 3 and 175

120 CYP2E1 and other CYP isoforms by turmeric might be responsible 4 received i.p. dose of dexamethasone 100 mg/kg/day for 3 days to 176

121 for alleviating the cisplatin induced-hepatotoxicity. Since, induce CYP3A1/2 [22] which started at 13th day in group 4. 177

122 increased expression of CYP2E1 had been indicated both in vivo

123 and in vitro to be a critical mediator or enhancer for cisplatin-
124 induced hepatotoxicity [21]. Moreover, inhibition of CYP3A1 by 2.4. Preparation of hepatic microsomes 178
125 several curcuminoids [37] could inhibit metabolic inactivation of
126 paclitaxel sufficiently to increase its effects, since paclitaxel metab- 24 h after the last treatment, rats from each group were sacri- 179
127 olites are inactive [30]. ficed by decapitation and liver samples were isolated quickly and 180
128 To our knowledge, there are no reports regarding the modula- rinsed with ice-cold saline, dried by blotting between filter papers. 181
129 tory effect of turmeric pretreatment on the inhibitory influence Preparation of hepatic microsomes was performed as described by 182
130 of cisplatin and paclitaxel on certain P450 enzymes in isolated Lake [15]. Briefly, liver samples were homogenized in 25 mM 183
131 rat hepatic microsomes. Thus, the aim of this study was directed Tris–HCl (pH 7.5) containing 1 mM EDTA and 0.25 M sucrose. The 184
132 to investigate the inhibitory effects of cisplatin and paclitaxel on homogenate was centrifuged at 18,000g for 20 min. The superna- 185
133 the activity of two CYP enzymes involved in drug metabolism tant was then centrifuged twice at 100,000g for 60 min. The result- 186
134 namely CYP2E1 and CYP3A1/2 in isolated hepatic microsomes ing microsomal pellets from each experimental group were pooled 187
135 from normal and turmeric pretreated rats, to explore the possible and resuspended in 0.1 M sodium phosphate buffer (pH 7.5) 188
136 modulatory effect of prior treatment with turmeric on the response containing 20% glycerol and stored at 80 °C until used. Protein 189
137 of these CYP enzymes to the inhibitory effect of these anticancer concentration in the microsomal fraction was determined as previ- 190
138 drugs. In addition, the kinetics potency of these drugs as CYP inhib- ously described by Lowry et al. [20] using bovine serum albumin as 191
139 itors was investigated by Lineweaver–Burk plot analysis. a standard. 192

Please cite this article in press as: E.M. Ahmed et al., Pretreatment with turmeric modulates the inhibitory influence of cisplatin and paclitaxel on CYP2E1
and CYP3A1/2 in isolated rat hepatic microsomes, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.05.007
 CBI 7048 No. of Pages 8, Model 5G
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E.M. Ahmed et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx 3

193 2.5. Measurement of CYP2E1 and CYP3A1/2 activities in presence of 2.7. Data analysis 256
194 different concentrations of cisplatin and paclitaxel
The results are presented as mean ± S.E. the values were 257
195 p-Nitrophenol hydroxylation assay was used to determine the obtained from the mean of triplicate incubations. The statistical 258
196 CYP2E1 activity as described by Alexidis et al. [1]. Briefly after analysis was conducted by using the SPSS program version 11.0 259
197 incubation of hepatic microsomes (1–1.5 mg protein) from either at P-value less than 0.05. An independent t-test, one-way ANOVA 260
198 untreated or turmeric pretreated animals with 5 mM p-nitrophe- and The strength of association between pairs of variables was 261
199 nol and in the presence of (10, 50, 100 lM) of either cisplatin or assessed by LSD comparison. 262
200 paclitaxel at 37 °C for 15 min, 20%trichloroacetic acid was added
201 to terminate the reaction, followed by addition of 10 mmol NaOH
202 to develop the color. The enzyme specific activities were 3. Results 263

203 determined by quantifying the production of p-nitrocatechol spec-

204 trophotometrically at 510 nm (UV-Unicam spectrophotometer) 3.1. The inhibitory effect of cisplatin and paclitaxel on the activity of 264

205 and expressed as lmol/min/mg protein. CYP2E1 in hepatic microsomes isolated from normal and turmeric 265

206 CYP3A1/2 activity was measured by colorimetric measurement pretreated rats 266

207 of formaldehyde liberated due to N-demethylation of erythromy-

208 cin based on the Hantzsch reaction as described by Alexidis et al. Results shown in Fig. 1(A and B) demonstrated that at 5 mM p- 267

209 [1]. Briefly after incubation of hepatic microsomes (1–1.5 mg pro- nitrophenol, addition of cisplatin or paclitaxel by the three concen- 268

210 tein) from either untreated or turmeric pretreated animals with trations (10, 50, 100 lM) resulted in a sustained gradual reduction in 269

211 5 mM erythromycin and in the presence of (10, 50, 100 lM) of hepatic normal microsomal CYP2E1 enzyme activity in concentra- 270

212 either cisplatin or paclitaxel at 37 °C for 15 min, together with tion dependant manner. However, incubation of CYP2E1 enzyme 271

213 20 mM semicarbazide and 1 mM NADPH to initiate the reaction. from the turmeric pretreated rats microsomes with cisplatin and 272

214 After 15 min, 25% ZnSO4 was added to terminate the reaction paclitaxel at the above concentrations resulted in less inhibition of 273

215 followed by the addition of Nash reagent. The color developed by its activity particularly at higher concentrations of the drug. 274

216 the reaction of Nash with the produced formaldehyde was

217 measured spectrophotometrically (UV-Unicam) at 415 nm. The 3.2. The inhibitory effect of cisplatin and paclitaxel on the activity of 275
218 enzyme specific activity was expressed as lmol/min/mg protein. CYP3A1/2 in hepatic microsomes isolated from normal and turmeric 276
pretreated rats 277
219 2.6. Determination of kinetic parameters of CYP2E1 and CYP3A1/2 in
220 presence and absence of cisplatin or paclitaxel with identification of Data compiled in Fig. 2(A and B) revealed that at substrate con- 278
221 the type of inhibition centration of 5 mM erythromycin, incubation of the normal hepatic 279
microsomes with cisplatin (10, 50 and 100 lM) showed gradual 280
222 The concentrations of cisplatin or paclitaxel which exhibited
223 the highest enzyme inhibition were selected from the above exper-
224 iments to determine the Michaelis–Menten kinetic parameters
225 including Vmax and Km for CYP2E1 and CYP3A1/2 and the type of
226 their inhibition by these drugs. The selected concentration of cis-
227 platin was 100 mM for both enzymes while that of paclitaxel
228 was 10 mM for CYP3A1/2 and 100 mM for CYP2E1. The
229 experiments were done by incubating different concentrations of
230 p-nitrophenol (1.25 mM, 2.5 mM, 5 mM and 10 mM) as substrate
231 for CYP2E1 or erythromycin (1.25 mM, 2.5 mM, 5 mM and
232 10 mM) as substrate for CYP3A1/2 in the presence or absence of
233 cisplatin or paclitaxel. The Km, Vmax in absence and the apparent
234 Vmax, Km values in presence of the inhibitor (I) as well as the mode
235 of inhibition were determined from double reciprocal plots
236 (Lineweaver–Burk). The Ki (inhibition constant) values were calcu-
237 lated using the following equations:
238 For competitive inhibition
239
K m ðIÞ
241
Ki ¼
K m  K m
242 For noncompetitive inhibition
243
V max ðIÞ
245
Ki ¼
V max  V max
246 For mixed type inhibition
247
V max  K m  ðIÞ
Ki ¼
249 ðV max K m  V max K m Þ
250
0 V max ðIÞ
252
Ki ¼
V max  V max
Fig. 1. The inhibitory effect of cisplatin (A) and paclitaxel (B) on the activity of
253 where values Km, Vmax are in the absence while the apparent CYP2E1 in hepatic microsomes isolated from normal (____) and turmeric pre-
254 K m ; V max are in the presence of inhibitor (I) which is the concentra- treated(——) rats at concentration 5 mM of p-nitrophenol. The values are expressed Q8
255 tion of the inhibitor. as mean ± S.E. (n = 3). a: significant difference from normal microsomes at p < 0.05.

Please cite this article in press as: E.M. Ahmed et al., Pretreatment with turmeric modulates the inhibitory influence of cisplatin and paclitaxel on CYP2E1
and CYP3A1/2 in isolated rat hepatic microsomes, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.05.007
 CBI 7048 No. of Pages 8, Model 5G
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4 E.M. Ahmed et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx

Fig. 2. The inhibitory effect of cisplatin (A) and paclitaxel (B) on the activity of
CYP3A1/2 in hepatic microsomes isolated from normal (____)and turmeric pre-
treated (———) rats at concentration 5 mM erythromycin. The values are expressed
as mean ± S.E. (n = 3). a: significant difference from normal microsomes at p < 0.05.

281 decline in CYP3A1/2 enzyme activity. While upon its incubation

282 with paclitaxel, the inhibitory effect of the drug appeared to
283 decrease by increasing the drug concentrations. Upon, addition of
284 cisplatin or paclitaxel by the above three concentrations to hepatic
285 microsomes from turmeric pretreated rats, the drugs exhibited
286 more inhibition in the activity of CYP3A1/2 compared to the nor-
287 mal microsomes values particularly at high drug concentration.

288 3.3. The kinetic parameters of CYP2E1 in hepatic microsomes isolated

289 from normal and turmeric pretreated rats in presence and absence of Fig. 3. Lineweaver–Burk plot showing the inhibition of CYP2E1 activity by cisplatin
290 cisplatin or paclitaxel (100 lM) in hepatic microsomes isolated from normal (A) and turmeric (B)
pretreated rats at concentrations 0, 1.25, 2.5, 5, and 10 mM of p-nitrophenol. The
values are expressed as mean ± S.E. (n = 3).
291 From the Lineweaver–Burk plot as shown in Fig. 3(A and B) and
292 Table 1, it is worthy noticed that, in the presence of the drug, pre- The results described in Fig. 4(A) and Table 1 showed that addi- 315
293 treatment with turmeric mildly affected the kinetic potency of tion of paclitaxel to hepatic microsomes isolated from normal rats 316
294 CYP2E1 compared to the normal microsomes with a negligible exerted a clear inhibition of CYP2E1 inducing marked decrement of 317
295 change in Vmax (10.8 lmol/min/mg protein versus 10 lmol/min/ the Vmax from 10 lmol/min/mg protein to 3.5 lmol/min/mg pro- 318
296 mg protein) and Km (2.7 lM versus 2.4 lM) (Table 1). Moreover, tein) together with mild increment of Km from 2.4 to 4 lM. These 319
297 the data revealed that addition of cisplatin to hepatic microsomes changes in both kinetic parameters of CYP2E1 by paclitaxel sug- 320
298 isolated from normal rats resulted in clear inhibition of CYP2E1 gested a mixed inhibition with Ki and Ki0 values (27 lM and 321
299 activity as demonstrated by lowering Vmax from 10 to 5 lmol/ 154 lM respectively). On the other hand, as shown in Fig. 4(B) in 322
300 min/mg protein and increasing Km from 2 up to 4 lM. These results hepatic microsomes isolated from turmeric pretreated rats, addi- 323
301 indicated that cisplatin inhibited CYP2E1 based on mixed type tion of paclitaxel exerted a less inhibition of CYP2E1 activity as 324
302 with Ki and Ki0 constants (43 lM and 209 lM respectively). More- indicated by an increase of Vmax from 3.5 to 5.38 lmol/min/mg 325
303 over, in hepatic microsomes isolated from turmeric pretreated rats protein and a decrease in Km from 4 lM to 3 lM compared to nor- 326
304 the activity of CYP2E1 was slightly affected by turmeric pretreat- mal microsomes incubated with paclitaxel. The pattern of inhibi- 327
305 ment with a mild change in Vmax and Km compared to normal tion revealed mixed type of inhibition with Ki and Ki0 constants 328
306 microsomal values (Table 1). However, Fig. 3(B) addition of cis- (77 lM and 199 lM respectively) suggesting a less inhibitory influ- 329
307 platin exerted less inhibition of CYP2E1 activity as indicated by ence of paclitaxel on CYP2E1 in response to turmeric pretreatment. 330
308 mild increase in V max (6 lmol/min/mg protein) and a mild
309 decrease in K m (2 lM) compared to normal hepatic microsomes 3.3. The kinetic parameters of CYP3A1/2 in hepatic microsomes 331
310 incubated with cisplatin. In response to turmeric pretreatment, isolated from normal and turmeric pretreated rats in presence and 332
311 the changes in both kinetic parameters suggested a mixed type absence of cisplatin or paclitaxel 333
312 of inhibition with Ki and Ki0 constants (228 lM and 229 lM respec-
313 tively) indicating a less inhibitory potency of cisplatin on CYP2E1 Analysis of Lineweaver–Burk plot shown in Fig. 5(A and B) as 334
314 activity in response to turmeric pretreatment. well as the data given in Table 2 revealed that the kinetic parame- 335

Please cite this article in press as: E.M. Ahmed et al., Pretreatment with turmeric modulates the inhibitory influence of cisplatin and paclitaxel on CYP2E1
and CYP3A1/2 in isolated rat hepatic microsomes, Chemico-Biological Interactions (2014), http://dx.doi.org/10.1016/j.cbi.2014.05.007
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E.M. Ahmed et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx 5

Table 1
The kinetic parameters of hepatic microsomal CYP2E1 activities from either normal or turmeric pretreated rats in presence and absence of cisplatin and paclitaxel.

Kinetic parameters Hepatic microsomes

Normal Turmeric Normal + cisplatin Turmeric Normal + paclitaxel Turmeric
pretreated pretreated + cisplatin pretreated + paclitaxel
Vmax (lmol/min/mg protein) 10.00 10.80 ———— ———— ———— ————
V max (lmol/min/mg ——— ——— 5.21 6.09 3.50 5.38
protein)
Km (lM) 2.40 2.70 ——— ——— ——— ———
K m (lM) ——— ——— 4.11 2.19 4.00 3.10
Ki (lM) ———— ——— 43.6 228.08 26.58 76.62
Ki0 (lM) ———— ——— 208.33 229.29 153.84 119.26
Type of inhibition ——— ——— Mixed Mixed Mixed Mixed

336 ters of CYP3A1/2 in hepatic microsomes isolated from turmeric hydroxylation more strongly than cisplatin. The inhibitory effect of 385
337 pretreated rats were lower than that from normal microsomes cisplatin on CYP2E1 was in line with the early observation of 386
338 (Vmax 3.2 lmol/min/mg protein versus 5 lmol/min/mg protein Masubuchi et al. [23] who observed a transient decrease in CYP2E1 387
339 and Km 2.6 lM versus 4.5 lM) suggesting uncompetitive mecha- activity after treatment with cisplatin. This transient inhibition 388
340 nism of inhibition by turmeric pretreatment. Moreover, addition may be due to a direct effect of cisplatin on CYP2E1. Moreover, 389
341 of cisplatin to hepatic microsomes isolated from normal rats Vaclavikova et al. [35] reported that CYP2E1 might be involved in 390
342 exerted a clear inhibition of the activity of CYP3A1/2 resulting in paclitaxel metabolism and cytotoxicity, since uninduced rat micro- 391
343 a decrement of Vmax from 5 lmol/min/mg protein to 3.5 lM/min/ somes did not change the effect of the drug, whereas CYP2E1- 392
344 mg protein without changing Km suggesting non-competitive type induced rat microsomes increased its cytotoxicity. Indeed, the 393
345 of inhibition with Ki constant (246.5 lM). However, addition of the inhibition of CYP2E1 by paclitaxel has not yet studied and defined. 394
346 drug to turmeric pretreated microsomes resulted in absence of the In the current study, addition of cisplatin and paclitaxel by the 395
347 drug in a greater inhibition of CYP3A1/2 as indicated by marked previously stated concentrations to turmeric pretreated micro- 396
348 reduction of Vmax (1.91 lmol/min/mg protein) without changing somes exhibited less inhibition to CYP2E1 activity, suggesting that 397
349 Km suggesting the same type of inhibition, with a smaller Ki con- turmeric pretreatment might modulates the response of CYP2E1 to 398
350 stant (148 lM) indicating a greater inhibitory influence of cisplatin these drugs. In previous work Volak et al. [37] reported that the 399
351 on CYP3A1/2 in response to turmeric pretreatment. activity of CYP2E1 in an in vitro study was less affected by curc- 400
352 On the other side, addition of paclitaxel to normal hepatic micro- uminoids and that less than 20% inhibition was observed for 401
353 somes, as shown in Fig. 6(A) and given in Table 2 exerted a clear inhi- CYP2E1 activity at the highest concentration (200 lM). In the con- 402
354 bition of CYP3A1/2 activity as indicated by a decrement of Vmax from trary, Sugiyama et al. [31] revealed that curcumin ingestion had no 403
355 5 lmol/min/mg protein to 3 lmol/min/mg protein and an incre- inhibitory influence on hepatic CYP activity in rats (mainly 404
356 ment of Km from 4.5 lM to 5.5 lM suggesting a mixed type of inhi- CYP2E1), indicating no pharmacokinetic interaction with the 405
357 bition with Ki and Ki0 values (12 lM and 32 lM respectively). co-administered drugs. 406
358 Moreover, data arisen from Fig. 6(B) and Table 2 showed that addi- Owing to the roles played by CYP2E1 in mediating carcinogenesis 407
359 tion of paclitaxel to hepatic microsomes isolated from turmeric pre- and chemical cytotoxicity, modulation of CYP2E1 is an important 408
360 treated rats switched the type of inhibition from mixed to issue in organ and tissue protection. Several studies have shown that 409
361 competitive one as indicated by a marked increase of Km from turmeric has anti-inflammatory and anticancer activities. Therefore, 410
362 2.6 lM up to 12.24 lM without any change in Vmax with a reduction the inhibition of CYP2E1 by turmeric, at least in part, may have a 411
363 of Ki to be 7.7 lM reflecting a greater inhibitory influence of crucial role in these pharmacological activities. CYP2E1 is a major 412
364 paclitaxel on CYP3A1/2 in response to turmeric pretreatment. isoenzyme involved in the bioactivation of chemicals and drugs into 413
toxic metabolites which may contribute to their tissue toxicity 414

365 4. Discussion [25,32]. Pretreatment with curcuminoids had been previously 415
reported to have a protective effect against carbon tetrachloride 416

366 Cytochromes P450 are major oxidative enzymes that metabo- and ethanol-induced hepatotoxicity [31,9,29]. Thus, the hepatopro- 417

367 lize xenobiotics including anticancer drugs. Modulation of these tective effect of turmeric may be related to inhibition or reduction of 418

368 enzymes can dramatically affect cytotoxicity and/or therapeutic CYP2E1 isoforms, via reducing the formation of toxic metabolites. 419

369 efficacy of these drugs. In this study, the primary aim was to inves- Moreover in this study, the interaction of either cisplatin or pac- 420

370 tigate the inhibitory potency of cisplatin and paclitaxel toward two litaxel with CYP2E1 in normal hepatic microsomes was in a mixed 421

371 P450 isoforms CYP2E1 and CYP3A1/2 occurring in differentially manner with a marked reduction of apparent Vmax and an increase 422

372 induced rat liver microsomes isolated from normal or turmeric of apparent Km. The reduction in V max of CYP2E1 was clearer in 423

373 pretreated rats. p-Nitrophenol hydroxylation and N-demethylation response to paclitaxel than to cisplatin. This was confirmed by 424

374 of erythromycin reactions were chosen for the study of the inhibi- smaller inhibition constants of paclitaxel of 48 and 24 lM than 425

375 tion of CYP2E1 and CYP3A1 activities respectively. In addition, the that for cisplatin 94 and 40 lM, as the smaller inhibition constant 426

376 kinetic potency of these drugs as CYP inhibitors was investigated is the greater strength of inhibition will be. Such interaction of cis- 427

377 by analysis of Lineweaver–Burk plot. platin or paclitaxel with CYP2E1 might be mediated via binding of 428

378 The results of the present study clearly revealed that the activ- either the drugs with the site on the enzyme molecules other than 429

379 ity of CYP2E1 was significantly inhibited by the three concentra- the active site. Interference of cisplatin or paclitaxel with this site 430

380 Q5 tions of cisplatin and paclitaxel (10, 50, 100 lM) after incubation on CYP2E1 could modify the conformation of the enzyme and pre- 431

381 of the hepatic microsomal fractions from normal rats with the vent the binding and subsequently the metabolism of concomi- 432

382 drugs. The inhibitory effects of both drugs increased with increas- tantly administered drugs [37]. On the other side, incubation of 433

383 ing the drug concentrations in a concentration dependent manner. either cisplatin or paclitaxel with turmeric pretreated rat liver 434

384 These results also indicated that paclitaxel inhibited p-nitrophenol microsomes resulted also in inhibition of CYP2E1 activity in a 435

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Fig. 4. Lineweaver–Burk plot showing the inhibition of CYP2E1 activity by Fig. 5. Lineweaver–Burk plot showing the inhibition of CYP3A1/2 activity by
paclitaxel (100 lM) in hepatic microsomes isolated from normal (A) and turmeric cisplatin (100 lM) in hepatic microsomes isolated from normal (A) and turmeric (B)
(B) pretreated rats at concentrations 0, 1.25, 2.5, 5, and 10 mM of p-nitrophenol. The pretreated rats at concentrations 0, 1.25, 2.5, 5, and 10 mM of erythromycin. The
values are expressed as mean ± S.E. (n = 3). values are expressed as mean ± S.E. (n = 3).

436 mixed manner. However the inhibitory effects of those drugs on inhibition of the enzyme activity as compared to the normal con- 459
437 CYP2E1 activity in response to turmeric pretreatment were lesser trol values. Moreover, incubation of normal microsomes with 460
438 than that observed in normal microsomes. Since, under turmeric (10, 50 and 100 lM) of paclitaxel in the current study caused a sig- 461
439 pretreatment the Ki constants of either cisplatin or paclitaxel were nificant inhibition in the activity of CYP3A1/2. However, the inhib- 462
440 increased than those observed in normal microsomes. Therefore itory effects of paclitaxel decreased with increasing the drug 463
441 the high Ki values were indicative of a weak interaction of the concentrations. These results are complying with the work of Kost- 464
442 drugs and CYP2E1 in turmeric pretreated microsomes. Its worthy rubsky et al. [13] who demonstrated that treatment of CYP3A1/2 465
443 noticed that the inhibitory effect of paclitaxel on CYP2E1 was more with concentrations of paclitaxel higher than 10 lM caused a 466
444 potent compared to that observed by cisplatin, and that pretreat- dose-dependent decrease in its activity and the amount of its 467
445 ment of rats with turmeric extract has limited effect on CYP2E1 enzyme protein. The same pattern of inhibition was also observed 468
446 activity. However, turmeric pretreatment may modify the protein upon the addition of paclitaxel in above concentrations to CYP3A1/ 469
447 conformation of the enzyme and/or its response to these antican- 2 of microsomes from turmeric pretreated rats. The observed inhi- 470
448 cer drugs as indicated by decreased inhibitory potency of cisplatin bition of the activity of CYP3A1/2 in response to turmeric pretreat- 471
449 and paclitaxel on CYP2E1 in the current study. ment in this study was in agreement with that reported by Appiah- 472
450 In the present study, the activity of CYP3A1/2 was significantly Opong et al. [3] who demonstrated that curcuma species possess- 473
451 inhibited by (10, 50 and 100 lM) of cisplatin after incubation of the ing 65–73% inhibitory activities toward CYP3A4. In the same direc- 474
452 microsomal fraction with the drug. The inhibitory effect of cis- tion, Yan et al. [40] and Zhang et al. [42] reported that curcumin 475
453 platin was increased with increasing the drug concentrations attenuated the activity of CYP3A enzymes in liver microsomes 476
454 (dose-dependent). These findings are in line with Ando et al. [2] and administration of curcumin was adequate to modify the 477
455 who reported that CYP3A1/2 activity was moderately inhibited expression of P-glycoprotein and CYP3A in rats. In the current 478
456 by cisplatin at 10 lM concentration. On the other hand, addition study, the rats were treated with turmeric at an oral dose of 479
457 of cisplatin by the above three concentrations to CYP3A1/2 in 100 mg/kg/day for 15 consecutive days. This higher dose and long 480
458 microsomes isolated from turmeric pretreated rat caused further term pretreatment were used in an attempt to obtain a more 481

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Table 2
The kinetic parameters of hepatic microsomal CYP3A1/2 activities from either normal or turmeric pretreated rats in presence and absence of cisplatin and paclitaxel.

Kinetic parameters Hepatic microsomes

Normal Turmeric pretreated Normal + cisplatin Turmeric pretreated + cisplatin Normal + paclitaxel Turmeric pretreated + paclitaxel
Vmax (lmol/min/mg protein) 5.00 3.20 ———— ———— ———— ————
V max (lmol/min/mg protein) ——— ——— 3.55 1.91 3.43 3.25
Km (lM) 4.50 2.60 ——— ——— ——— ———
K m (lM) ——— ——— 4.90 2.80 5.58 12.24
Ki (lM) ———— ——— 246.56 148.06 12.24 7.72
Ki0 (lM) ———— ——— ———— ——— 31.84 ———
Type of inhibition ——— ——— Non competitive Non competitive Mixed Competitive

482 obvious response to turmeric. Thus, the accumulative absorption of CYP3A1/2 activity in a non-competitive manner based on 490
483 turmeric components into the general circulation was adequate to decreased Vmax and unchanged Km together with higher Ki value. 491
484 modify the expression of CYP3A in these rats. Indeed, turmeric The higher Ki value was indicative of the weakness of the interac- 492
485 pretreatment has been shown to attenuate the activity of tion between cisplatin and CYP3A1/2. This finding is compatible 493
486 CYP3A1/2 in liver microsomes in uncompetitive manner as with that demonstrated by Kamataki et al. [11] who found that 494
487 indicated by reduction of Vmax and Km of this enzyme. the inhibition of CYP3A activity by cisplatin was in a non-compet- 495
488 The results of the present study revealed also that incubation of itive manner. Although they did not verify the mechanism of inhi- 496
489 the normal microsomes with cisplatin resulted in inhibition of bition, yet they postulated that platinum compounds might inhibit 497
the activity of CYP3A by interacting with its heme moiety, which is 498
critical to the activation of molecular oxygen to oxidize substrates. 499
In the present study, incubation of turmeric pretreated rat 500
microsomes with cisplatin resulted in greater inhibition of CYP3A 501
activity in a non-competitive manner with a lower apparent Vmax 502
and a lower Ki values than in normal microsomes. These results 503
indicate that cisplatin inhibited CYP3A1/2 in turmeric pretreated 504
microsomes more strongly than in normal microsomes. Lee et al. 505
[16] and Kimura et al. [12] reported that curcumin showed com- 506
petitive, non-competitive and mixed type of inhibition patterns 507
for CYP3A4 and that human CYP3A4 and rat CYP3A1 had 73% pro- 508
tein homology. 509
Moreover the current study clearly revealed that paclitaxel 510
seemed to be a potent inhibitor of CYP3A1/2. The pharmacokinetic 511
studies showed that the inhibition was in a mixed manner with a 512
reduction of Vmax and an increase of Km along with smaller Ki, Ki0 513
values (12 lM, 22 lM). These smaller inhibition constants were 514
indicative of a strong interaction between paclitaxel and CYP3A1/ 515
2. On the other side, addition of paclitaxel to turmeric pretreated 516
rat microsomes interestingly resulted in switching the type of inhi- 517
bition of CYP3A1/2 from mixed to competitive manner with much 518
smaller Ki value. The increased inhibitory potency of paclitaxel on 519
CYP3A1/2 in response to turmeric pretreatment could be attrib- 520
uted to the uncompetitive inhibition of the enzyme by turmeric 521
extract where the turmeric components may bind the enzyme 522
active site with a smaller Km as shown in Table 2. These observa- 523
tions are in accordance with Volak et al. [37] who reported that 524
inhibition of microsomal CYP3A activity by curcuminoids was both 525
time-dependent and competitive. The observation of time depen- 526
dency of inhibition at high curcuminoid concentrations but not 527
at low concentrations is likely complex and might involve low 528
affinity enzymatic production of curcuminoid metabolites with 529
increased CYP3A inhibition potency. Thus co-administration of tur- 530
meric extract with these drugs raises concern about possible drug 531
interactions. 532

5. Conclusions 533

This in vitro study showed that turmeric pretreatment modu- 534

lates the activity of CYP450 enzymes, as it had strong inhibitory 535
influence on CYP3A1/2 with a limited effect on CYP2E1. 536
Pretreatment with turmeric attenuated the inhibitory effects of cis- 537
Fig. 6. Lineweaver–Burk plot showing the inhibition of CYP3A1/2 activity by
paclitaxel (10 lM) in hepatic microsomes isolated from normal (A) and turmeric (B)
platin and paclitaxel on CYP2E1 based on the increase in the Ki con- 538
pretreated rats at concentrations 0, 1.25, 2.5, 5, and 10 mM of erythromycin. The stants of these drugs. However, this pretreatment enhanced the 539
values are expressed as mean ± S.E. (n = 3). inhibitory influence of these drugs on CYP3A1/2 with a marked 540

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8 E.M. Ahmed et al. / Chemico-Biological Interactions xxx (2014) xxx–xxx

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