doi: 10.1111/fcp.12071
Fundamental & Clinical Pharmacology
ORIGINAL ARTICLE
Cetuximab increases concentrations of irinotecan and of its active metabolite SN-38 in plasma and tumour of human colorectal carcinoma-bearing mice Celine Chua,b, Chadi Abbaraa,c*, Mahamadou Tandiaa,b, Melanie Polrotd, Patrick Gonind, Robert Farinottib, Laurence Bonhomme-Faivrea,b a
Laboratory of Pharmacology, Service Pharmacie, H^opital Paul Brousse AP-HP, 14 avenue Paul Vaillant-Couturier, 94800 Villejuif, France b UPRES EA 4123 Faculty of Pharmaceutical Sciences, Universite Paris Sud XI, 5 rue Jean-Baptiste Clement, 92296 Ch^atenay-Malabry cedex, France c Department of Pharmacology and Toxicology, Centre Hospitalier Universitaire d’Angers, 4 rue Larrey, 49100 Angers, France d Animal and Veterinary Resources, IFR 54, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France
Keywords cetuximab, irinotecan, P-glycoprotein, pharmacokinetics
Received 16 September 2013; revised 12 December 2013; accepted 17 February 2014
*Correspondence and reprints: [email protected]
ABSTRACT
In a previous study, we showed that cetuximab, a monoclonal antibody directed towards epidermal growth factor receptor, could inhibit P-glycoprotein (P-gp), an efflux protein of ATP-binding cassette family, and lead to an increased P-gp substrate intracellular concentration. Cetuximab is given with irinotecan to patients with metastasis colorectal cancer who did not respond to irinotecan-based therapy. The mechanism of this successful clinical reversion remains unknown. As irinotecan is a P-gp substrate, we tested here whether cetuximab could modify irinotecan concentration in mice. Therefore, concentrations of irinotecan and of its active metabolite SN-38 were measured by HPLC in plasma and tumour of mice bearing a human colorectal carcinoma xenograft when irinotecan is given orally alone or after a pretreatment with cetuximab. Pharmacokinetic analysis showed no significant modification of irinotecan concentrations but a significant increase (1.7-fold) in SN-38 AUCs in plasma and in tumour after a pretreatment with cetuximab. Those results suggest that cetuximab influence irinotecan distribution into tissues probably due to inhibition of P-gp. As SN-38 is 200-fold more potent than irinotecan, cetuximab could reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour can lead to pharmacokinetic modification and a higher anticancer efficacy.
INTRODUCTION Irinotecan (Campto), a topoisomerase I inhibitor, is widely used in first and second line of treatment of advanced colorectal cancer. Irinotecan is converted into its active metabolite SN-38, which is approximately 100- to 1000-fold more cytotoxic than the parent drug [1]. Over the past decade, response rate and overall
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survival of patients treated for a metastatic colorectal cancer have been significantly increased due to the development of targeted therapies. Cetuximab (Erbitux) is an IgG1 chimeric monoclonal antibody directed against the epidermal growth factor receptor (EGFR). Signalling pathways of EGFR are involved in controlling cell survival, angiogenesis, migration and cell invasion, and metastatic potential of cells. Cetuximab is given to te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
Cetuximab increases SN-38 AUC in mice
patients treated for metastatic colorectal cancer in combination with irinotecan when irinotecan-based therapy has failed [2,3]. However, the mechanism of this clinical reversion remains unknown. Irinotecan and SN-38 are substrates of various polymorphic enzymes and transporters leading to very complicated pharmacokinetics and thus have been the subject of intensive investigation in recent years [4–6]. Irinotecan is metabolized into SN-38 by carboxylesterases types 1 and 2. Competing with the formation of SN-38 is the oxidation of irinotecan into the inactive metabolites by CYP3A4 and CYP3A5. SN-38 is further conjugated by UDP-glucuronosyltransferase isoforms to form an inactive b-glucuronic acid conjugate, SN-38G to be eliminated by biliary and renal excretion. In addition to the liver, activation of irinotecan into SN-38 could be done in intestines as b-glucuronidase and carboxylesterase activities were also observed in normal colorectal and tumour tissue [5,7]. Finally, elimination of irinotecan is dependent on drug-transporting proteins from the ATP-binding cassette drug transporters family, notably P-glycoprotein (P-gp), multidrug resistance-associated protein gene (MRP) and breast cancer resistance protein (BCRP) [8]. Drug-transporting protein such as P-gp can cause multidrug resistance in tumour cells by decreasing intracellular drug levels. Enhanced expression of P-gp is considered to be a major mechanism of chemotherapeutics resistance [9,10]. Conversely, inhibitors of P-gp may increase oral absorption and distribution of P-gp transported drugs [9–11]. For example, verapamil enhances bioavailability of irinotecan and decreases its biliary excretion [11]. In a previous study, we showed that cetuximab can inhibit P-gp in vitro and thus increase doxorubicin (P-gp substrate) intracellular accumulation [12]. Considering it as an interesting hypothesis to explain resistance reversion of irinotecan by cetuximab, we compared pharmacokinetic studies in mice bearing human colorectal carcinoma to evaluate cetuximab effect on irinotecan and its active metabolite SN-38 plasma and tumour disposition.
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Mice bearing human colorectal carcinoma xenograft model In this study was used a xenograft model originating from a human tumour collection, established under the CReMEC project: the CR-IGR016P primary xenograft. The patient, from whom the tumour originated, was a woman, with an adenocarcinoma of the sigmoid colon with ovarian metastases. The sample was directly derived from the primary tumour. The CR-IGR-016P tissue was cut into small pieces. These tissues were then subcutaneously implanted into Female Foxn1nu CD-1 nude mice mouse flanks. These mice were purchased from Animal and Veterinary Resources, Institut Gustave Roussy, IFR54 (Villejuif, France). Mice were housed under standard laboratory sterile conditions, with sterile water and regular sterile (gamma-irradiated) chow provided ad libitum in a 12h/12-h light/dark cycle at a 21–23 °C temperature. Anaesthesia was induced with 5% isoflurane and maintained with 2.5% isoflurane in air. When the tumour had reached 2–3 cm in diameter, it was sampled and cut into small pieces to obtain the second-passage model, which was used in the study. The animals were treated in accordance with the European committee standards concerning the care and use of laboratory animals. The experimental protocol was approved by Local Animal Experimentation Committee (N°26, Ministere de la Recherche et de l’Enseignement Superieur).
MATERIALS AND METHODS
Irinotecan and SN-38 pharmacokinetic studies Mice were randomized into two groups. The first group orally received irinotecan at the dose of 40 mg/kg. The second group intraperitoneally received cetuximab at the dose of 90 mg/kg on days 1 and 3 then orally received irinotecan at 40 mg/kg 1 h after cetuximab administration. For irinotecan and SN-38 assay, blood samples (0.5 mL on average) were withdrawn from retro-orbital plexus and collected in heparinized tubes at 0.25, 0.5, 1, 2, 3, 4 and 8 h after irinotecan administration, three mice per point. Blood samples were centrifuged for 10 min at 6200 g. Plasma were harvested into clean tubes and immediately analysed as described below. After blood sampling, mice were sacrificed. Tumours were collected and stored at 80 °C until analysis.
Drugs Irinotecan (Campto) was purchased from Pfizer (Montrouge, France). Cetuximab (Erbitux) was purchased from Merck Serono (Lyon, France).
Irinotecan and SN-38 quantification in plasma Irinotecan and SN-38 plasma concentrations were quantified using a HPLC method coupled with
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fluorescence detector. Quantification followed solidphase extraction. For analysis, 100 µL of plasma was mixed with 200 µL of acetonitrile acidified (to 1 mL acetonitrile add 50 lL acetic acid). The mixture was vortexed for 10 s and centrifuged for 10 min at 5000 rpm. Supernatants were harvested into clean tubes and then evaporated under a gentle stream of nitrogen at 30 °C. The residue was reconstituted in acetonitrile/acetic acid mixture (70/30) acidified with trifluoroacetic acid (to 10 mL of mixture add 30 lL trifluoroaceticacid) and vortexed for 20 s. An aliquot of 100 µL was then injected into the chromatographic system. Chromatographic analysis was accomplished using a Nucleosil C18 column (4.6 * 125 mm, 3 lm) (Interchim, Montlucßon, France) with a mobile phase (acetonitrile/methanol/tetra butyl ammonium 0.0025 M pH = 3.2, 22/5/73, v/v/v) delivered at a flow rate of 1.2 mL/min. The eluent was monitored at 260 nm. The irinotecan and SN-38 standard curves were correctly described by unweighted least-square linear regression. Over the irinotecan (or SN-38) plasma concentration range of 10–2500 ng/mL (5–1250 ng/mL), the determination coefficient (R2) of the calibration curves remained >0.99. Based on quality control samples, the overall relative SD (an index of precision) was less than 12%. The irinotecan and SN-38 lower limits of quantification were 10 and 5 ng/mL. Three irinotecan and SN-38 quality controls were prepared: low (30 and 15 ng/mL), medium (750 and 375 ng/mL) and high (2000 and 1000 ng/mL).
Data analysis Because each animal provided only one sample of blood, data from animals of the same group were pooled using a naive averaging data approach [13]. Data were analysed separately for each treatment, blood and tumour group using the average concentration at each point. The noncompartmental analysis was performed using WINNONLINE professional version 5.2 software (Pharsight, Mountain View, CA, USA). Main pharmacokinetics parameters were selected from previous published studies [14–16]. The mean maximum concentrations (Cmax) and the times necessary to reach it (Tmax) were evaluated from experimental curves. Irinotecan and SN-38 terminal half-lives (t1/2) were calculated from the respective terminal rate constants (Ke), estimated as the slope of the log-linear terminal portion of the mean matrix concentration vs. time curve, by linear regression analysis. The mean areas under the concentration–time curves (AUC) were calculated by the trapezoidal method from 0 to the last concentration–time point. Irinotecan and SN-38 trough concentrations were defined as the last quantifiable concentrations. Irinotecan and SN-38 AUC of both groups were compared using Bailer’s method [17]. Cmax were compared using a Student’s t-test with a significant level at 0.05. As Tmax and half-lives are unique values, their variation after a cetuximab pretreatment was described but statistical test could not be carried out.
Irinotecan and SN-38 quantification in tumour For analysis, each weighted tumour was mixed first with 50 µL of acetonitrile and 50 µL of water. Irinotecan and SN-38 were then extracted according to the extraction protocol as described above. The extraction was repeated four times. Irinotecan and SN-38 tumour concentrations were quantified using the HPLC with fluorescence detector as described above. Calibration standards of irinotecan and SN-38 were prepared in drug-free tumours by spiking with concentrated standards to obtain a concentration range of irinotecan (or SN-38) between 0.5 and 50 ng/g (0.25–25 ng/g). Three irinotecan and SN-38 quality controls were prepared in drug-free tumours by spiking concentrated standards: low (1.5 and 0.75 ng/g), medium (7.5 and 3.75 ng/g) and high (40 and 20 ng/g). Irinotecan and SN-38 lower limit of quantification were 0.5 and 0.25 ng/g.
Cetuximab administration modifies irinotecan plasma and tumour pharmacokinetics In Figures 1 and 2 are reported plasma and tumour concentrations vs. time curves of irinotecan following oral administration alone or after a pretreatment with cetuximab. Plasma and tumour pharmacokinetic parameters of irinotecan are reported in Tables I and II. In plasma, Tmax were observed 2 h after administration of irinotecan for both groups; cetuximab does not affect the time required for the plasma concentration to reach its maximum value. Irinotecan AUC was 1.3-fold higher in cetuximab-treated mice group, but this increase is not statistically significant. However, enhanced irinotecan concentration at the sampling time at 4 h was observed in mice treated with cetuximab. The terminal half-lives were similar, and trough concentrations were not significantly different between both groups.
RESULTS
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Table I Plasma pharmacokinetic parameters of irinotecan after oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Cmax
Mice received
irinotecan and
irinotecan alone
cetuximab
835.9 266.4
657.8 146.3
(ng/mL) SD
NS
Tmax(h) AUC
Figure 1 Irinotecan plasma concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
2 1660.2 368.7
2 2218.5 630.0
(ng*h/mL) SD
Z = 0.74 < 1.96 NS
Ratio AUC Trough
P = 0.59 < 1.96
1.3 23.2 13.5
17.0 3.15
concentration
P = 0.48 > 0.05 NS
(ng/mL) SD Terminal
1.4
1.3
half-life (h)
Table II Tumour pharmacokinetic parameters of irinotecan after oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Cmax
Figure 2 Irinotecan tumour concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
Cetuximab administration modifies SN-38 plasma and tumour pharmacokinetics Plasma and tumour concentrations vs. time curves of SN-38 following oral administration in both groups of te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
irinotecan and
irinotecan alone
cetuximab
2336.8 1018.3
3541.6 1564.2
(ng/g) SD Ratio Cmax Tmax (h) AUC
Trough concentration
P = 0.55 > 0.05 NS
1.5 2 7070.8 1505.2
4 13861.0 3976.2
(ng*h/g) SD Ratio AUC
In tumours, irinotecan AUC was twofold higher in cetuximab-treated mice group than in irinotecan alone treated group, but this increase was not statistically significant. As shown on Figure 2, in the group of irinotecan alone, the time necessary to reach the maximal concentration was achieved at 2 h after administration. In cetuximab pretreated group, the time necessary to reach the maximal concentration was at 4 h after administration reflecting a decrease in the absorption speed of irinotecan. Trough concentrations were not significantly different between both groups.
Mice received
Z = 1.60 < 1.96 NS
2 507.4 85.7
442.8 229.1
P = 0.67 > 0.05 NS
(ng/g) SD
treatment are shown in Figures 3 and 4. Plasma and tumour pharmacokinetic parameters of SN-38 are mentioned in Tables III and IV. In plasma, as previously described for irinotecan, enhanced SN-38 plasma concentration at 4 h was observed in mice treated with cetuximab. On contrary, a 1.7-fold significant increase in SN-38 AUC was observed in cetuximab-treated mice group (Bailer’s method, P < 0.05). Terminal half-lives were similar, and a 1.2-fold increase in trough concentration was observed in cetuximab-treated mice group but it was not statistically significant.
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Table III Plasma pharmacokinetic parameters of SN-38 after irinotecan oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received Mice received
irinotecan and
irinotecan alone
cetuximab
726.3 217.7
Cmax
884.3 210.7
(ng/mL) SD Ratio Cmax
NS 1.2 1644.7 190.1
AUC
Figure 3 SN-38 plasma concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
P = 0.63 > 0.05
2805.1 441.8
Z = 2.26 > 1.96 S
(ng*h/mL) SD Ratio AUC Trough
1.7 29.5 11.1
34.7 4.2
concentration
P = 0.69 > 0.05 NS
(ng/mL) SD Terminal
1.8
1.7
half-life (h)
Table IV Tumour pharmacokinetic parameters of SN-38 after irinotecan oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Figure 4 SN-38 tumour concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
Mice received
irinotecan and
irinotecan alone
cetuximab
363.4 87.7
Cmax
423.3 81.0
P = 0.67 > 0.05 NS
(ng/g) SD Ratio Cmax
1.2 1297.4 133.0
AUC
2219.7 233.6
Z = 3.40 > 1.96 S
(ng*h/g) SD
In tumours, a 1.7-fold significant increase in SN-38 AUC was observed in cetuximab-treated mice group (Bailer’s method, P < 0.05). Trough concentrations were not significantly different between both groups. Relationship between plasma concentrations of irinotecan and of SN-38 in mice treated by cetuximab For each point, irinotecan plasma mean value of mice treated with irinotecan and cetuximab was divided by irinotecan plasma mean value of mice treated with irinotecan alone. SN-38 plasma mean value of mice treated with irinotecan and cetuximab was divided by SN-38 plasma mean value of mice treated with irinotecan alone. Figure 5 indicates that plasma concentration mean ratios of irinotecan and SN-38 follow the same trend.
Ratio AUC Trough
1.7 110.7 56.3
208.2 107.8
P = 0.24 > 0.05 NS
concentration (ng/g) SD
Higher irinotecan and SN-38 plasma mean concentration ratios were observed at three different points: 0.25, 0.5 and 4 h. Relationship between plasma and tumour irinotecan concentrations in mice treated by cetuximab For each point, irinotecan plasma and tumour mean values of mice treated with irinotecan and cetuximab were divided by irinotecan plasma and tumour mean values of mice treated with irinotecan alone. As shown te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
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Cetuximab increases SN-38 AUC in mice
Figure 5 Average plasma value ratio of irinotecan and SN-38 (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
Figure 7 Average plasma and tumour value ratios of SN-38 (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
Figure 8 Average ratios of SN-38 mean plasma concentrations divided by irinotecan mean plasma concentrations in the absence and presence of cetuximab. Figure 6 Average plasma and tumour value ratios of irinotecan (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
in Figure 6, irinotecan plasma and tumour mean value ratios follow the same trend resulting in a correlation between plasma and tumour irinotecan values. Relationship between plasma and tumoral SN-38 concentrations in mice treated by cetuximab SN-38 plasma and tumour mean values of mice treated with irinotecan and cetuximab were divided by SN-38 plasma and tumour mean values of mice treated with irinotecan alone. As shown in Figure 7, higher SN-38 plasma value ratios were observed at two points: 0.25 and 4 h. SN-38 tumour value ratios slightly changed at each point (from 1.1 to 2.5 with an average of 1.95). Effect of cetuximab on irinotecan metabolism into SN-38 SN-38 plasma mean concentrations were divided by irinotecan plasma mean concentrations in mice treated or not with cetuximab. These plasma ratios are shown te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
in Figure 8. The same ratios were then extracted from tumour and represented in Figure 9. The effect of cetuximab on SN-38 formation was not constant in plasma and tumour. In plasma, higher SN-38 formation was observed in the absence of cetuximab in the early sampling times then the ratios decreased in the late sampling times reflecting higher SN-38 formation in the presence of cetuximab. In tumour, higher SN-38 formation was observed in the presence of cetuximab in the early sampling times and then the ratios decreased at the late sampling time to reach the ratio values observed in the group nontreated with cetuximab. DISCUSSION In a previous study, we showed that cetuximab could directly interact with P-gp and inhibit its functionality leading to doxorubicin (a P-gp substrate) intracellular accumulation increase in two cancerous cell lines over expressing P-gp [12]. As irinotecan and SN-38 are known to be P-gp substrates [8], we suspected that cetuximab could increase their concentration in vivo.
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Figure 9 Average ratios of SN-38 mean tumour concentrations divided by irinotecan mean tumour concentrations in the absence and presence of cetuximab.
We presented here pharmacokinetics of irinotecan and of its active metabolite SN-38 in plasma and tumours after irinotecan oral administration in mice bearing human colorectal cancer xenograft and compared them with those obtained after a pretreatment with cetuximab. In most of studies, irinotecan had been administered intravenously [18,19] although it has been administered orally in early clinical trials [20,21]. But as P-gp is present in intestinal epithelial membrane and biliary canalicular, oral route was used to evaluate cetuximab effect on irinotecan because it can affect both intestinal and biliary P-gp. The pharmacokinetic comparisons allowed us to evaluate the effect of cetuximab on irinotecan oral bioavailability. Also, SN-38 pharmacokinetics allowed studying cetuximab effect on the distribution and elimination of the parent drug ‘irinotecan’. We observed a nonsignificant increase in irinotecan AUC in plasma and tumours (1.3-fold and twofold, respectively) but a significant increase in SN-38 AUC in plasma and tumours (1.7-fold for both). Irinotecan AUC tumour increase, although statistically nonsignificant, can be due to an increase in the distribution into tissues, which can also explain SN-38 plasma concentrations increase and hence the increase in tumour AUC. P-glycoprotein, as a drug-transporting protein, is known to affect all steps of drug pharmacokinetics: absorption, distribution, metabolism and elimination. Absorption is the most affected step in case of modulation of P-gp activity, particularly after oral administration. Elimination of substrates of P-gp is, in general, the step that is less affected by the modulation of P-gp activity, and is, in general, due to the effect on the distribution in
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the excretion organs such as kidneys and biliary gland. The effect on metabolism is limited to effect on the distribution in the organ of metabolism, the liver [15,22]. As it had been shown in previous published studies [15,16], P-gp inhibition will result in an increase in Cmax and in the AUC without an important effect on the terminal half-life. Meanwhile, in our study, the effect of cetuximab on the absorption step seems to be less important probably due to the delay between the administration of cetuximab and of irinotecan. Actually, irinotecan Cmax and AUC in mice treated or not with cetuximab were not significantly different. All the results obtained in this study suggest that irinotecan distribution seemed to be the most affected step by the pretreatment with cetuximab. This can be confirmed by results of SN-38 pharmacokinetic analysis. SN-38 appears in plasma due to metabolism of irinotecan in plasma, liver and kidney. This transformation occurs after metabolism of irinotecan by carboxyl esterase. Carboxyl esterase activity is widely distributed in mammalian tissues, with the highest levels present in liver microsomes. Rapid hydrolysis of irinotecan by mouse liver and kidney carboxyl esterase (M-LK) had been reported [23], and this property ranks this carboxyl esterase as one of the most efficient esterases known to hydrolyse this prodrug. Hence, it can be considered that the major part of SN-38 is formed after distribution of irinotecan into these tissues (liver and kidney). In case of transformation in liver and kidney, SN-38 distribution to plasma is controlled by a passive diffusion mechanism [23]. Hence, achieving of 1.7-fold SN-38 AUC increase in plasma can be greatly explained by a higher disposition of irinotecan in liver and kidney. 1.7-fold of SN-38 in plasma can be the result of equivalent disposition of irinotecan in liver, which can strengthen the trend observed between both molecules. To document whether cetuximab may modify irinotecan metabolism through the formation of SN-38, we evaluated the plasma and tumour average concentration ratio of mice treated by irinotecan with or without cetuximab (Figures 8 and 9). This evaluation showed that in plasma, at first sampling times, cetuximab pretreatment led to a decrease in SN-38 formation while it led to its increase in the tumour. One hour later in the plasma and 3 h in the tumour, the values are comparable. It may also be observed that in plasma at 4 h, SN-38 concentration in cetuximab-treated mice increased may be due to SN-38 diffusion from the tiste Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
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sues. The results can be explained by the fact that cetuximab inhibit P-gp, facilitating irinotecan diffusion into tissues such as liver and kidneys and lead to lower SN-38 formation in plasma. Once SN-38 was formed in tissue, it diffused into plasma and thus SN-38 formation increased in late sampling times in the group pretreated with cetuximab. In parallel, SN-38 formation in tumour decreased. A higher distribution of irinotecan and SN-38 due to cetuximab particularly led to higher concentrations of both molecules at the 4-h sampling time, which can be the result of an enterohepatic cycle. Hence, cetuximab effect on irinotecan and SN-38 pharmacokinetics was the result of the cetuximab effect on the distribution of both molecules (mainly by P-gp inhibition) rather than an effect on irinotecan metabolism. In addition, no published data described an induction of carboxylesterase by cetuximab. The effect of cetuximab in plasma on SN-38 formation was not the same at each sampling time (decreasing in the first times then increasing), which suggests that cetuximab did not affect irinotecan metabolism. Finally, SN-38 tumour value ratios were SN-38 mean concentrations in mice treated with irinotecan and cetuximab divided by SN-38 mean concentrations in mice treated with irinotecan alone. Their increase explained the significant increase in SN-38 tumour AUC and slight variations at each sampling time (from 1.1 to 2.5 with an average of 1.95) even when SN-38 plasma value ratios decreased. The increase observed in SN-38 AUC in tumour is due to an increase in SN-38 AUC in plasma, and probably also to an inhibition of SN-38 efflux from tumour caused by cetuximab. All these results can explain clinically obtained outcomes. Cetuximab effect on the pharmacokinetics of irinotecan after IV administration should be more important on the distribution step, hence affecting formation of SN-38 in plasma. As SN-38 is 200-fold more potent than irinotecan, our results are interesting and could explain how cetuximab may reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour could lead to pharmacokinetic modifications resulting in a higher antitumour efficacy. CONCLUSION Cetuximab concentration significantly increased SN-38 plasma and tumour AUC in mice bearing human colote Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
rectal carcinoma. Our results suggest that cetuximab can reverse irinotecan resistance by increasing its active metabolite disposition by inhibition of efflux transporters such as P-gp in intestines, biliary system and tumour. ACKNOWLEDGEMENT We thank the Cremec consortium that permitted the establishment of the xenograft model CR-IGR016P (‘Projet C.Re.M.E.C.’) and Dr. Simone Orbach for her kind help. CONFLICT OF INTEREST The authors declare no conflict of interest. ABBREVIATIONS P-gp – P-glycoprotein AUC – area under the curve Cmax – maximal concentration Tmax – time to reach maximal concentration t1/2 – terminal half-life REFERENCES 1 Xu Y., Villalona-Calero M.A. Irinotecan: mechanisms of tumor resistance and novel strategies for modulating its activity. Ann. Oncol. (2002) 13 1841–1851. 2 Cunningham D., Humblet Y., Siena S. et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecanrefractory metastatic colorectal cancer. New. Engl. J. Med. (2004) 351 337–345. 3 Prewett M.C., Hooper A.T., Bassi R. et al. Enhanced antitumor activity of anti-epidermal growth factor receptor monoclonal antibody IMC-C225 in combination with irinotecan (CPT-11) against human colorectal tumor xenografts. Clin. Cancer Res. (2002) 8 994–1003. 4 Smith N.F., Figg W.D., Sparreboom A. Pharmacogenetics of irinotecan metabolism and transport: an update. Toxicol. In Vitro (2006) 20 163–175. 5 Van der Bol J.M., Loos W.J., de Jong F.A. et al. Effect of omeprazole on the pharmacokinetics and toxicities of irinotecan in cancer patients: a prospective cross-over drug-drug interaction study. Eur. J. Cancer (2011) 47 831–838. 6 Van der Bol J.M., Visser T.J., Loos W.J. et al. Effects of methimazole on the elimination of irinotecan. Cancer Chemother. Pharmacol. (2011) 67 231–236. 7 Xu G., Zhang W., Ma M.K. et al. Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with
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8
9
10
11
12
13
14
15
16
activation of irinotecan. Clin. Cancer Res. (2002) 8 2605– 2611. Arimori K., Kuroki N., Hidaka M. et al. Effect of Pglycoprotein modulator, cyclosporin A, on the gastrointestinal excretion of irinotecan and its metabolite SN-38 in rats. Pharm. Res. (2003) 20 910–917. El-Readi M.Z., Hamdan D., Farrag N. et al. Inhibition of Pglycoprotein activity by limonin and other secondary metabolites from Citrus species in human colon and leukaemia cell lines. Eur. J. Pharmacol. (2010) 626 139–145. Goldstein L.J., Galski H., Fojo A. et al. Expression of a multidrug resistance gene in human cancers. J. Natl. Cancer Inst. (1989) 81 116–124. Bansal T., Mishra G., Jaggi M. et al. Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats. Eur. J. Pharm. Sci. (2009) 36 580–590. Chu C., Abbara C., No€el-Hudson M.S. et al. In vivo and in vitro evidence of the effect of Erbitux on drug substrates of ABCB1. FIRN. (2012) Salzburg, Austria. Burtin P., Mentre F., van Bree J., et al. Sparse sampling for assessment of drug exposure in toxicological studies. Eur. J. Drug Metab. Pharmacokinet. (1996) 21 105–111. Cattaneo D., Meraviglia P., Cozzi V., et al. Atypical pharmacokinetics of atazanavir in an HIV-1-infected patient. Fun. Clin. Pharm. (2012) 26 204–206. Bonhomme-Faivre L., Benyamina A., Reynaud M., et al. Disposition of Delta tetrahydrocannabinol in CF1 mice deficient in mdr1a P-glycoprotein. Addict. Biol. (2008) 13 295–300. Kovarick J.M., Beyer D., Bizot M.N., et al. Pharmacokinetic interaction between verapamil and everolimus in
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20
21
22
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healthy subjects. Br. J. Clin. Pharmacol. (2005) 60 434–437. Bailer A.J. Testing for the equality of area under the curves when using destructive measurement techniques. J. Pharmacokinet. Biopharm. (1998) 16 303–309. Czejka M., Gruenberger B., Kiss A., et al. Pharmacokinetics of irinotecan in combination with biweekly cetuximab in patients with advanced colorectal cancer. Anticancer Res. (2010) 30 2355–2360. Pierga J.Y., Dieras V., Faivre S., et al. Pharmacokinetic profile of cetuximab (Erbitux) alone and in combination with irinotecan in patients with advanced EGFR-positive adenocarcinoma. Eur. J. Cancer (2005) 41 1739–1745. Schoemaker N.E., Kuppens I.E., Huinink W.W., et al. Phase I study of an oral formulation of irinotecan administered daily for 14 days every 3 weeks in patients with advanced solid tumours. Cancer Chemother. Pharmacol. (2005) 55 263– 270. Soepenberg O., Dumez H., Verweij J., et al. Phase I and pharmacokinetic study of oral irinotecan given once daily for 5 days every 3 weeks in combination with capecitabine in patients with solid tumors. J. Clin. Oncol. (2005) 23 889– 898. Chu C., Abbara C., Noel-Hudson M.S., et al. Disposition of everolimus in mdr1a-/1b- mice and after a pre-treatment of lapatinib in Swiss mice. Biochem. Pharmacol. (2009) 77 1629–1634. Xie M., Yang D., Wu M., et al. Mouse liver and kidney carboxylesterase (M-LK) rapidly hydrolyzes antitumor prodrug irinotecan and the N-terminal three quarter sequence determines substrate selectivity. Drug Metab. Dispos. (2003) 31 21–27.
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Fundamental & Clinical Pharmacology
ORIGINAL ARTICLE
Cetuximab increases concentrations of irinotecan and of its active metabolite SN-38 in plasma and tumour of human colorectal carcinoma-bearing mice Celine Chua,b, Chadi Abbaraa,c*, Mahamadou Tandiaa,b, Melanie Polrotd, Patrick Gonind, Robert Farinottib, Laurence Bonhomme-Faivrea,b a
Laboratory of Pharmacology, Service Pharmacie, H^opital Paul Brousse AP-HP, 14 avenue Paul Vaillant-Couturier, 94800 Villejuif, France b UPRES EA 4123 Faculty of Pharmaceutical Sciences, Universite Paris Sud XI, 5 rue Jean-Baptiste Clement, 92296 Ch^atenay-Malabry cedex, France c Department of Pharmacology and Toxicology, Centre Hospitalier Universitaire d’Angers, 4 rue Larrey, 49100 Angers, France d Animal and Veterinary Resources, IFR 54, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94800 Villejuif, France
Keywords cetuximab, irinotecan, P-glycoprotein, pharmacokinetics
Received 16 September 2013; revised 12 December 2013; accepted 17 February 2014
*Correspondence and reprints: [email protected]
ABSTRACT
In a previous study, we showed that cetuximab, a monoclonal antibody directed towards epidermal growth factor receptor, could inhibit P-glycoprotein (P-gp), an efflux protein of ATP-binding cassette family, and lead to an increased P-gp substrate intracellular concentration. Cetuximab is given with irinotecan to patients with metastasis colorectal cancer who did not respond to irinotecan-based therapy. The mechanism of this successful clinical reversion remains unknown. As irinotecan is a P-gp substrate, we tested here whether cetuximab could modify irinotecan concentration in mice. Therefore, concentrations of irinotecan and of its active metabolite SN-38 were measured by HPLC in plasma and tumour of mice bearing a human colorectal carcinoma xenograft when irinotecan is given orally alone or after a pretreatment with cetuximab. Pharmacokinetic analysis showed no significant modification of irinotecan concentrations but a significant increase (1.7-fold) in SN-38 AUCs in plasma and in tumour after a pretreatment with cetuximab. Those results suggest that cetuximab influence irinotecan distribution into tissues probably due to inhibition of P-gp. As SN-38 is 200-fold more potent than irinotecan, cetuximab could reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour can lead to pharmacokinetic modification and a higher anticancer efficacy.
INTRODUCTION Irinotecan (Campto), a topoisomerase I inhibitor, is widely used in first and second line of treatment of advanced colorectal cancer. Irinotecan is converted into its active metabolite SN-38, which is approximately 100- to 1000-fold more cytotoxic than the parent drug [1]. Over the past decade, response rate and overall
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survival of patients treated for a metastatic colorectal cancer have been significantly increased due to the development of targeted therapies. Cetuximab (Erbitux) is an IgG1 chimeric monoclonal antibody directed against the epidermal growth factor receptor (EGFR). Signalling pathways of EGFR are involved in controlling cell survival, angiogenesis, migration and cell invasion, and metastatic potential of cells. Cetuximab is given to te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
Cetuximab increases SN-38 AUC in mice
patients treated for metastatic colorectal cancer in combination with irinotecan when irinotecan-based therapy has failed [2,3]. However, the mechanism of this clinical reversion remains unknown. Irinotecan and SN-38 are substrates of various polymorphic enzymes and transporters leading to very complicated pharmacokinetics and thus have been the subject of intensive investigation in recent years [4–6]. Irinotecan is metabolized into SN-38 by carboxylesterases types 1 and 2. Competing with the formation of SN-38 is the oxidation of irinotecan into the inactive metabolites by CYP3A4 and CYP3A5. SN-38 is further conjugated by UDP-glucuronosyltransferase isoforms to form an inactive b-glucuronic acid conjugate, SN-38G to be eliminated by biliary and renal excretion. In addition to the liver, activation of irinotecan into SN-38 could be done in intestines as b-glucuronidase and carboxylesterase activities were also observed in normal colorectal and tumour tissue [5,7]. Finally, elimination of irinotecan is dependent on drug-transporting proteins from the ATP-binding cassette drug transporters family, notably P-glycoprotein (P-gp), multidrug resistance-associated protein gene (MRP) and breast cancer resistance protein (BCRP) [8]. Drug-transporting protein such as P-gp can cause multidrug resistance in tumour cells by decreasing intracellular drug levels. Enhanced expression of P-gp is considered to be a major mechanism of chemotherapeutics resistance [9,10]. Conversely, inhibitors of P-gp may increase oral absorption and distribution of P-gp transported drugs [9–11]. For example, verapamil enhances bioavailability of irinotecan and decreases its biliary excretion [11]. In a previous study, we showed that cetuximab can inhibit P-gp in vitro and thus increase doxorubicin (P-gp substrate) intracellular accumulation [12]. Considering it as an interesting hypothesis to explain resistance reversion of irinotecan by cetuximab, we compared pharmacokinetic studies in mice bearing human colorectal carcinoma to evaluate cetuximab effect on irinotecan and its active metabolite SN-38 plasma and tumour disposition.
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Mice bearing human colorectal carcinoma xenograft model In this study was used a xenograft model originating from a human tumour collection, established under the CReMEC project: the CR-IGR016P primary xenograft. The patient, from whom the tumour originated, was a woman, with an adenocarcinoma of the sigmoid colon with ovarian metastases. The sample was directly derived from the primary tumour. The CR-IGR-016P tissue was cut into small pieces. These tissues were then subcutaneously implanted into Female Foxn1nu CD-1 nude mice mouse flanks. These mice were purchased from Animal and Veterinary Resources, Institut Gustave Roussy, IFR54 (Villejuif, France). Mice were housed under standard laboratory sterile conditions, with sterile water and regular sterile (gamma-irradiated) chow provided ad libitum in a 12h/12-h light/dark cycle at a 21–23 °C temperature. Anaesthesia was induced with 5% isoflurane and maintained with 2.5% isoflurane in air. When the tumour had reached 2–3 cm in diameter, it was sampled and cut into small pieces to obtain the second-passage model, which was used in the study. The animals were treated in accordance with the European committee standards concerning the care and use of laboratory animals. The experimental protocol was approved by Local Animal Experimentation Committee (N°26, Ministere de la Recherche et de l’Enseignement Superieur).
MATERIALS AND METHODS
Irinotecan and SN-38 pharmacokinetic studies Mice were randomized into two groups. The first group orally received irinotecan at the dose of 40 mg/kg. The second group intraperitoneally received cetuximab at the dose of 90 mg/kg on days 1 and 3 then orally received irinotecan at 40 mg/kg 1 h after cetuximab administration. For irinotecan and SN-38 assay, blood samples (0.5 mL on average) were withdrawn from retro-orbital plexus and collected in heparinized tubes at 0.25, 0.5, 1, 2, 3, 4 and 8 h after irinotecan administration, three mice per point. Blood samples were centrifuged for 10 min at 6200 g. Plasma were harvested into clean tubes and immediately analysed as described below. After blood sampling, mice were sacrificed. Tumours were collected and stored at 80 °C until analysis.
Drugs Irinotecan (Campto) was purchased from Pfizer (Montrouge, France). Cetuximab (Erbitux) was purchased from Merck Serono (Lyon, France).
Irinotecan and SN-38 quantification in plasma Irinotecan and SN-38 plasma concentrations were quantified using a HPLC method coupled with
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fluorescence detector. Quantification followed solidphase extraction. For analysis, 100 µL of plasma was mixed with 200 µL of acetonitrile acidified (to 1 mL acetonitrile add 50 lL acetic acid). The mixture was vortexed for 10 s and centrifuged for 10 min at 5000 rpm. Supernatants were harvested into clean tubes and then evaporated under a gentle stream of nitrogen at 30 °C. The residue was reconstituted in acetonitrile/acetic acid mixture (70/30) acidified with trifluoroacetic acid (to 10 mL of mixture add 30 lL trifluoroaceticacid) and vortexed for 20 s. An aliquot of 100 µL was then injected into the chromatographic system. Chromatographic analysis was accomplished using a Nucleosil C18 column (4.6 * 125 mm, 3 lm) (Interchim, Montlucßon, France) with a mobile phase (acetonitrile/methanol/tetra butyl ammonium 0.0025 M pH = 3.2, 22/5/73, v/v/v) delivered at a flow rate of 1.2 mL/min. The eluent was monitored at 260 nm. The irinotecan and SN-38 standard curves were correctly described by unweighted least-square linear regression. Over the irinotecan (or SN-38) plasma concentration range of 10–2500 ng/mL (5–1250 ng/mL), the determination coefficient (R2) of the calibration curves remained >0.99. Based on quality control samples, the overall relative SD (an index of precision) was less than 12%. The irinotecan and SN-38 lower limits of quantification were 10 and 5 ng/mL. Three irinotecan and SN-38 quality controls were prepared: low (30 and 15 ng/mL), medium (750 and 375 ng/mL) and high (2000 and 1000 ng/mL).
Data analysis Because each animal provided only one sample of blood, data from animals of the same group were pooled using a naive averaging data approach [13]. Data were analysed separately for each treatment, blood and tumour group using the average concentration at each point. The noncompartmental analysis was performed using WINNONLINE professional version 5.2 software (Pharsight, Mountain View, CA, USA). Main pharmacokinetics parameters were selected from previous published studies [14–16]. The mean maximum concentrations (Cmax) and the times necessary to reach it (Tmax) were evaluated from experimental curves. Irinotecan and SN-38 terminal half-lives (t1/2) were calculated from the respective terminal rate constants (Ke), estimated as the slope of the log-linear terminal portion of the mean matrix concentration vs. time curve, by linear regression analysis. The mean areas under the concentration–time curves (AUC) were calculated by the trapezoidal method from 0 to the last concentration–time point. Irinotecan and SN-38 trough concentrations were defined as the last quantifiable concentrations. Irinotecan and SN-38 AUC of both groups were compared using Bailer’s method [17]. Cmax were compared using a Student’s t-test with a significant level at 0.05. As Tmax and half-lives are unique values, their variation after a cetuximab pretreatment was described but statistical test could not be carried out.
Irinotecan and SN-38 quantification in tumour For analysis, each weighted tumour was mixed first with 50 µL of acetonitrile and 50 µL of water. Irinotecan and SN-38 were then extracted according to the extraction protocol as described above. The extraction was repeated four times. Irinotecan and SN-38 tumour concentrations were quantified using the HPLC with fluorescence detector as described above. Calibration standards of irinotecan and SN-38 were prepared in drug-free tumours by spiking with concentrated standards to obtain a concentration range of irinotecan (or SN-38) between 0.5 and 50 ng/g (0.25–25 ng/g). Three irinotecan and SN-38 quality controls were prepared in drug-free tumours by spiking concentrated standards: low (1.5 and 0.75 ng/g), medium (7.5 and 3.75 ng/g) and high (40 and 20 ng/g). Irinotecan and SN-38 lower limit of quantification were 0.5 and 0.25 ng/g.
Cetuximab administration modifies irinotecan plasma and tumour pharmacokinetics In Figures 1 and 2 are reported plasma and tumour concentrations vs. time curves of irinotecan following oral administration alone or after a pretreatment with cetuximab. Plasma and tumour pharmacokinetic parameters of irinotecan are reported in Tables I and II. In plasma, Tmax were observed 2 h after administration of irinotecan for both groups; cetuximab does not affect the time required for the plasma concentration to reach its maximum value. Irinotecan AUC was 1.3-fold higher in cetuximab-treated mice group, but this increase is not statistically significant. However, enhanced irinotecan concentration at the sampling time at 4 h was observed in mice treated with cetuximab. The terminal half-lives were similar, and trough concentrations were not significantly different between both groups.
RESULTS
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Table I Plasma pharmacokinetic parameters of irinotecan after oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Cmax
Mice received
irinotecan and
irinotecan alone
cetuximab
835.9 266.4
657.8 146.3
(ng/mL) SD
NS
Tmax(h) AUC
Figure 1 Irinotecan plasma concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
2 1660.2 368.7
2 2218.5 630.0
(ng*h/mL) SD
Z = 0.74 < 1.96 NS
Ratio AUC Trough
P = 0.59 < 1.96
1.3 23.2 13.5
17.0 3.15
concentration
P = 0.48 > 0.05 NS
(ng/mL) SD Terminal
1.4
1.3
half-life (h)
Table II Tumour pharmacokinetic parameters of irinotecan after oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Cmax
Figure 2 Irinotecan tumour concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
Cetuximab administration modifies SN-38 plasma and tumour pharmacokinetics Plasma and tumour concentrations vs. time curves of SN-38 following oral administration in both groups of te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
irinotecan and
irinotecan alone
cetuximab
2336.8 1018.3
3541.6 1564.2
(ng/g) SD Ratio Cmax Tmax (h) AUC
Trough concentration
P = 0.55 > 0.05 NS
1.5 2 7070.8 1505.2
4 13861.0 3976.2
(ng*h/g) SD Ratio AUC
In tumours, irinotecan AUC was twofold higher in cetuximab-treated mice group than in irinotecan alone treated group, but this increase was not statistically significant. As shown on Figure 2, in the group of irinotecan alone, the time necessary to reach the maximal concentration was achieved at 2 h after administration. In cetuximab pretreated group, the time necessary to reach the maximal concentration was at 4 h after administration reflecting a decrease in the absorption speed of irinotecan. Trough concentrations were not significantly different between both groups.
Mice received
Z = 1.60 < 1.96 NS
2 507.4 85.7
442.8 229.1
P = 0.67 > 0.05 NS
(ng/g) SD
treatment are shown in Figures 3 and 4. Plasma and tumour pharmacokinetic parameters of SN-38 are mentioned in Tables III and IV. In plasma, as previously described for irinotecan, enhanced SN-38 plasma concentration at 4 h was observed in mice treated with cetuximab. On contrary, a 1.7-fold significant increase in SN-38 AUC was observed in cetuximab-treated mice group (Bailer’s method, P < 0.05). Terminal half-lives were similar, and a 1.2-fold increase in trough concentration was observed in cetuximab-treated mice group but it was not statistically significant.
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Table III Plasma pharmacokinetic parameters of SN-38 after irinotecan oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received Mice received
irinotecan and
irinotecan alone
cetuximab
726.3 217.7
Cmax
884.3 210.7
(ng/mL) SD Ratio Cmax
NS 1.2 1644.7 190.1
AUC
Figure 3 SN-38 plasma concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
P = 0.63 > 0.05
2805.1 441.8
Z = 2.26 > 1.96 S
(ng*h/mL) SD Ratio AUC Trough
1.7 29.5 11.1
34.7 4.2
concentration
P = 0.69 > 0.05 NS
(ng/mL) SD Terminal
1.8
1.7
half-life (h)
Table IV Tumour pharmacokinetic parameters of SN-38 after irinotecan oral administration (40 mg/kg) obtained by a noncompartimental analysis in nude mice bearing human colorectal carcinoma xenograft, which received or not cetuximab (90 mg/kg). Mice received
Figure 4 SN-38 tumour concentrations after oral administration of irinotecan (40 mg/kg) either given alone or after a pretreatment by cetuximab (90 mg/kg) in nude mice bearing human colorectal carcinoma xenograft (n = 3 per group).
Mice received
irinotecan and
irinotecan alone
cetuximab
363.4 87.7
Cmax
423.3 81.0
P = 0.67 > 0.05 NS
(ng/g) SD Ratio Cmax
1.2 1297.4 133.0
AUC
2219.7 233.6
Z = 3.40 > 1.96 S
(ng*h/g) SD
In tumours, a 1.7-fold significant increase in SN-38 AUC was observed in cetuximab-treated mice group (Bailer’s method, P < 0.05). Trough concentrations were not significantly different between both groups. Relationship between plasma concentrations of irinotecan and of SN-38 in mice treated by cetuximab For each point, irinotecan plasma mean value of mice treated with irinotecan and cetuximab was divided by irinotecan plasma mean value of mice treated with irinotecan alone. SN-38 plasma mean value of mice treated with irinotecan and cetuximab was divided by SN-38 plasma mean value of mice treated with irinotecan alone. Figure 5 indicates that plasma concentration mean ratios of irinotecan and SN-38 follow the same trend.
Ratio AUC Trough
1.7 110.7 56.3
208.2 107.8
P = 0.24 > 0.05 NS
concentration (ng/g) SD
Higher irinotecan and SN-38 plasma mean concentration ratios were observed at three different points: 0.25, 0.5 and 4 h. Relationship between plasma and tumour irinotecan concentrations in mice treated by cetuximab For each point, irinotecan plasma and tumour mean values of mice treated with irinotecan and cetuximab were divided by irinotecan plasma and tumour mean values of mice treated with irinotecan alone. As shown te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
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Cetuximab increases SN-38 AUC in mice
Figure 5 Average plasma value ratio of irinotecan and SN-38 (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
Figure 7 Average plasma and tumour value ratios of SN-38 (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
Figure 8 Average ratios of SN-38 mean plasma concentrations divided by irinotecan mean plasma concentrations in the absence and presence of cetuximab. Figure 6 Average plasma and tumour value ratios of irinotecan (in mice treated by cetuximab and irinotecan divided by mice treated by irinotecan alone).
in Figure 6, irinotecan plasma and tumour mean value ratios follow the same trend resulting in a correlation between plasma and tumour irinotecan values. Relationship between plasma and tumoral SN-38 concentrations in mice treated by cetuximab SN-38 plasma and tumour mean values of mice treated with irinotecan and cetuximab were divided by SN-38 plasma and tumour mean values of mice treated with irinotecan alone. As shown in Figure 7, higher SN-38 plasma value ratios were observed at two points: 0.25 and 4 h. SN-38 tumour value ratios slightly changed at each point (from 1.1 to 2.5 with an average of 1.95). Effect of cetuximab on irinotecan metabolism into SN-38 SN-38 plasma mean concentrations were divided by irinotecan plasma mean concentrations in mice treated or not with cetuximab. These plasma ratios are shown te Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
in Figure 8. The same ratios were then extracted from tumour and represented in Figure 9. The effect of cetuximab on SN-38 formation was not constant in plasma and tumour. In plasma, higher SN-38 formation was observed in the absence of cetuximab in the early sampling times then the ratios decreased in the late sampling times reflecting higher SN-38 formation in the presence of cetuximab. In tumour, higher SN-38 formation was observed in the presence of cetuximab in the early sampling times and then the ratios decreased at the late sampling time to reach the ratio values observed in the group nontreated with cetuximab. DISCUSSION In a previous study, we showed that cetuximab could directly interact with P-gp and inhibit its functionality leading to doxorubicin (a P-gp substrate) intracellular accumulation increase in two cancerous cell lines over expressing P-gp [12]. As irinotecan and SN-38 are known to be P-gp substrates [8], we suspected that cetuximab could increase their concentration in vivo.
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Figure 9 Average ratios of SN-38 mean tumour concentrations divided by irinotecan mean tumour concentrations in the absence and presence of cetuximab.
We presented here pharmacokinetics of irinotecan and of its active metabolite SN-38 in plasma and tumours after irinotecan oral administration in mice bearing human colorectal cancer xenograft and compared them with those obtained after a pretreatment with cetuximab. In most of studies, irinotecan had been administered intravenously [18,19] although it has been administered orally in early clinical trials [20,21]. But as P-gp is present in intestinal epithelial membrane and biliary canalicular, oral route was used to evaluate cetuximab effect on irinotecan because it can affect both intestinal and biliary P-gp. The pharmacokinetic comparisons allowed us to evaluate the effect of cetuximab on irinotecan oral bioavailability. Also, SN-38 pharmacokinetics allowed studying cetuximab effect on the distribution and elimination of the parent drug ‘irinotecan’. We observed a nonsignificant increase in irinotecan AUC in plasma and tumours (1.3-fold and twofold, respectively) but a significant increase in SN-38 AUC in plasma and tumours (1.7-fold for both). Irinotecan AUC tumour increase, although statistically nonsignificant, can be due to an increase in the distribution into tissues, which can also explain SN-38 plasma concentrations increase and hence the increase in tumour AUC. P-glycoprotein, as a drug-transporting protein, is known to affect all steps of drug pharmacokinetics: absorption, distribution, metabolism and elimination. Absorption is the most affected step in case of modulation of P-gp activity, particularly after oral administration. Elimination of substrates of P-gp is, in general, the step that is less affected by the modulation of P-gp activity, and is, in general, due to the effect on the distribution in
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the excretion organs such as kidneys and biliary gland. The effect on metabolism is limited to effect on the distribution in the organ of metabolism, the liver [15,22]. As it had been shown in previous published studies [15,16], P-gp inhibition will result in an increase in Cmax and in the AUC without an important effect on the terminal half-life. Meanwhile, in our study, the effect of cetuximab on the absorption step seems to be less important probably due to the delay between the administration of cetuximab and of irinotecan. Actually, irinotecan Cmax and AUC in mice treated or not with cetuximab were not significantly different. All the results obtained in this study suggest that irinotecan distribution seemed to be the most affected step by the pretreatment with cetuximab. This can be confirmed by results of SN-38 pharmacokinetic analysis. SN-38 appears in plasma due to metabolism of irinotecan in plasma, liver and kidney. This transformation occurs after metabolism of irinotecan by carboxyl esterase. Carboxyl esterase activity is widely distributed in mammalian tissues, with the highest levels present in liver microsomes. Rapid hydrolysis of irinotecan by mouse liver and kidney carboxyl esterase (M-LK) had been reported [23], and this property ranks this carboxyl esterase as one of the most efficient esterases known to hydrolyse this prodrug. Hence, it can be considered that the major part of SN-38 is formed after distribution of irinotecan into these tissues (liver and kidney). In case of transformation in liver and kidney, SN-38 distribution to plasma is controlled by a passive diffusion mechanism [23]. Hence, achieving of 1.7-fold SN-38 AUC increase in plasma can be greatly explained by a higher disposition of irinotecan in liver and kidney. 1.7-fold of SN-38 in plasma can be the result of equivalent disposition of irinotecan in liver, which can strengthen the trend observed between both molecules. To document whether cetuximab may modify irinotecan metabolism through the formation of SN-38, we evaluated the plasma and tumour average concentration ratio of mice treated by irinotecan with or without cetuximab (Figures 8 and 9). This evaluation showed that in plasma, at first sampling times, cetuximab pretreatment led to a decrease in SN-38 formation while it led to its increase in the tumour. One hour later in the plasma and 3 h in the tumour, the values are comparable. It may also be observed that in plasma at 4 h, SN-38 concentration in cetuximab-treated mice increased may be due to SN-38 diffusion from the tiste Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
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sues. The results can be explained by the fact that cetuximab inhibit P-gp, facilitating irinotecan diffusion into tissues such as liver and kidneys and lead to lower SN-38 formation in plasma. Once SN-38 was formed in tissue, it diffused into plasma and thus SN-38 formation increased in late sampling times in the group pretreated with cetuximab. In parallel, SN-38 formation in tumour decreased. A higher distribution of irinotecan and SN-38 due to cetuximab particularly led to higher concentrations of both molecules at the 4-h sampling time, which can be the result of an enterohepatic cycle. Hence, cetuximab effect on irinotecan and SN-38 pharmacokinetics was the result of the cetuximab effect on the distribution of both molecules (mainly by P-gp inhibition) rather than an effect on irinotecan metabolism. In addition, no published data described an induction of carboxylesterase by cetuximab. The effect of cetuximab in plasma on SN-38 formation was not the same at each sampling time (decreasing in the first times then increasing), which suggests that cetuximab did not affect irinotecan metabolism. Finally, SN-38 tumour value ratios were SN-38 mean concentrations in mice treated with irinotecan and cetuximab divided by SN-38 mean concentrations in mice treated with irinotecan alone. Their increase explained the significant increase in SN-38 tumour AUC and slight variations at each sampling time (from 1.1 to 2.5 with an average of 1.95) even when SN-38 plasma value ratios decreased. The increase observed in SN-38 AUC in tumour is due to an increase in SN-38 AUC in plasma, and probably also to an inhibition of SN-38 efflux from tumour caused by cetuximab. All these results can explain clinically obtained outcomes. Cetuximab effect on the pharmacokinetics of irinotecan after IV administration should be more important on the distribution step, hence affecting formation of SN-38 in plasma. As SN-38 is 200-fold more potent than irinotecan, our results are interesting and could explain how cetuximab may reverse irinotecan resistance by an effect on its active metabolite. Inhibiting SN-38 efflux by P-gp drug transporters in biliary system and tumour could lead to pharmacokinetic modifications resulting in a higher antitumour efficacy. CONCLUSION Cetuximab concentration significantly increased SN-38 plasma and tumour AUC in mice bearing human colote Francßaise de Pharmacologie et de The rapeutique ª 2014 Socie Fundamental & Clinical Pharmacology 28 (2014) 652–660
rectal carcinoma. Our results suggest that cetuximab can reverse irinotecan resistance by increasing its active metabolite disposition by inhibition of efflux transporters such as P-gp in intestines, biliary system and tumour. ACKNOWLEDGEMENT We thank the Cremec consortium that permitted the establishment of the xenograft model CR-IGR016P (‘Projet C.Re.M.E.C.’) and Dr. Simone Orbach for her kind help. CONFLICT OF INTEREST The authors declare no conflict of interest. ABBREVIATIONS P-gp – P-glycoprotein AUC – area under the curve Cmax – maximal concentration Tmax – time to reach maximal concentration t1/2 – terminal half-life REFERENCES 1 Xu Y., Villalona-Calero M.A. Irinotecan: mechanisms of tumor resistance and novel strategies for modulating its activity. Ann. Oncol. (2002) 13 1841–1851. 2 Cunningham D., Humblet Y., Siena S. et al. Cetuximab monotherapy and cetuximab plus irinotecan in irinotecanrefractory metastatic colorectal cancer. New. Engl. J. Med. (2004) 351 337–345. 3 Prewett M.C., Hooper A.T., Bassi R. et al. Enhanced antitumor activity of anti-epidermal growth factor receptor monoclonal antibody IMC-C225 in combination with irinotecan (CPT-11) against human colorectal tumor xenografts. Clin. Cancer Res. (2002) 8 994–1003. 4 Smith N.F., Figg W.D., Sparreboom A. Pharmacogenetics of irinotecan metabolism and transport: an update. Toxicol. In Vitro (2006) 20 163–175. 5 Van der Bol J.M., Loos W.J., de Jong F.A. et al. Effect of omeprazole on the pharmacokinetics and toxicities of irinotecan in cancer patients: a prospective cross-over drug-drug interaction study. Eur. J. Cancer (2011) 47 831–838. 6 Van der Bol J.M., Visser T.J., Loos W.J. et al. Effects of methimazole on the elimination of irinotecan. Cancer Chemother. Pharmacol. (2011) 67 231–236. 7 Xu G., Zhang W., Ma M.K. et al. Human carboxylesterase 2 is commonly expressed in tumor tissue and is correlated with
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9
10
11
12
13
14
15
16
activation of irinotecan. Clin. Cancer Res. (2002) 8 2605– 2611. Arimori K., Kuroki N., Hidaka M. et al. Effect of Pglycoprotein modulator, cyclosporin A, on the gastrointestinal excretion of irinotecan and its metabolite SN-38 in rats. Pharm. Res. (2003) 20 910–917. El-Readi M.Z., Hamdan D., Farrag N. et al. Inhibition of Pglycoprotein activity by limonin and other secondary metabolites from Citrus species in human colon and leukaemia cell lines. Eur. J. Pharmacol. (2010) 626 139–145. Goldstein L.J., Galski H., Fojo A. et al. Expression of a multidrug resistance gene in human cancers. J. Natl. Cancer Inst. (1989) 81 116–124. Bansal T., Mishra G., Jaggi M. et al. Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats. Eur. J. Pharm. Sci. (2009) 36 580–590. Chu C., Abbara C., No€el-Hudson M.S. et al. In vivo and in vitro evidence of the effect of Erbitux on drug substrates of ABCB1. FIRN. (2012) Salzburg, Austria. Burtin P., Mentre F., van Bree J., et al. Sparse sampling for assessment of drug exposure in toxicological studies. Eur. J. Drug Metab. Pharmacokinet. (1996) 21 105–111. Cattaneo D., Meraviglia P., Cozzi V., et al. Atypical pharmacokinetics of atazanavir in an HIV-1-infected patient. Fun. Clin. Pharm. (2012) 26 204–206. Bonhomme-Faivre L., Benyamina A., Reynaud M., et al. Disposition of Delta tetrahydrocannabinol in CF1 mice deficient in mdr1a P-glycoprotein. Addict. Biol. (2008) 13 295–300. Kovarick J.M., Beyer D., Bizot M.N., et al. Pharmacokinetic interaction between verapamil and everolimus in
C. Chu et al.
17
18
19
20
21
22
23
healthy subjects. Br. J. Clin. Pharmacol. (2005) 60 434–437. Bailer A.J. Testing for the equality of area under the curves when using destructive measurement techniques. J. Pharmacokinet. Biopharm. (1998) 16 303–309. Czejka M., Gruenberger B., Kiss A., et al. Pharmacokinetics of irinotecan in combination with biweekly cetuximab in patients with advanced colorectal cancer. Anticancer Res. (2010) 30 2355–2360. Pierga J.Y., Dieras V., Faivre S., et al. Pharmacokinetic profile of cetuximab (Erbitux) alone and in combination with irinotecan in patients with advanced EGFR-positive adenocarcinoma. Eur. J. Cancer (2005) 41 1739–1745. Schoemaker N.E., Kuppens I.E., Huinink W.W., et al. Phase I study of an oral formulation of irinotecan administered daily for 14 days every 3 weeks in patients with advanced solid tumours. Cancer Chemother. Pharmacol. (2005) 55 263– 270. Soepenberg O., Dumez H., Verweij J., et al. Phase I and pharmacokinetic study of oral irinotecan given once daily for 5 days every 3 weeks in combination with capecitabine in patients with solid tumors. J. Clin. Oncol. (2005) 23 889– 898. Chu C., Abbara C., Noel-Hudson M.S., et al. Disposition of everolimus in mdr1a-/1b- mice and after a pre-treatment of lapatinib in Swiss mice. Biochem. Pharmacol. (2009) 77 1629–1634. Xie M., Yang D., Wu M., et al. Mouse liver and kidney carboxylesterase (M-LK) rapidly hydrolyzes antitumor prodrug irinotecan and the N-terminal three quarter sequence determines substrate selectivity. Drug Metab. Dispos. (2003) 31 21–27.
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