Simultaneous analysis of mitotane and its main metabolites in human blood and urine samples by SPE-HPLC technique
Ana Mornarª*, Miranda Sertića, Nikša Turkb,c, Biljana Nigovića and Mirko Koršićb,d
ABSTRACT: Adrenocortical carcinoma (ACC) is a rare malignancy with an incompletely understood pathogenesis and a poor prognosis. The adrenalytic activity of mitotane has made it the most important single drug in the treatment of ACC. Unfor- tunately, the exact mechanism of mitotane action is still unknown. It is believed that mitotane belongs to the class of drugs that require metabolic transformation by cytochrome P450 for therapeutic action; therefore determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment. The objective of this work was to develop and validate an SPE-HPLC method for simultaneous determination of mitotane and its metabolites in different biological fluids. The sample preparation consisted of a solid-phase extraction on a Discovery DSC18 cartridge, while analy- sis of extracts was performed on a Symmetry C18 column. The usefulness of the proposed method was confirmed by analysis of plasma, red cell and urine samples from patient chronically treated with 1.5 g of mitotane. The patient involved in this study had a high plasma concentration of mitotane and none of the investigated metabolites were found. In order to inves- tigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed. Copyright @ 2012 John Wiley & Sons, Ltd.
Keywords: mitotane; metabolites; SPE-HPLC; RT-PCR; CYP450
Introduction
Adrenocortical carcinoma (ACC) is a rare but aggressive malig- nancy with an incompletely understood pathogenesis and a poor prognosis. Only complete surgical resection is potentially curable; however, even after apparently successful excision, local or metastatic recurrence is frequent. Unfortunately, treatment options for advanced ACC are severely limited (Fassnacht and Allolio, 2009; Ng and Libertino, 2003; Phan, 2007). Mitotane [1,1-(o,p’-dichlorodiphenyl)-2,2-dichoroethane; o,p’-DDD] has been used for 50 years as the first-line drug in the treatment of disseminated ACC. It reduces local recurrence of the disease and development of metastases even after a seemingly total surgical removal of the tumor (Wooten and King, 1993; Hahner and Fassnacht, 2005). Unfortunately, the exact mechanism of mitotane action is still unknown. It is presumed that mitotane is hydroxylated at the x- or ß-carbon and afterwards ß-hydroxylation is transformed by dehydrochlorination into an acyl chloride. The acyl chloride either convalently binds to bionucleophiles in the cancer cells or is transformed to an acetic acid derivative for renal excretion (Fig. 1). The proposed bioactivation of mitotane is carried out in the mitochondria and is mediated by cytochrome P450, giving adrenal selectivity to the action of mitotane. Owing to var- iability of cancer cells regarding the ability to metabolize mitotane, only some tumors respond to mitotane therapy (Schteingart, 2007; Scripture et al., 2005). Another important limitation of mitotane therapy is its marked toxicity. Most of patients receiving mitotane experience gastrointestinal disturbances, which consist of an- orexia, nausea or vomiting and in some cases diarrhea. Central nervous system side effects occur in about half of the patients.
These consist primarily of depression as manifested by lethargy, somnolence, dizziness or vertigo (Schulick and Brennan, 1999). Transient skin rashes, which do not seem to be dose-related, have been observed in approximately 15% of the patients. According to most of studies, mitotane plasma concentrations of 14-20 µg/mL are considered therapeutic with acceptable toxicity (Haak et al., 1994; Baudin et al., 2001). As described above, mitotane belongs to the class of drugs that require metabolic transformation for therapeutic action; therefore, determination of plasma levels of not only mitotane but also its metabolites would help in carrying out the treatment.
* Correspondence to: Ana Mornar, University of Zagreb, Faculty of Phar- macy and Biochemistry, A. Kovačića 1, 10 000 Zagreb, Croatia. E-mail: amornar@pharma.hr
a University of Zagreb, Faculty of Pharmacy and Biochemistry, A. Kovačića 1, 10 000 Zagreb, Croatia
b School of Medicine, University of Zagreb, Salata 3b, 10 000 Zagreb, Croatia
” Department of Gastroenterology, University Department of Medicine, Zagreb University Hospital Center, Kišpatičeva 12, 10 000 Zagreb, Croatia
d Department of Endocrinology, University Department of Medicine, Zagreb University Hospital Center, Kišpatičeva 12, 10 000 Zagreb, Croatia
Abbreviations used: ACC, adrenocortical carcinoma; DAD, diode array detection; DDA, 4,4’-dichlorodiphenylacetate; DDD, mitotane; DDE, 4,4’- dichlrodiphenylethene; o,p’-DDD, 1,1-(o,p’-dichlorodiphenyl)-2,2-dichoroethane; SPE, solid-phase extraction.
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acylation of bionucleophiles
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Although some HPLC methods have been reported for analysis of mitotane and its main metabolites in human blood samples, no SPE-HPLC methods have been reported for simultaneous analysis of mitotane and its main metabolites in different biological fluids after oral administration of mitotane (Garg et al., 2011; De Francia et al., 2006; Andersen et al., 1999).
Therefore, the aim of our work was to develop and validate a simple, fast and reliable method for the simultaneous determi- nation of mitotane and its main metabolites in human plasma, red cell and urine samples using a solid-phase extraction (SPE) technique and high-performance liquid chromatography (HPLC) with diode array (DAD) detection. Furthermore, the pro- posed method was applied for identification and quantification of mitotane and its metabolites in plasma, red cell and urine samples from an adrenocortical carcinoma patient chronically treated with 1.5 g/day of mitotane.
Experimental
Chemicals and standards
Mitotane (DDD), 4,4’-dichlorodiphenylacetate (DDA) and 4,4’-dichlro- diphenylethene (DDE) were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile, methanol and formic acid, HPLC grade (Merck, Darmstadt, Germany), were used. Ultrapure water was prepared with a Milli-Q purification system (Millipore, Bedford, MA, USA).
Sample preparation
Stock solutions (1 mg/ml) of the studied compound were prepared in acetonitrile. Working solutions were made by dilution with acetonitrile and used to prepare spiked plasma, red cell and urine samples.
Blood and urine samples used for method development were obtained from five healthy volunteers (after obtaining their written con- sent), who were free of dietary restrictions. Blood samples were stored in glass tubes containing potassium citrate as the anticoagulant. Plasma and red cells were separate after centrifugation of 15 min at 600 g and 4°℃ (within 2 h from collection). Red cells were then lysed by suspension in a triple volume of ultrapure water. Urine samples were collected in glass tubes. Untreated plasma, red cell and urine samples were stored
at -70℃ and processed using the protocol outlined below upon thaw- ing at room temperature.
DDA, DDD, and DDE were extracted from spiked and patient samples by vortex mixing of 500 µL samples (plasma, red cells and urine) with 500 uL acetonitrile followed by centrifugation at 2500 g for 5 min. In order to avoid problems associated with clogging of the solid-phase extraction cartridges, organic phase was filtered through a 0.20 um filter (Gelman, Ann Arbor, MI, USA) prior to the purification process.
The solid-phase extraction procedure utilized reversed-phase solid- phase extraction cartridges (Discovery DSC-18, 500 mg, 3 mL, Supelco, Bellefonte, PA, USA) in conjunction with a Supelco extraction manifold system (12 position manifold with a 10 x 75 mm test tube rack). The vacuum pressure on the manifold was maintained at -15 mmHg throughout the duration of the SPE protocol.
The cartridges were activated and conditioned using 1 mL of metha- nol and 2 mL of acetonitrile. A 500 µL aliquot of organic phase obtained in the first extraction were transferred onto SPE column. Analyte elution was carried out with 3 mL of acetonitrile. Additionally, SPE of a mixture of standard solutions was conducted following the above methodology to ensure no interference from the cartridge materials.
Chromatographic conditions
The chromatography was performed on an Agilent 1100 Series LC system (Agilent Technologies, Waldbronn, Germany) consisting of a vacuum degassing unit, a quaternary pump, a standard automatic sam- ple injector, a column oven and a diode array detector. A chromato- graphic column Symmetry C18, 150 x 4.6 mm, particle size 3.5 um (Waters, Miliford, MA, USA) was used in separation and quantification of mitotane and its metabolites in plasma, red cells and urine, respec- tively. The mobile phase consisted of acetonitrile, ultrapure water and formic acid at the ratio of 90:10:0.1 (v/v/v). The mobile phase was filtered through a Sartorius (Goettingen, Germany) cellulose nitrate filter (47 mm nylon membrane, 0.45 um). The purified samples were transferred to amber autosampler vials and stored at 4℃. The 5 µL ali- quots were injected onto the HPLC system for analysis. The column temperature was fixed at 25 ℃ and a constant flow-rate of 1 mL/min was employed throughout. The DAD detector recorded UV spectra in the range from 190 to 400 nm and chromatogram was obtained at 230 nm. All data acquisition and processing were performed using ChemStation (Agilent Technologies, Waldbronn, Germany).
Validation
Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonization (ICH). The method was validated for selectivity, linearity, extraction efficiency, precision and accuracy. The limits of determination and quantification as well as stability of analytes in acetonitrile, plasma, red cells and urine were also tested (ICH, 2006).
Results and discussion
Method development
In the method described by De Francia et al. (2006), sample preparation and chromatographic analysis of mitotane and DDE in plasma and red cells separated them from DDA. Garg et al. (2011) proposed a new HPLC method for simultaneous determination of mitotane and its main metabolites only in human plasma using a simple procedure for sample purification. The aim of our work was to propose a novel HPLC method for simultaneous determination of mitotane and both of its meta- bolites in different biological matrices (human plasma, red cell and urine samples). The three analytes, mitotane and its main metabolites, have different physical-chemical properties. The lipophilicity of mitotane predicted by ADME Boxes program (Japertas et al., 2002) was 6.55, for DDE the value was 6.66 while for DDA it significantly lower at 4.39. As a consequence, finding
a suitable sample purification procedure and chromatographic conditions was not easy. Owing to the very low concentrations of the analytes in samples and the complexity of the matrix, a very efficient sample pre-treatment procedure was needed to obtain satisfactory results. In preliminary experiments, metha- nol, ethanol and acetonitrile were tested as protein precipitant agents. The methanol and ethanol induced the formation of clusters containing the drug, so the chosen precipitant agent was acetonitrile. Unfortunately, the chromatograms were still rather poor owing to serious matrix effect and high noise. Despite the cost, solid-phase extraction was chosen for sample preparation, because it allows good sample purification and high extraction yields to be obtained. Different protocols were tested to optimize extraction of analytes from biological fluids. Various SPE sorbents, conditioning and elution solvents were tested. Using the above described procedure, not only were the interfering substances removed from samples, but good recoveries of all three analytes were also achieved.
Furthermore, the challenge was to develop simple chromato- graphic method for simultaneous identification and quantification of mitotane and both metabolites, more polar DDA and less polar DDE. Representative chromatograms of blank plasma, red cell and urine samples, as well as, plasma, red cell and urine samples spiked with 500 µL (10 µg/mL) of mitotane, DDE and DDA are presented in Fig. 2. There were no co-eluting peaks in the vicinity of the three analytes in the chromatograms of blank plasma, red
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cells or urine. The retention times of DDA, DDD and DDE were 2.160± 0.020, 3.790± 0.016 and 5.977 ± 0.028 min (n=6), respec- tively. Furthermore, the optimized conditions yielded sharp and symmetrical peaks of all analytes. Resolution factors were greater than 20.1, indicating satisfactory separation of resolved analytes. The total analysis time for each run was 10 min. A complete sep- aration between mitotane and its metabolites was achieved dur- ing first 6 min, but an additional 4 min were required in order to eliminate any potential sample carry-over prior to the next injection.
Comparing our method with those proposed by De Francia et al. (2006), some advantages of our method should be pointed out. De Francia and co-workers developed two different sample preparation procedures and two different chromatographic methods for mitotane and its main metabolites determination in human plasma and red cell samples. In order to shorten analysis time, SPE procedures for simultaneous extraction of all analytes from biological fluids were proposed. Furthermore, a unique HPLC method for simultaneous determination of all analytes was devel- oped. In addition, our method was applied for determination of mitotane and its metabolites not only in human plasma and red cell samples but also in human urine samples.
Method validation
Selectivity. No endogenous inferences were observed in extracts from drug-free human blood and urine. Figure 2 shows the representative chromatograms of a drug-free plasma, and red cell and urine samples, demonstrating that the developed SPE-HPLC method is highly selective.
Extraction efficiency. The efficiency and reproducibility of the extraction procedure were evaluated by measuring the mean re- covery of mitotane, DDA and DDE from spiked plasma, red cells and urine. The determination of the extraction efficiency in all samples was made by adding 500 µL of working standard solu- tions (1 and 20 µg/mL) to the 500 uL of sample in replicate (n=6). The analyte chromatographic peak areas thus obtained were compared with those obtained from standard solutions at the same concentration, injected directly into the liquid chro- matographic apparatus, and the percentage extraction yield was calculated. The percentage of spiked analytes recovered from samples was 83.64-107.92% in plasma, 81.26-106.69% in red cells and 82.70-105.74% in urine. It should be pointed out that, despite the wide variability in matrix compositions, adequate extraction yields for all analytes were achieved in all biofluids tested.
Stability. The stability of standard solutions (100 µg/ml) of mitotane, DDA and DDE was tested. Samples were stored at room temperature for 12h and frozen at -70℃ for 30 days. The recoveries ranged from 97.50 to 101.57%, which indicates that investigated compounds were stable for at least 12h at room temperature and stored frozen at -70℃ for at least 30 days. Furthermore, the stability testing of mitotane, DDA and DDE was performed in plasma, red cells and urine by adding 500 µL of working standard solution (20 µg/mL) to 500 µL of sample in replicate (n =3). Samples were analyzed immediately after preparation and after storage at room temperature for 12 h and frozen at -70℃ after 30 days of storage. The percent- age of spiked analytes recovered from samples ranged from 91.19 to 102.59%. These results indicated that DDD, DDA and DDE were stable both at room temperature for up to 12 h and stored frozen for 30 days.
Linearity. Aliquots of 500 µL of analyte standard solutions at seven different concentrations (0.5-50 µg/mL) were added to 500 µL of blank plasma, red cell and urine samples, subjected to the SPE procedure and analyzed. Equations were obtained by least-squares linear regression analysis of the peak area vs analyte concentration. The calibration curve was linear over the concentration range studied (/2>0.991) for all investigated compounds. The detection (LOD) and the quantification (LOQ) limits were established from the calibration curve as LOD= 3.3 s/b and LOQ = 10 s/b, where s is the standard deviation of the intercept and b is the slope of the calibration curve (Table 1).
Precision. For plasma, red cell and urine samples precision experiments for DDD, DDA and DDE were performed by adding 500 µL of standard working solutions (20 µg/mL) to 500 µL of blank sample. The samples were than processed according to the above described SPE method. The procedure was repeated six times within the same day to obtain the intra-day precision while the inter-day precision was assessed by replicate analysis on three continual days. The results of precision measurements, expressed as recovery and relative standard deviation (RSD), are reported in Table 2. The RSD values of intra- and inter-day preci- sion were less than 2.38% for all the analytes, indicating that the precision was acceptable for all the compounds of interest in all investigated biological fluids.
Accuracy. In order to obtain analyte additions corresponding to the lower limit, the middle point and a high level of the re- spective calibration curve, 500 µL aliquots of working standard solutions at three different concentrations were added to 500 uL of plasma, red cells and urine. The procedure was repeated three times at each concentration level to obtain relative stan- dard deviation values (RSD). The accuracy of measurements was expressed in terms of recovery. It was 90.62-109.60% in plasma, 90.99-105.35% in red cells and 91.89-105.89% in urine. As expected, improved values were observed at higher analyte spike concentrations. Complete detailed results are given in Table 3.
Application of the method to real plasma, red cell and urine samples
The usefulness of the elaborated method was confirmed by iden- tification and quantification of mitotane and its main metabolites DDA and DDE in plasma, red cell and urine samples from an ad- renocortical carcinoma patient (52 years, female) chronically trea- ted with 1.5 g of mitotane (500 mg taken every 8 h). In 2005, the patient was diagnosed with carcinoma of the left suprarenal gland after diagnostic evaluation. Afterwards left nefrectomy was performed. Two years later metastasis in the left side of the lungs was detected and mitotane therapy was prescribed. As pa- tient suffered from meaningful and painful side effects caused by mitotane therapy, gradually titration of dosage was performed. Three months prior to our investigation the patient was chroni- cally treated with 1.5g of mitotane. Additionally, the patient was treated with simvastatine (20 mg), flucortisone (0.1 mg), hydrocortisone (10 mg), pantoprazole (40 mg) and ß-glucan (500 mg). Blood samples were collected at 1, 2, 4 and 8 h after consumption of 500 mg of mitotane. Urine samples were col- lected as four different aliquots of urine collected after the drug administration, in sequence from 0 to 1 h, from 1 to 2 h, from 2 to 4 h and from 4 to 8 h. Chromatograms from plasma and red cell samples, obtained 2 h after oral administration of mitotane,
| Table 1. Linearity parameters for mitotane and metabolites on spiked blank plasma, red cell and urine samples. | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Analyte | Linearity concentration range (µg/mL)* | Equation | Plasma | LOQ | Equation | Red cell | LOQ (µg/mL) | Equation | Urine | ||||
| r | LOD | r | LOD | r | LOD (µg/mL) | LOQ (µg/mL) | |||||||
| (µg/mL) | (µg/mL) | (µg/mL) | |||||||||||
| DDA | 0.5-50 | y = 41.036x - 2.152 | 0.991 | 0.130 | 0.390 | y = 42.021x+9.519 | 0.992 | 0.125 | 0.380 | y = 40.012x+2.519 | 0.991 | 0.132 | 0.399 |
| DDD | 0.5-50 | y = 28.509 x - 0.963 | 0.996 | 0.050 | 0.152 | y = 29.864x - 4.551 | 0.995 | 0.050 | 0.152 | y = 31.425 x+ 2.375 | 0.997 | 0.048 | 0.145 |
| DDE | 0.5-50 | y = 31.550 x - 0.145 | 0.993 | 0.090 | 0.272 | y = 32.368x - 9.570 | 0.992 | 0.089 | 0.280 | y = 31.995x+2.573 | 0.991 | 0.090 | 0.275 |
*Linearity range values were identical for all matrices. LOD, Limit of detection; LOQ, limit of quantitation; DDA, 4,4’-dichlorodiphenylacetate; DDD, mitotane; DDE, 4,4’- dichlrodiphenylethene.
| Analyte | Plasma Red cell | Urine | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Intra-day precision (n=6) | Inter-day precision (n = 18) | Intra-day precision (n= 6) | Inter-day precision (n= 18) | Intra-day precision (n=6) | Inter-day precision (n = 18) | |||||||
| Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | |
| DDA | 96.96 | 1.10 | 96.98 | 1.65 | 97.85 | 2.35 | 96.35 | 1.41 | 96.54 | 1.25 | 95.25 | 1.35 |
| DDD | 97.93 | 1.18 | 97.92 | 0.42 | 96.20 | 1.24 | 95.15 | 1.42 | 97.33 | 2.15 | 96.15 | 2.38 |
| DDE | 98.29 | 2.28 | 98.23 | 1.35 | 97.35 | 1.28 | 95.75 | 2.38 | 98.12 | 1.17 | 97.59 | 2.34 |
ªAll samples were spiked with 500 µL of standard working solutions (20 µg/mL). DDA, 4,4’-dichlorodiphenylacetate; DDD, mitotane; DDE, 4,4’-dichlrodiphenylethene.
| Table 3. Accuracy data of mitotane and its metabolites in plasma, red cells and urine (n=3) | |||||||
|---|---|---|---|---|---|---|---|
| Analyte | Spiked | Plasma | Red cell | Urine | |||
| concentration (µg/mL) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | Recovery (%) | RSD (%) | |
| DDA | 1 | 90.62 | 3.52 | 105.35 | 2.55 | 91.89 | 1.45 |
| 10 | 100.01 | 1.44 | 98.96 | 0.41 | 99.25 | 1.31 | |
| 50 | 101.29 | 0.29 | 101.15 | 0.24 | 100.35 | 1.22 | |
| DDD | 1 | 109.60 | 2.41 | 90.99 | 1.95 | 105.89 | 1.01 |
| 10 | 100.03 | 1.80 | 99.97 | 0.80 | 102.25 | 0.25 | |
| 50 | 99.97 | 0.50 | 101.35 | 0.50 | 101.25 | 0.46 | |
| DDE | 1 | 103.27 | 2.13 | 92.25 | 1.56 | 104.52 | 2.89 |
| 10 | 100.19 | 1.03 | 100.59 | 0.95 | 99.12 | 1.91 | |
| 50 | 99.30 | 0.80 | 98.89 | 0.70 | 99.21 | 1.75 | |
| DDA, 4,4'-dichlorodiphenylacetate; DDD, mitotane; DDE, 4,4'-dichlrodiphenylethene. | |||||||
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are shown in Fig. 3(A and B), respectively. A chromatogram from a urine sample, collected from 1 to 2 h after drug administration, is given in Fig. 3(C). In Fig. 4 is given the time profile of plasma and red cell samples. As can be seen from chromatograms, only unchanged mitotane was found in plasma, red cell and urine samples, indicating that the drug did not undergo to bio- transformation. The maximum concentration of drug in plasma was found 1 h after administration, indicating an adequate ab- sorption. It should be pointed out that plasma level of mitotane was higher than therapeutic concentrations. Although the highest concentration of mitotane was found in plasma samples, a signif- icant concentration was found in red cells. The urinary extrac- tion-time profile of mitotane after oral administration of 500 mg mitotane is shown in Fig. 5. The maximum excretion was observed between 1 and 2 h after drug administration. Considering the total amount of urinary excretion over the 8h, the concentration of mitotane detected in urine was low with respect to intake dosage (0.016% of the administered dose of mitotane), suggesting the biliary and fecal excretion of the drug.
As discussed above, the exact anticancer molecular mecha- nism of mitotane is still unclear. It is believed that the adrenolytic effect of mitotane depends on its metabolic activation through metabolism mediated by cytochrome P450. Also, the inability of developing cancer cells to metabolize mitotane can result in
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plasma concentrations higher than therapeutic levels, with a higher risk of significant undesirable toxicity. The specific cyto- chrome P450 isoenzyme involved in mitotane metabolism is still unknown (Scripture et al., 2005).
According to our results, the female patient involved in this study had high plasma concentration of mitotane and none of investigated metabolites were found. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 enzymes could be related to the metabolism of mitotane, real-time PCR analysis was performed. The detection of CYP2C9*1, CYP2C9*2, CYP2C9*3, CYP2C19*1, CYP2C19*2 and CYP2C19*3 alleles was performed according to a previously described procedure (Božina et al., 2003, 2005). An intermediate metabolic phenotype of CYP2C9 (*1/*3) and a normal metabolic phenotype of CYP2C19 (*1/*1) were found. Although further detailed investiga- tions are required, our results indicate the possibility that genetic variation in CYP2C9 isoenzyme might be associated with the plasma concentrations of mitotane and its metabolites, and there- fore might influence the pharmacological and toxicological effects of mitotane. In addition, none of the co-administered drugs were metabolized by CYP2C19 isoenzyme (Human P450 Metabolism Database, 2011).
Conclusion
In this study, an SPE-HPLC method for the simultaneous determi- nation of mitotane and its main metabolites in human plasma, red cell and urine samples was developed and validated. The pro- posed method was successfully applied for the identification and quantification of mitotane and its main metabolites in plasma, red cell and urine samples from an adrenocortical carcinoma female patient chronically treated with 1.5 g of mitotane. A high plasma concentration of mitotane and none of investigated metabolites were found in all samples. In order to investigate whether the polymorphism of CYP2C9 and CYP2C19 isoenzymes could be related to the metabolism of mitotane, RT-PCR analysis was performed. The obtained results indicate the possibility that genetic variation in CYP2C9 isoenzyme might be associated with the high plasma concentrations of mitotane and therefore might influence the toxicological effects of mitotane.
Acknowledgments
This work was supported through a grant (Investigation of new methods in analysis of drugs and bioactive substances, no. 006-0061117-1240) from the Ministry of Science, Education and Sports of the Republic of Croatia. Furthermore, the authors would like thank to the members of the University Institute of Laboratory Diagnosis, University Hospital Center and School of Medicine, Zagreb, Croatia for RT-PCR analysis.
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