A High-Pressure Liquid Chromatographic Method for Measuring Mitotane [1,1-(o,p’-Dichlorodiphenyl)-2,2-dichloroethane] and Its Metabolite 1,1-(o,p’-Dichlorodiphenyl)-2,2-dichloroethene in Plasma
Anders Andersen, David J. Warren, *Ole Nome, tLisbeth Vesterhus, and Lars Slørdal Department of Clinical Pharmacology, *Department of Oncology and tHospital Pharmacy, The Norwegian Radium Hospital, Oslo, Norway
Summary: The adrenolytic agent mitotane [o,p’-DDD or 1,1-(o,p’-dichloro- diphenyl)-2,2-dichloroethane] has been employed in the nonsurgical treatment of patients with adrenal carcinoma for several decades. Its use is hampered by serious side effects, which may be limited by analytically guided dose modifi- cations in the individual patient. Mitotane analyses have previously been un- dertaken by gas chromatography with electron capture detection. A sensitive high-pressure liquid chromatographic method for measuring mitotane in plasma is described. After protein precipitation with 1.5 vol of acetone, mito- tane and its metabolite 1,1-(o,p’-dichlorodiphenyl)-2,2-dichloroethene (o,p’- DDE) are resolved by isocratic elution from a C18 reversed-phase support and quantified by ultraviolet detection at 230 nm. Recoveries of mitotane and o,p’- DDE after deproteinization were quantitative. Within-run and between-day coefficients of variation were <4% over the entire therapeutic range. The limit of detection was 0.25 mol/L and the standard curve was linear in the 1-100 umol/L range. The method has been evaluated using samples obtained from an adolescent girl who had metastatic adrenocortical carcinoma. Data from this single patient may suggest that systemic absorption of mitotane is adequate, and toxicity possibly decreased, when mitotane is administered by the rectal route. Key Words: Mitotane-Adrenocortical carcinoma-High-pressure liquid chromatography.
Adrenocortical carcinomas are rare tumors with a poor prognosis. The worldwide annual incidence of this particular cancer is in the 0.5-2 per million range (1,2). In addition to the problems associated with tumor burden and frequent metastases, the ad- renocortical carcinomas are often endocrinologi-
cally active and may present as Cushing-like syn- dromes. The therapy of choice is surgical, but cure is rarely achievable. Patients with differentiated carcinoma may live for considerable periods with metastatic disease (1).
Mitotane [o,p’-DDD or 1,1-(o,p’-dichlorodiphe- nyl)-2,2-dichloroethane], an adrenolytic agent that has been in clinical use for the last 3 decades, is the only drug that has been associated with cure or re- mission, metastatic regression, and increased sur- vival in these patients. The drug may also be effec- tive in nonsurgical treatment of Cushing’s disease
(3,4) and Leydig cell tumors (5). In the commonly recommended standard doses of up to 10 g daily, mitotane therapy is associated with severe gastro- intestinal and CNS adverse effects. Based on cor- relations with clinical toxicities and laboratory pa- rameters, several investigators have suggested that mitotane should be administered with therapeutic drug monitoring guidance, and adrenolytic activity in the absence of severe side effects has been asso- ciated with steady-state therapeutic windows in the 16-63 µmol/L (5-20 µg/ml) range (4-6). Both the severity of side effects and the slow elimination ki- netics, with half-lives approaching 1 month or more (7), would seem to underscore the need for analyt- ically based dose modifications during treatment.
Mitotane analysis has been undertaken in a lim- ited number of laboratories by a labor-intensive procedure using gas chromatography with electron capture detection (8,9). The main advantage of these methods, i.e., the high sensitivity, is coun- tered by laborious sample handling procedures and the limited response range of the detectors. We have devised a very simple, fast, and accurate HPLC method for assaying mitotane and its metab- olite o,p’-DDE in plasma. The assay has been eval- uated clinically.
MATERIALS AND METHODS
Reagents
Mitotane and its metabolite 1,1-(o,p’-dichloro- diphenyl)-2,2-dichoroethene (o,p’-DDE) were pur- chased from Sigma Chemical Co., St. Louis, MO, and Supelco Inc., Bellefonte, PA, USA, respec- tively. High-pressure liquid chromatography (HPLC) grade acetone was obtained from E. Merck, Darmstadt, Germany, and HPLC grade methanol was from Rathburn Chemicals Ltd., Walkerburn, UK. All aqueous reagents and mobile phases were made up in water purified by reversed osmosis followed by polishing with a Milli-Q UF- PLUS system (Millipore Corporation, Bedford, MA, U.S.A.).
Apparatus
Chromatographic equipment was produced by Shimadzu Corp., Tokyo, Japan. The solvent deliv- ery system consisted of a DGU-3A on-line degasser coupled to a LC-9A quaternary gradient pump. Col- umn temperature was maintained using a CT0-6A
column oven and on-line solvent preheater. Sam- ples were injected with a SIL-9A autoinjector main- tained at ambient temperature and detected by a SPD-6AV variable wavelength ultraviolet (UV) de- tector. Peak area integrations were performed by a Chromatopac C-R6A integrator.
Chromatography
Chromatography was performed on a Supelcosil LC-18 column (4.6 × 150 mm, particle size 3 um; Supelco Inc.) protected by a 20-mm Supelguard. The mobile phase consisted of a 50 mM KH2PO4 buffer (adjusted to pH 7.0 by addition of KOH): methanol mixture (20:80, vol:vol). The mobile phase was delivered at a rate of 1.25 ml/min and the column temperature maintained at 50℃. The UV detector was operated at 230 nm. Fifty microliters of sample was injected.
Standard Solutions
Standard solutions (2 mmol/L) of mitotane and metabolite were made up in ethanol, aliquoted, and stored at - 70°℃ until used. Further dilutions of pri- mary drug standards were subsequently made in plasma or acetone-H2O mixtures.
Sample Preparation
Protein was precipitated by the addition of 300 ul of acetone to 200 ul of plasma sample in 1.8 ml Eppendorf tubes, followed by immediate vortex mixing. The precipitate was pelleted by centrifuga- tion at 17,000 r/min (26,000 × g) for 5 min in a Hermle Z252MK centrifuge (Maschinenfabrik Berthold Hermle AG, Gosheim, Germany). The re- sultant supernatant was transferred to borosilicate glass autosampler vials (Chromacol Ltd., London, England) before analysis.
RESULTS
Chromatography
Chromatograms obtained after processing of plasma spiked with known levels of mitotane and o,p’-DDE, plasma from a patient given mitotane therapy, and blank plasma are shown in Fig. 1. With the chromatographic system described, mitotane and o,p’-DDE eluted as symmetrical peaks with re- tention times of 6.2 and 9.3 min, respectively. No
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interference has been observed from endogenous components present in plasma from healthy, unex- posed individuals.
The calibration curve, prepared from drug-free plasma samples spiked with known amounts of mi- totane and o,p’-DDE, is shown in Fig. 2. The assay is linear in the 1-100 umol/L range with a limit of detection for mitotane (S:N ratio > 5:1) consis- tently found to be 0.25 umol/L.
Analytical Variables
Drug recovery after protein precipitation was de- termined by comparing spiked plasma samples with
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samples produced by the direct addition of mitotane and o,p’-DDE to a solution of 60% acetone in wa- ter. Mean drug recoveries after removal of protein were quantitative and independent of mitotane and o,p’-DDE concentrations in the samples (Table 1). Attempts to precipitate plasma proteins with either methanol or acetonitrile gave recoveries of ~20% (data not shown).
The within-run and between-run coefficients of variation (CVs) of independently processed spiked plasma samples were also independent of drug lev- els and found to be <4% (Table 2). Carryover be- tween injections was <0.25%.
Assay performance was independent of whether serum, lithium heparin plasma, or potassium EDTA plasma was used (data not shown).
Analytical Stability of Mitotane and o,p’-DDE
Plasma samples were spiked to final concentra- tions of mitotane and o,p’-DDE of 1 and 100 umol/ L, processed, analyzed and left in the SIL-9A au- toinjector for 24 h before reanalysis. Compared with the initial results, the concentrations measured on reanalyses were 103 + 3% for mitotane and 116 ± 11% for o,p’-DDE (means ± SD, n = 10).
Unprocessed plasma samples have been found to be stable for at least 1 week both refrigerated and at room temperature, irrespective of being exposed to light or not. Reanalysis of patient samples have given similar (<10% variation) results after storage at - 70°℃ for >12 months (data not shown).
DISCUSSION
No consensus has been reached concerning the role of mitotane in therapy for adrenocortical car- cinomas. In a review of the English literature that included a total of 1,891 cases, Wooten and King (1) reported that these tumors are rarely curable and that only one-third of the cases not cured by surgery
| Concentration (pmol/L) | o,p'-DDD | o,p'-DDE |
|---|---|---|
| 1 | 117 | 110 |
| 10 | 107 | 109 |
| 100 | 105 | 105 |
| Concentration (umol/L) | o,p'-DDD o,p'-DDE | ||
|---|---|---|---|
| Within-day CV (n = 5) | |||
| 1 | 2.9 | 2.5 | |
| 10 | 1.8 | 1.8 | |
| 100 | 0.6 | 1.1 | |
| Between-day CV (n = 4) | |||
| 1 | 3.7 | 3.5 | |
| 10 | 2.0 | 2.6 | |
| 100 | 1.3 | 1.8 | |
are responsive to mitotane treatment. In a study of 105 patients who had been referred to a single French center in the years 1963-1987, Luton et al. (2) reported objective tumor regression in 8 mito- tane-treated individuals. A study of 19 patients treated at M. D. Anderson Cancer Center between 1988 and 1991 found that neither disease-free inter- val nor survival was improved by the drug (10). However, these reports do not describe adminis- tered drug doses and blood levels.
In a study of 34 patients with adrenocortical car- cinoma, van Slooten et al. reported mitotane plasma levels >14 µg/ml (44 µmol/L) and <10 µg/ ml (31 µmol/L) in 7 of 8 responders and 19 of 20 nonresponders, respectively (6). These investiga- tors also stated that dose-dependently progressive side effects, usually in the form of neuromuscular symptoms, appeared in patients with mitotane se- rum levels exceeding 20 µg/ml (62 umol/L). Based on a more recent study of a total of 96 patients, the investigators found that mitotane treatment with high (>14 µg/ml) serum levels had a significant and independently favorable influence on patient sur- vival (11). A Swedish study has reported 60% dis- ease-free survival (mean observation period 1.5 years, range 3 months-21 years) in 10 patients treated with surgery and, with two exceptions, mi- totane. Notably, a majority (five of eight) of these patients had received the drug with laboratory- guided dose adjustments to serum levels in the 13- 20 µg/ml (41-62 umol/L) range (12). These reports may indeed suggest that mitotane blood levels should be monitored during therapy, and raises the possibility that some further advances in the treat- ment of adrenal carcinomas may lie in the improved use of this established drug. This would be facili-
tated by assay methodologies that are easily imple- mented in the clinical laboratory.
The present method is based on precipitation of sample in acetone, reversed-phase chromatogra- phy, and UV detection. The addition of 1.5 vol ace- tone has been found to precipitate >99% of plasma proteins (13). Moolenaar et al. have previously re- ported that precipitation with acetone increased the analytical recoveries of mitotane and o,p’-DDE to >93% with a method that employed an additional extraction step with heptane (8). Chromatograms (Fig. 1) demonstrate excellent separation of the compounds of interest with a run time of 12 min. Peak identities have been confirmed by the appear- ance of solitary mitotane and o,p’-DDE peaks de- spite alterations of both pH (from 7 to 3) and meth- anol content in the mobile phase. Because mitotane is believed to confer little or no therapeutic activity at serum concentrations <16-31 µmol/L (4,6,11), sensitivity (limit of detection 0.25 pmol/L) is more than adequate. Assay linearity is satisfactory (Fig. 2), and the method is highly reproducible over a wide concentration range (Tables 1 and 2). Mitotane and o,p’-DDE are stable in plasma for at least 1 week at room temperature and, if frozen, for long periods.
Others (14) have reported high levels of the me- tabolite o,p’-DDA [1,1-(o,p’-dichlorodiphenyl) ace- tic acid] in mitotane-treated subjects. We have been unable to obtain this compound from the cited (and other) sources, and must presume that this compar- atively polar metabolite does not interfere with the chromatographic separation of the nonpolar com- pounds mitotane and o,p’-DDE.
The method has been evaluated in a single pa- tient. After a 6-8-month-long history of weight loss, fatigue, and signs of hyperadrenalism, a previously healthy 14-year-old girl was diagnosed with a hor- monally active adrenal adenocarcinoma after lapa- rotomy with subsequent histology. Local tumor me- tastases were excised from the liver and the intralu- minal vena cava. The patient had lung metastases as judged by radiographic examination. Oral mitotane therapy with a daily dose of 3,500 mg was initiated postoperatively. In addition, the patient was treated with cortisone acetate (25 mg daily) and antiemetic drugs.
Four weeks after surgery, she received the first of a total of four cycles of chemotherapy, consisting of carboplatin (350 mg/m2) and etoposide (100 mg/m2 intravenously on day 1, 200 mg/m2 orally on days 2 and 3). Chemotherapy was discontinued because of
disease progression. After another 2 months of re- ceiving oral mitotane, steroid substitution, and anti- emetic drugs only, nausea and vomiting still af- fected the patient so extensively that she wanted the mitotane therapy to be discontinued. This was discouraged, and the patient was offered mitotane in the form of suppositories. The suppositories were made by the hospital pharmacy by dissolving 1,000 mg of mitotane in hard fat (a mixture of mono-, di-, and triglycerides of the C9_17 fatty acids) to a total weight of 1.8 g. The patient was administered a dose of 3,000 mg (three suppositories) daily. During the following 2 weeks, nausea abated considerably. Blood samples for mitotane and o,p’-DDE measure- ments were obtained at the time when rectal mito- tane was substituted for the oral preparation, and at two later time points (Fig. 3). However, the pa- tient’s lung metastases increased to cause progres- sive dyspnea, and she died at the age of 15, ~8 months after the time of diagnosis. Autopsy was not performed.
This patient is typical insofar as representing the quantitatively dominating among subjects with this infrequent disease (1): female, young, and affected with a functional tumor of poor prognosis. She was seriously troubled by nausea and vomiting, presum- ably at least in part induced by the oral mitotane. At the time when the daily mitotane dose was reduced from 3,500 to 3,000 mg and rectal administration was initiated, the mitotane level in plasma was 78 umol/L (25 µg/ml), i. e., in the range reported to be associated with severe side effects (6). Subsequent measurements would suggest that both mitotane and o,p’-DDE levels were subsequently approach- ing steady state (Fig. 3), with the last mitotane con-
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centration measured (38 µmol/L or 12 µg/ml) within or very close to the assumed therapeutic range. The association between rectal administration, reduced plasma mitotane levels, and cessation of emesis is, of course, speculative. We do, however, believe that mitotane assays of blood samples from this sin- gle patient support the contention that mitotane is systemically absorbed when administered by the rectal route, and speculate whether some of the emetic properties of the compound may, at least in part, be due to local effects.
An unexpected observation was the increased o,p’-DDE concentrations concomitant with declin- ing mitotane levels during measurements after the altered scheduling of drug (Fig. 3). Altered metab- olism due to partial circumvention of first passage through the portal vein and liver may explain the observation. Mitotane may also act as both an in- hibitor or inducer on metabolic enzymes (6), and dose-dependent autoinhibitory effects on metabo- lism cannot be ruled out. The question is further compounded by our lack of insight into basic as- pects of mitotane pharmacology; the parent com- pound may be a prodrug insofar as conferring little adrenolytic activity by itself, its mechanism of ac- tion has not been resolved, and the metabolic pat- tern and identity of active biotransformation prod- uct(s) remains obscure (15,16).
Acknowledgment: This work was financially supported by the Norwegian Cancer Society, the Grete Harbitz Leg- acy, and the Astrid and Birger Torsted Cancer Legacy. D.J.W. is a Norwegian Cancer Society Senior Re- searcher.
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