A simplified method for therapeutic drug monitoring of mitotane by gas chromatography-electron ionization-mass spectrometry.

Mitotane is a key drug for the treatment of adrenal cortical carcinoma. Due to its narrow therapeutic window, 14-20 µg/mL, monitoring its concentration is crucially important. In this study, a simplified method for measuring mitotane in plasma using gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) was developed. Through deproteination and liquid-liquid extraction, mitotane and an internal standard (IS) were extracted from plasma samples. GC-EI-MS yielded retention times of 8.2 and 8.7 min, for mitotane and the IS, respectively, with a total run time of 12 min. Selectivity and intra-/inter-batch accuracy and precision analyses provided a lower limit of quantification of 0.25 µg/mL, and a calibration curve between 0.25 and 40 µg/mL had good linearity (coefficient of determination = 0.992). The matrix effect factor and percent recovery of the method had good precision. Additionally, long-term sample stability was observed below 4°C. In a clinical setting, mitotane levels in plasma from an adrenal cortical carcinoma patient were within calibration range. Therefore, this simplified method can be applied to routine therapeutic drug monitoring of mitotane, which may contribute to improved treatment of adrenal cortical carcinoma.

Therapeutic drug monitoring of mitotane: Analytical assay and patient follow-up.

Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal gland. Mitotane (o,p'-DDD) is the most effective chemotherapy for ACC. According to the literature, mitotane plasma trough concentrations within 14-20 mg L-1 are correlated with a higher response rate with acceptable toxicity. Therapeutic drug monitoring (TDM) of mitotane is therefore recommended. The aim of this study was to propose a robust and simple method for mitotane quantification in plasma. The validation procedures were based on international guidelines. Sample preparation consisted of a single protein precipitation with methanol using 100 µL of plasma. The supernatant was submitted to liquid chromatography coupled with ultra-violet detection at 230 nm. Mitotane retention time was 7.1 min. The limit of detection was 0.1 mg L-1 and the limit of quantification was 0.78 mg L-1 . The assay demonstrated a linear range of 0.78-25 mg L-1 with correlation coefficients (r2 ) at 0.999. Inter- and intra-assay precision was <4.85%. Evaluation of accuracy showed a deviation <13.69% from target concentration at each quality control level. This method proved easy and rapid to perform mitotane TDM and required a small volume of sample. It was successfully applied to routine TDM in our laboratory.

Development of a pharmacokinetic model of mitotane: toward personalized dosing in adrenocortical carcinoma.

BACKGROUND: Mitotane is the drug of choice in medical treatment of adrenocortical carcinoma. The antineoplastic effect seems to be correlated with a minimum plasma level of 14 mg/L, but plasma concentration build-up is in general slow due to the long elimination half-life. Consequently, the therapeutic effect sets in after weeks or even months. The objective of this study was to develop a pharmacokinetic model that enables clinicians to adjust dosing based on a target drug exposure, which facilitates personalized therapy. METHODS: Data on dosing and plasma level measurements performed throughout mitotane therapy were retrospectively collected in a population of 29 patients from 2 hospitals. A population pharmacokinetic model was constructed based on data from 20 patients using iterative 2-stage Bayesian fitting (MWPharm). The model was validated in an independent sample of 9 patients. RESULTS: The concentration-time data were best described by a 3-compartment model. The model estimated mitotane clearance at 0.94 ± 0.37 L/h and a volume of distribution in the steady state at 161 ± 68 L/kg of lean body mass. The mean prediction error was 14% ± 13%. CONCLUSIONS: A pharmacokinetic model was developed, which characterized mitotane by slow clearance and large volume of distribution. The model seems to be able to predict mitotane levels in individual patients with an error margin of 14%. The model enables one to adapt dosing based on individual plasma level measurements in prospective setting, which improves the accuracy of the prediction. We expect that individualization of mitotane dosing leads to anticipated and more rapid attainment of the therapeutic levels and potentially to improved clinical management of mitotane treatment.

Influence of the CYP2B6 polymorphism on the pharmacokinetics of mitotane.

OBJECTIVE: The aim of this study was to assess the potential impact of the pharmacogenetic variability of CYP2B6 and ABCB1 genes on the pharmacokinetics of mitotane. METHODS: A retrospective analysis was carried out on 27 patients with adrenocortical carcinoma on postoperative adjunctive mitotane. CYP2B6 and ABCB1 polymorphisms were genotyped and tested for an association with plasma trough concentration after 3, 6, 9, and 12 months of therapy. RESULTS: Patients with the GT/TT genotype had higher mitotane plasma concentrations compared with patients with GG at 3 months (14.80 vs. 8.01 µg/ml; P=0.008) and 6 months (17.70 vs. 9.75 µg/ml; P=0.015). Multivariate logistic regression analysis showed that only the CYP2B6 rs3745274GT/TT genotype (odds ratio=10.7; P=0.017) was a predictor of mitotane plasma concentrations of at least 14 µg/ml after 3 months of treatment. Mitotane concentrations were not influenced by the polymorphisms of the ABCB1 gene. CONCLUSION: Evaluation of the CYP2B6 polymorphism enabled prediction of the individual response to adjuvant mitotane treatment.

Mitotane treatment for adrenocortical carcinoma: an overview.

Adrenocortical carcinoma (ACC) is a rare aggressive endocrine neoplasm characterized by a 5-year survival of less than 50%. Due to the widespread use of imaging techniques in clinics, ACC is increasingly recognized as an incidentally discovered tumor. Mostly characterized by poor prognosis, ACC is often diagnosed at an advanced stage of disease. Early diagnosis is uncommon; when diagnosed, ACCs are usually large and have invaded adjacent organs, even if metastatic spread to distant sites can be absent. Complete surgical resection is the only potentially curative treatment for patients with localized disease; however, due to a recurrence rate of 50-70% after apparent radical surgery, there is a strong rationale for a concomitant systemic treatment. Adrenolytic therapy with mitotane (o,p>-DDD), administered alone or in combination with others antineoplastic agents, is the primary treatment for patients with advanced ACC and is increasingly used also in an adjuvant setting, even if controversy exists on this issue due to the limitations of the available literature. Despite being in use for many years, the rarity of ACC precluded the organization of randomized trials; thus, many areas of uncertainty and controversy remain regarding the role of this old drug in the clinical management of patients with ACC. The purpose of this paper is to review the current evidence on mitotane treatment in patients with advanced disease and in ACC patients after complete surgical resection as adjuvant treatment.

Drug interactions with mitotane by induction of CYP3A4 metabolism in the clinical management of adrenocortical carcinoma.

Mitotane [1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, (o,p'-DDD)] is the only drug approved for the treatment for adrenocortical carcinoma (ACC) and has also been used for various forms of glucocorticoid excess. Through still largely unknown mechanisms, mitotane inhibits adrenal steroid synthesis and adrenocortical cell proliferation. Mitotane increases hepatic metabolism of cortisol, and an increased replacement dose of glucocorticoids is standard of care during mitotane treatment. Recently, sunitinib, a multityrosine kinase inhibitor (TKI), has been found to be rapidly metabolized by CYP3A4 during mitotane treatment, indicating clinically relevant drug interactions with mitotane. We here summarize the current evidence concerning mitotane-induced changes in hepatic monooxygenase expression, list drugs potentially affected by mitotane-related CYP3A4 induction and suggest alternatives. For example, using standard doses of macrolide antibiotics is unlikely to reach sufficient plasma levels, making fluoroquinolones in many cases a superior choice. Similarly, statins such as simvastatin are metabolized by CYP3A4, whereas others like pravastatin are not. Importantly, in the past, several clinical trials using cytotoxic drugs but also targeted therapies in ACC yielded disappointing results. This lack of antineoplastic activity may be explained in part by insufficient drug exposure owing to enhanced drug metabolism induced by mitotane. Thus, induction of CYP3A4 by mitotane needs to be considered in the design of future clinical trials in ACC.

The Challenging Pharmacokinetics of Mitotane: An Old Drug in Need of New Packaging.

Adrenocortical carcinoma (ACC) is a malignant tumor originating from the adrenal gland cortex with a heterogeneous but overall dismal prognosis in advanced stages. For more than 50 years, mitotane has remained a cornerstone for the treatment of ACC as adjuvant and palliative therapy. It has a very poor aqueous solubility of 0.1 mg/l and high partition coefficient in octanol/water (log P) value of 6. The commercially available dosage form is 500 mg tablets (Lysodren®). Even at doses up to 6 g/day (12 tablets in divided doses) for several months, > 50% patients do not achieve therapeutic plasma concentration > 14 mg/l due to poor water solubility, large volume of distribution and inter/intra-individual variability in bioavailability. This article aims to give a concise update of the clinical challenges associated with the administration of high-dose mitotane oral therapy which encompass the issues of poor bioavailability, difficult-to-predict pharmacokinetics and associated adverse events. Moreover, we present recent efforts to improve mitotane formulations. Their success has been limited, and we therefore propose an injectable mitotane formulation instead of oral administration, which could bypass many of the main issues associated with high-dose oral mitotane therapy. A parenteral administration of mitotane could not only help to alleviate the adverse effects but also circumvent the variable oral absorption, give better control over therapeutic plasma mitotane concentration and potentially shorten the time to achieve therapeutic drug plasma concentrations considerably.

Mitotane as tablet form is currently the standard treatment for adrenocortical carcinoma. It has been used for 5 decades but suffers from highly variable responses in patients, subsequent adverse effects and overall lower response rate. This can be fundamentally linked to the exceedingly poor water solubility of mitotane itself. In terms of enhancing water solubility, a few research groups have attempted to develop better formulations of mitotane to overcome the issues associated with tablet dosage form. However, the success rate was limited, and these formulations did not make it into the clinics. In this article, we have comprehensively reviewed the properties of these formulations and discuss the reasons for their limited utility. Furthermore, we discuss a recently developed mitotane nanoformulation that led us to propose a novel approach to mitotane therapy, where intravenous delivery supplements the standard oral administration. With this article, we combine the current state of knowledge as a single piece of information about the various problems associated with the use of mitotane tablets, and herein we postulate the development of a new injectable mitotane formulation, which can potentially circumvent the major problems associated to mitotane's poor water solubility.

How close are we to personalized mitotane dosing in the treatment of adrenocortical carcinoma? State of the art and future perspectives.

INTRODUCTION: Mitotane is the only drug registered specifically for adrenocortical carcinoma. Finding the optimal dose for a patient is difficult due to large differences in bioavailability, toxicity and effect. We therefore look to improve personalized dosing of mitotane. AREAS COVERED: We searched PubMed for studies related to mitotane dosing, pharmacokinetics, pharmacogenetics and combination therapy. Comparison of different dosing strategies have not resulted in an optimal advice. Several computerized pharmacokinetic models have been proposed to predict plasma levels. The current pharmacokinetic models do not explain the full variance in plasma levels. Pharmacogenetics have been proposed to find the unexplained variance. Studies on combination therapy have not yet led to a potential dose adjustment for mitotane. EXPERT OPINION: Computerized pharmacokinetics models are promising tools to predict plasma levels, further validation is needed. Pharmacogenetics are introduced in these models, but more research is required before clinical application. We believe that in the near future, personalized mitotane dosage will be aided by a validated web-based pharmacokinetic model with good predictive ability based primarily on clinical characteristics, adjustable for actual plasma levels and dosage.

Population Pharmacokinetic and Pharmacogenetic Analysis of Mitotane in Patients with Adrenocortical Carcinoma: Towards Individualized Dosing.

BACKGROUND: Mitotane is the only approved treatment for patients with adrenocortical carcinoma (ACC). A better explanation for the variability in the pharmacokinetics (PK) of mitotane, and the optimization and individualization of mitotane treatment, is desirable for patients. OBJECTIVES: This study aims to develop a population PK (PopPK) model to characterize and predict the PK profiles of mitotane in patients with ACC, as well as to explore the effect of genetic variation on mitotane clearance. Ultimately, we aimed to facilitate mitotane dose optimization and individualization for patients with ACC. METHODS: Mitotane concentration and dosing data were collected retrospectively from the medical records of patients with ACC taking mitotane orally and participating in the Dutch Adrenal Network. PopPK modelling analysis was performed using NONMEM (version 7.4.1). Genotypes of drug enzymes and transporters, patient demographic information, and clinical characteristics were investigated as covariates. Subsequently, simulations were performed for optimizing treatment regimens. RESULTS: A two-compartment model with first-order absorption and elimination best described the PK data of mitotane collected from 48 patients. Lean body weight (LBW) and genotypes of CYP2C19*2 (rs4244285), SLCO1B3 699A>G (rs7311358) and SLCO1B1 571T>C (rs4149057) were found to significantly affect mitotane clearance (CL/F), which decreased the coefficient of variation (CV%) of the random inter-individual variability of CL/F from 67.0 to 43.0%. Fat amount (i.e. body weight - LBW) was found to significantly affect the central distribution volume. Simulation results indicated that determining the starting dose using the developed model is beneficial in terms of shortening the period to reach the therapeutic target and limit the risk of toxicity. A regimen that can effectively maintain mitotane concentration within 14-20 mg/L was established. CONCLUSIONS: A two-compartment PopPK model well-characterized mitotane PK profiles in patients with ACC. The CYP2C19 enzyme and SLCO1B1 and SLCO1B3 transporters may play roles in mitotane disposition. The developed model is beneficial in terms of optimizing mitotane treatment schedules and individualizing the initial dose for patients with ACC. Further validation of these findings is still required.

Development of microemulsion of mitotane for improvement of oral bioavailability.

BACKGROUND: Mitotane (o,p'-DDD) is considered to be the drug of choice in the treatment of nonresectable and metastasized adrenocortical carcinoma. However, mitotane has poor solubility in the gastrointestinal tract and very low bioavailability. Consequently, to achieve therapeutic plasma level, high cumulative doses (4-6 g/day) of mitotane were usually used during 3-5 months. To shorten this equilibration time and reduce gastrointestinal side effects, a self-microemulsifying drug delivery system (SMEDDS) of mitotane has been developed. METHOD: First time, the solubility of mitotane was determined in various oils and surfactants; then, the influence of oils, surfactants, and cosurfactants on the formation of SMEDDS was investigated by constructing ternary phase diagrams. SMEDDS was characterized by morphological observations and droplet size measurements. Intestinal drug permeation of SMEDDS of mitotane (3 mM) was assessed in an Ussing-type apparatus and the bioavailability was determined in a rabbit model. RESULTS: The optimum formulation consisted of a mixture of Capryol, Tween, and Cremophor EL (33:33:33). The formulation was found to pass through the intestinal barrier much faster than a solution of mitotane (14.85 +/- 0.8 versus 3.03 +/- 0.2 micromol/cm(2)). Moreover, after oral administration in rabbits, the relative bioavailability was 3.4, compared with that of the conventional form (Lysodren). CONCLUSION: This SMEDDS can now be considered as a very good candidate to optimize the administration of mitotane.

A Micellar Mitotane Formulation with High Drug-Loading and Solubility: Physico-Chemical Characterization and Cytotoxicity Studies in 2D and 3D In Vitro Tumor Models.

Adrenocortical carcinoma (ACC) is a rare tumor and prognosis is overall poor but heterogeneous. Mitotane (MT) has been used for treatment of ACC for decades, either alone or in combination with cytotoxic chemotherapy. Even at doses up to 6 g per day, more than half of the patients do not achieve targeted plasma concentration (14-20 mg L-1 ) even after many months of treatment due to low water solubility, bioavailability, and unfavorable pharmacokinetic profile. Here a novel MT nanoformulation with very high MT concentrations in physiological aqueous media is reported. The MT-loaded nanoformulations are characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry, and powder X-ray diffraction which confirms the amorphous nature of the drug. The polymer itself does not show any cytotoxicity in adrenal and liver cell lines. By using the ACC model cell line NCI-H295 both in monolayers and tumor cell spheroids, micellar MT is demonstrated to exhibit comparable efficacy to its ethanol solution. It is postulated that this formulation will be suitable for i.v. application and rapid attainment of therapeutic plasma concentrations. In conclusion, the micellar formulation is considered a promising tool to alleviate major drawbacks of current MT treatment while retaining bioactivity toward ACC in vitro.

Pharmacokinetics of the adrenocorticolytic compounds 3-methylsulphonyl-DDE and o,p'-DDD (mitotane) in Minipigs.

The pharmacokinetics of the adrenocorticolytic drug candidate 3-Methylsulphonyl-DDE (3-MeSO2-DDE) and the anticancer drug o,p'-DDD (mitotane) were studied in Göttingen minipigs. The animals were given 3-MeSO2-DDE or o,p'-DDD as single oral doses (30 mg/kg). Concentrations in plasma and subcutaneous fat were measured by gas chromatography at different time points during 180 days. Maximal plasma concentrations appeared within 24 h for both compounds, but were about 2 times higher for 3-MeSO2DDE. o,p'-DDD plasma concentrations declined rapidly to low levels during 4 days. 3-MeSO2-DDE also decreased rapidly, but remained at high concentrations throughout the study. In fat, 3-MeSO2-DDE reached about 25-fold higher levels than o,p'-DDD at 30 days, and both substances were eliminated slowly from this tissue. 3-MeSO2-DDE liver concentrations were about 18-fold higher than those in plasma at 180 days. In contrast, o,p'-DDD liver and plasma levels were about equal at 180 days. o,p'-DDD had roughly 45 times larger CL/F than 3-MeSO2-DDE, confirming that the elimination of this compound was more rapid. Both compounds were characterised by their localisation and retention in fat tissue, and the individual size of the fat stores clearly determined the plasma concentrations. It is concluded that although 3-MeSO2-DDE is an interesting candidate for therapeutic use due to its potential characteristics to specifically target adrenocortical tumour cells the slow elimination of the compound might make it challenging to design appropriate dosage regimes.

Enzyme autoinduction by mitotane supported by population pharmacokinetic modelling in a large cohort of adrenocortical carcinoma patients.

OBJECTIVE: Mitotane is used for the treatment of adrenocortical carcinoma. High oral daily doses of typically 1- 6 g are required to attain therapeutic concentrations. The drug has a narrow therapeutic index and patient management is difficult because of a high volume of distribution, very long elimination half-life, and drug interaction through induction of metabolizing enzymes. The present evaluation aimed at the development of a population pharmacokinetic model of mitotane to facilitate therapeutic drug monitoring. METHODS: Appropriate dosing information, plasma concentrations (1137 data points) and covariates were available from therapeutic drug monitoring (TDM) of 76 adrenocortical carcinoma patients treated with mitotane. Using nonlinear mixed effects modeling, a simple structural model was first developed, with subsequent introduction of metabolic autoinduction. Covariate data were analyzed to improve overall model predictability. Simulations were performed to assess the attainment of therapeutic concentrations with clinical dosing schedules. RESULTS: A one-compartment pharmacokinetic model with first order absorption was found suitable to describe the data, with an estimated central volume of distribution of 6086 L related to a high interindividual variability of 81.5%. Increase in clearance of mitotane during treatment could be modeled by a linear enzyme autoinduction process. Body mass index was found to have an influence upon disposition kinetics of mitotane. Model simulations favor a high dose regimen to rapidly attain therapeutic concentrations, with the first TDM suggested on day 16 of treatment to avoid systemic toxicity. CONCLUSION: The proposed model describes mitotane pharmacokinetics and can be used to facilitate therapy by predicting plasma concentrations.

Circannual variation of mitotane and its metabolites plasma levels in patients with adrenocortical carcinoma.

OBJECTIVES: Mitotane is the reference drug for the adrenocortical carcinoma treatment; its pharmacological activity seems to depend on drug transformation in two active metabolites: o,p'-DDE (dichlorodiphenylethene) and o,p'-DDA (dichlorodiphenylacetate). Mitotane and metabolites are lipophilic agents; thus, they tend to accumulate into adipose tissues (white and brown), which change their prevalence seasonally. Aim of the work was to evaluate mitotane and metabolites plasma levels variation over the year, in adrenocortical cancer patients treated with Lysodren® for at least 6 months. METHODS: We enrolled a group of 86 adrenocortical carcinoma diagnosed patients, who underwent radical surgery and started mitotane as adjuvant treatment. For drug and metabolites plasma level (from samples collected ~12 h after the dose administration of mitotane, just before the subsequent administration) determination, a validated chromatographic method was used. KEY FINDINGS: Results showed an evidence of a seasonal trend for the three substance (o,p'-DDD, o,p'-DDE and o,p'-DDA) plasma levels, in terms of acrophases and lower values. Furthermore, it came out that male patients need a higher significant mitotane drug dose than female patients to reach mitotane therapeutic window. CONCLUSIONS: In conclusion, this is the first study assessing a mitotane plasma level variation over the year, but further studies in larger cohorts are required.

RRM1 modulates mitotane activity in adrenal cancer cells interfering with its metabolization.

The anti-proliferative activity of mitotane (o,p'DDD) in adrenocortical cancer is mediated by its metabolites o,p'DDE and o,p'DDA. We previously demonstrated a functional link between ribonucleotide reductase M1(RRM1) expression and o,p'DDD activity, but the mechanism is unknown. In this study we assessed the impact of RRM1 on the bioavailability and cytotoxic activity of o,p'DDD, o,p'DDE and o,p'DDA in SW13 and H295R cells. In H295R cells, mitotane and its metabolites showed a similar cytotoxicity and RRM1 expression was not influenced by any drug. In SW13 cells, o,p'DDA only showed a cytotoxic activity and did not modify RRM1 expression, whereas the lack of sensitivity to o,p'DDE was associated to RRM1 gene up-modulation, as already demonstrated for o,p'DDD. RRM1 silencing in SW13 cells increased the intracellular transformation of mitotane into o,p'DDE and o,p'DDA. These data demonstrate that RRM1 gene interferes with mitotane metabolism in adrenocortical cancer cells, as a possible mechanisms of drug resistance.

Developing treatment for adrenocortical carcinoma.

Cancer of the adrenal cortex (ACC) is a rare endocrine malignancy with limited treatment options. Patients typically present with autonomous hormonal overproduction and/or a large abdominal mass. Hormonal assays and medical imaging can be diagnostic, but urinary steroid profiling might be a more sensitive technique to assess malignancy in adrenal tumours. The stage of the disease at diagnosis is the most important prognostic factor. The current staging system needs refinement, especially to separate aggressive from indolent disease in stage IV patients and to select patients who need adjuvant treatment after complete surgical resection. Regarding the latter, assessing the proliferation index Ki-67 seems the best tool currently available. Genomic profiling is expected to become of clinical relevance in the future. Medical therapy is centred on the adrenolytic drug mitotane, which carries considerable toxicity and is not easy to manage. Its tolerability and long plasma level build-up phase may be improved by therapeutic drug monitoring based on pharmacokinetic modelling and intensive counselling of patients. Current chemotherapy regimens can offer disease stabilization in about 50% of patients, but an objective response should be expected in <25%. Research on targeted therapy and immunotherapy is difficult in this rare disease with often heavily pre-treated patients and has not yet been successful. Quality of care should be ensured by treating patients in centres with established experience in multidisciplinary oncologic care, who adhere to prevailing guidelines and state-of-the-art in diagnostic and treatment concepts. International collaboration in fundamental research and clinical trials is the key to further elucidate the pathogenesis and to improve patient care.

Systemic availability of o,p'-DDD in normal dogs, fasted and fed, and in dogs with hyperadrenocorticism.

The systemic availability of o,p'-DDD was studied in 12 normal dogs and seven dogs with pituitary-dependent hyperadrenocorticism (PDH). The drug was given by mouth at 50 mg kg-1 and plasma o,p'-DDD concentrations were determined by gas-liquid chromatography. First, six normal dogs were given the drug three times at intervals of one week in a Latin square pattern. Systemic drug availability was found to be very poor from intact tablets in fasted dogs, better with pure drug dissolved in maize oil given by stomach tube, and best with ground tablets mixed in oil poured on dog food. Then six normal dogs and five with PDH were given one dose of o,p'-DDD as intact tablets in dog food. Systemic drug availability was good in the normal animals and, for unknown reasons, better in dogs with PDH. The half-time of elimination was shorter in dogs with PDH than in normal ones. There was evidence of a gradual rise in plasma o,p'-DDD concentrations in seven dogs with PDH treated with 25 mg kg-1 every 12 hours for 14 or 20 days. The interaction between food and o,p'-DDD probably contributes to the variation in clinical response of dogs treated with the drug. The efficiency of therapy with o,p'-DDD should be improved considerably by administering the drug with food.

Short-term variation in plasma mitotane levels confirms the importance of trough level monitoring.

OBJECTIVE: Mitotane is the drug of choice in patients with adrenocortical carcinoma. The anti-neoplastic effect is correlated with mitotane plasma levels, which render it crucial to reach and maintain the concentration above 14 mg/l. However, mitotane pharmacokinetics is poorly understood. The aim of this study was to investigate the variation in plasma mitotane levels during the day and the influence of a single morning dose. DESIGN: A prospective case-control study was conducted to investigate the variation in plasma mitotane levels. METHODS: Patients who had been treated for at least 24 weeks and had reached the therapeutic plasma level (14 mg/l) at least once were eligible. In the first group, mitotane levels were determined hourly for the duration of 8 h after administration of a single morning dose. In the second group, mitotane levels were assessed similarly without administration of a morning dose. RESULTS: Ten patients were included in this study, and three patients participated in both groups. Median plasma level at baseline was 16.2 mg/l (range 11.3-23.3 mg/l) in the first group (n=7) and 17.0 mg/l (13.7-23.8) in the second group (n=6). Plasma levels displayed a median increase compared with baseline of 24% (range 6-42%) at t=4 after morning dose and a change of 13% (range -14 to 33%) at t=4 without morning dose (P=0.02). CONCLUSION: A substantial increase in mitotane plasma levels was observed in steady-state patients within a period of 8 h after morning dosing. Without morning dose, mitotane curves showed a variable profile throughout the day. This implies that random sampling could yield incidentally high levels. For this reason, we recommend early-morning trough sampling as standard management in monitoring mitotane treatment.