Contents
  1. Introduction 5
  2. Chemistry 7
  3. Pharmacological aspects 8
  4. Pharmacokinetics 8
  5. Absorption 8
  6. Distribution 9
  7. Metabolism 11
  8. Elimination 14
  9. Pharmacodynamics and pharmacological effects 15
  10. Toxicity 20
  11. New perspectives for mitotane treatment 22
  12. Conclusions and future directions 25
Introduction
Adrenocortical carcinoma (ACC) is a rare endocrine malignancy arising from one of the three cortical layers of the adrenal gland. ACCs can cause an increase in the production of one or more steroid hormones, such as cortisol, androgens, and aldosterone, resulting in clinical manifestations of Cushing’s syndrome, virilization (facial acne, pubic pilification, hirsutism, increase in muscle mass), and high blood pressure, respectively. Clinical manifestations are variable, and the prognosis of ACC is often unfavorable, especially at older ages [1,2]. The general incidence of ACC is 1.7–2.0 cases/million/year among adults, whereas it is even rarer among children in most countries (0.2-0.3 cases/million/year); it is more common among women [2]. One exception is Southern Brazil, wherein the ACC incidence is higher in children than in adults [3], reaching a rate of 3.4–4.2 per million children versus an estimated worldwide incidence of 0.3 per one million children younger than 15 years [4].
Currently, mitotane is the only approved drug, as single or adjuvant treatment, for post-operative or inoperable ACC. The daily dose usually ranges between 2 and up to 10 g to reach the ideal plasma concentration with tolerable toxicity, which is 14–20 mg L-1[5,6]. The mean dose is 4 g day-1 for adults and 4 g m²/day-1 for children for ACC stage 3/4 and recurrence of stage 1/2 [7,8]. In most patients, the steady state is reached after 3 months of daily treatment [6,9]. Nevertheless, patients experienced several adverse events of the gastrointestinal (diarrhea, nausea, vomiting, and anorexia) and central nervous (confusion, ataxia, and dizziness) systems [10] and many other adverse effects that were not well documented (ClinicalTrials.gov Identifier: NCT00568139), limiting its therapeutic use mainly among pediatric ACC patients.
Mitotane [1-(o-chlonophenyl)-1-(p-chIorophenyl) 2,2-dichioroethane], also known as o,p′-DDD, was isolated in 1940 from the insecticide dichlorodiphenyltrichloroethane (DDT). The adrenolytic effect of mitotane was first reported in dogs for inducing selective necrosis in the zona fasciculata and zona reticularis of the adrenal cortex [11,12]. The first clinical evidence was published in 1959, when the efficacy of mitotane for the treatment of ACC was reported in male patients [13]. Thereafter, even though the mechanism of action was unclear, mitotane was rapidly approved by the Food and Drug Administration (FDA) for ACC treatment, and it became commercially available in a tablet form as the reference product Lysodren® [14].
The first line treatment for ACC after surgery is mitotane monotherapy for less progressive ACC or mitotane plus chemotherapy, such as etoposide, doxorubicin, and cisplatin (mitotane + EDC), to treat more aggressive forms [15,16]. However, when first line therapy fails, mitotane can also be employed in combination with streptozotocin, vincristine, gemcitabine + capecitabine, and avelumab [15]. A recent meta-analysis revealed that mitotane monotherapy with or without radiotherapy, decreased the recurrence rate and mortality after tumor resection among patients without distant metastasis [17]. However, the efficacy of mitotane combined with chemotherapy is still unclear. To evaluate if adjuvant mitotane + EDC, after resection of ACC, has survival benefits for patients, we searched for “mitotane AND survival AND adrenocortical carcinoma OR adrenal tumor” in PubMed, Cochrane, Web of Science, and Scopus between November and December of 2019. We identified 851 records in the databases. Of these, 298 were duplicated and 553 were screened by title and abstract evaluation. Thirty-one full-text articles were assessed for eligibility. None of these articles met the inclusion criteria for meta-analysis of the benefits of adjuvant mitotane plus chemotherapy in prolonging the survival of patients. Thus, the clinical efficacy of mitotane in prolonging patient survival is still controversial due to a lack of randomized controlled studies. However, some clinical trials of mitotane + EDC for advanced ACC, recently registered in the Cochrane library, may provide useful data for the evaluation of patient survival in the future. Among the seven trials evaluating mitotane therapy plus chemotherapy identified in our study, only two have monitored the plasma mitotane levels [18,19] (Table 1).
Even after five decades of the mitotane approval by FDA, few studies have addressed pharmacological aspects of mitotane, probably because ACC is a rare disease. Therefore, better understanding of the effects of mitotane on ACC is needed to improve the clinical monitoring of its efficacy and development of new drugs with specific action against ACC with fewer toxic effects. Thus, this review summarized the chemistry, pharmacokinetic and pharmacodynamic features, and therapeutic and toxic effects of mitotane and the new formulations containing this drug.
Chemistry
Unlike DDT, the mitotane molecule exists in two configurations depending on the position of the chlorine element in the benzene ring: the position ortho in one ring and the position para in another ring. These mitotane configurations are characterized by the presence of an asymmetric chiral carbon atom that gives rise to two enantiomeric (R and S ) molecules. Considering that mitotane is available as a racemic mixture, both enantiomers, (S)-(-)-o,p′-DDD and (R)-(+)-o,p′-DDD (Fig. 1A) are present in the commercial drug. Furthermore, it has been demonstrated that mitotane metabolism involves two reactions via α- and β-hydroxylation. α-hydroxylation forms the metabolite o,p’-DDE (DDE), whereas β-hydroxylation forms o,p’-dichlorodiphenyl acyl chloride (DDAC). DDAC has a strong affinity for biological nucleophiles, which can acylate different cellular molecules; otherwise, DDAC could be rapidly converted to the metabolite o,p′-DDA (DDA) in the presence of water (Fig. 1B).
  1. Pharmacological aspects
  2. Pharmacokinetics
3.1.1. Absorption
Mitotane has poor water solubility (0.1 mg mL-1 at 25ºC), being better soluble in organic solvents such as ethanol (20 mg mL-1), dimethylsulfoxide (DMSO) (30 mg mL-1), and dimethyl formamide (DMF) (PubChem; Cayman Chemical). In pre-clinical studies, mitotane was often dissolved in olive oil or DMSO followed by aqueous buffer, to reach ideal plasma concentrations [24,25]. In clinical tests, mitotane reached the maximum absorption when administered together with dietary lipids such as high fat milk, chocolate, or oil emulsions, which increased its absorption by 5-fold compared to that of mitotane tablets [26].
Although mitotane (single dose of 2 g) reached the maximal plasma concentration of 0.0016 mg mL-1 (data from 9 patients) 10 h after administration [26], the time to reach the ideal plasma concentration (14 mg L-1) among ACC patients was around 116 days with an accumulative dose of approximately 626 g (data from 53 patients) [27]. However, it is difficult to determine mitotane bioavailability since there is no data about in vitro intestinal absorption, bioavailability of intravenously administered mitotane, or hepatic extraction to determine whether mitotane has first-pass metabolism. Despite the lack of information about the pharmacokinetics of mitotane, there is no doubt that the oral bioavailability is low, probably due to poor absorption and extensive metabolism. Indeed, patients required high doses (2–10 g day-1) to reach the therapeutic plasma concentration (14-20 mg L-1) and the steady state was reached only about 3 months after treatment initiation [5,6]. In addition, high variation in plasma concentration was observed with different doses among patients probably due to the basic individual variability in body mass index, sex, age, and metabolic enzyme activity. Metabolic cytochrome P450 (CYP) polymorphisms might play an important role in mitotane absorption. In fact, D’Avolio et al. (2013) [27] demonstrated that the polymorphism of CYP2B6 was positively correlated with higher plasma mitotane concentration. In patients with any stage ACC treated with mitotane monotherapy, the polymorphism of CYP2W1 was associated with a reduced probability of reaching the mitotane therapeutic range and with lower response rates, whereas the polymorphism of CYP2B6 was correlated with high plasma levels of mitotane [28]. It was documented that mitotane is a CYP2B inducer (see section 3.1.3); however, the roles of CYP2B6 and CYP2W1 in mitotane functions are not yet clear. Thus, Kerkhofs and colleagues (2015) [29] proposed a new pharmacokinetic model adapted to individual patients in daily clinical practice. The 3-compartment model was proposed to measure mitotane levels through the volumes of distribution and clearance [29]. Therefore, this model can be used as a tool to elucidate the absorption of mitotane and the variability among patients.
3.1.2. Distribution
The distribution of mitotane in various body compartments has been studied for a long time. The estimated volume of distribution was relatively high. Thus, the plasma concentration was more influenced by mitotane distribution than the elimination process [29]. The maximum concentration of mitotane can be detected in the plasma of patients 2–8 hours after a single dose of 2 g [26]. Nevertheless, DDA levels were higher than mitotane levels in the plasma, whereas DDE was barely detected after the administration of repeated doses [30]. However, the mitotane concentration in adipose tissue was 200-fold higher than the plasma concentration in patients during chemotherapy [31]. In rats, after 80 days of diet containing 1% mitotane, mitotane tended to accumulate mainly in adipose tissue, as well as in the liver, kidney, brain, and adrenal tissue [32]. Mitotane labeled with14C was localized mainly in the zona fasciculata and zona reticularis in human adrenocortical tumor and mouse adrenal tissue [33,34].
Distribution of mitotane involved chylomicron and lipoprotein binding as a higher proportion of plasma mitotane was bound to lipoproteins [35]. Under normolipidemic conditions, a substantial amount of mitotane was bound to high density lipoprotein and albumin, whereas under hypertriglyceridemic conditions, mitotane was bound mainly to chylomicron and to very low-density lipoproteins (VLDL) [36]. The distribution of mitotane involves cellular diffusion by adhering to LDL. Interestingly, mitotane promoted LDL formation [37], and probably promoted its self-uptake into adrenals cells. However, in vitro , lipoprotein binding inhibited the activity of mitotane, suggesting that lipoprotein-free mitotane was its therapeutically active fraction [35,38]. Higher antiproliferative and proapoptotic effects of mitotane were shown in H295R cells grown in lipoprotein-free medium, and a higher rate of tumor control was demonstrated in patients with ACC treated with mitotane and statins [38]. Under hypertriglyceridemic conditions, the bound mitotane-VLDL complex does not enter into the adrenal cells [36], and the patient may be unresponsive to the effects of mitotane. High concentrations of DDA and DDE were also found bound to chylomicrons. However, it is important to note that DDA is not too lipophilic due to the presence of the carboxylic acid group, whereas mitotane and DDE have a high affinity for lipids [35]. Therefore, to estimate toxicity and efficacy and improve patient care, it is important to quantify lipoprotein-free and lipoprotein-bound mitotane [35].
Regarding the enantiomers of mitotane, different elimination ratios were suggested in the exposure of enantiomers to human placentas as measured by gas chromatography [39]. S(-)-o,p′-DDD was dominant in the plasma of two minipigs whereas the enantiomer R-(+)-o,p′-DDD was dominant in the plasma of three minipigs [40]. Besides, the two isomers were equally potent in decreasing H295R cell viability, (S)-(-)-o,p′-DDD affected hormone secretion (dehydroepiandrosterone [DHEA] and cortisol) slightly less than the (R)-(+)-o,p′-DDD and the racemic mixture [41]. Thus, it is also suggested that the racemic mixture is important for the therapeutic effects of mitotane. However, the pharmacological features of each enantiomer in ACC treatment are not yet clear.
3.1.3. Metabolism
The metabolism of mitotane was previously described as occurring only in adrenal cells [42]. This drug has two reactions in the mitochondria of adrenal cells: α-hydroxylation which yields the end-product DDE, and β-hydroxylation which gives rise to the end-product DDA (Fig. 1B). Although measurements of plasma DDA for estimating the effects of mitotane suggested that it was an active metabolite [30], other authors claimed that DDA was an inactive metabolite [38]. In fact, DDA is not so lipophilic and its presence is further found in the urine (see section 3.1.4), suggesting that DDA is an end-product of the mitotane deactivation pathway. In contrast, it has been speculated that DDE could be an active metabolite, since it was not extensively found in plasma, urine, or feces (see section 3.1.4) and had a cytotoxic effect on the H295R adrenocortical cell line [43]. However, no additional studies have been conducted yet to confirm this hypothesis.
The β-hydroxylation of mitotane has another important function beyond DDA formation, which is the generation of an intermediate reactive metabolite, an acyl chloride derivative (DDAC), which might be responsible for covalent binding to mitochondrial macromolecules in the adrenals [44]. It is important to point out that the covalent bond is not permanent and can be reversed by the addition of glutathione reductase (GSH), suggesting that GSH can inactivate mitotane [45]. The reaction for the covalent bond is mediated through a specific CYP450 responsible for steroidogenesis in the adrenal cells, such as CYP11A1 (see section 3.2) [46]. In the presence of a P450 inhibitor (erythromycin), mitotane and DDA tend to accumulate in fat tissue and plasma, whereas the levels of DDE decrease [32], supporting the theory that P450 is responsible for α-hydroxylation of mitotane. In addition, accumulation of 14C labeled mitotane in the adrenals was partially reversed by metyrapone, a known inhibitor of CYP11B1 [33,34], suggesting that both CYP11A1 and CYP11B1 are involved in the formation of the DDAC.
Other CYPs are also related to the effects of mitotane. Recently, Murtha and colleagues demonstrated that silencing of CYP2A6 mRNA in H295R cells promoted higher sensitivity of this cell line to the effects of mitotane [47]. This enzyme is a known metabolizer of some xenobiotics; however, the exact function of CYP2A6 in ACC is still unclear. Moreover, high expression level of CYP2W1 mRNA was observed in both normal and neoplastic adrenal glands and was related to a better response to mitotane treatment among patients [48]. It was suggested that CYP2W1 might play a role in mitotane metabolism and could be a potential predictive marker of sensitivity to mitotane treatment [49]; however, the tissue expression and function of CYP2W1 are still under debate.
When mitotane was administered to mice, via the intraperitoneal route (440 mg kg-1, dissolved in olive oil, for 48 days), the antitumor effects remained for 34 days, compared to those achieved by oral administration, which yielded antitumor effects for only 13 days [25]. This suggests that mitotane undergoes first pass metabolism when administered orally and/or is largely eliminated via feces. Furthermore, the intestinal microbiome could also influence mitotane absorption and metabolism. The hypothesis of intestinal mitotane metabolism is supported by high concentrations of DDA and DDE found in chyle. Moreover, DDE might be metabolized further in the liver, which would explain why this compound was barely detected in the plasma [35]. This indicates that mitotane might be metabolized in the liver via enzymatic (i.e. via CYP450) and non-enzymatic (i. e., glutathione [GSH]) reactions since DDT is metabolized in the liver in the same way [50]. However, no preclinical studies have investigated the metabolism of mitotane, DDA, and DDE in the liver. Moreover, the role of the intestinal microbiome in mitotane metabolism should also be considered, since the gut microbiota plays a role in drug metabolism prior to absorption or during enterohepatic circulation, via various microbial enzymatic reactions in the intestine [51]. Therefore, understanding the effects of liver and gut microbiota on mitotane metabolism is crucial to explaining the changes in its pharmacokinetics.
Mitotane has a narrow therapeutic index; thus, the possibility of pharmacokinetic drug–drug interactions should not be discarded. This drug has a long-lasting inducing effect on CYP3A4 and CYP2B and a potent inhibitory effect on CYP2C19, which result in clinical interactions with many drugs metabolized by these enzymes [52,53,54]. Therefore, when associative treatment of mitotane in combination with such drugs (i.e. doxorubicin, etoposide, hydrocortisone, cyclophosphamide, omeprazole, and clopidogrel) is necessary, the therapeutic effects and toxicity of these drugs need to be monitored closely.
3.1.4. Elimination
After an oral intake of a single mitotane dose in tablet form (2 g), around 40% of unchanged mitotane could be detected in the feces after 12 h, whereas mitotane in milk or emulsion decreased the amount of drug excreted to less than 10% [26]. Furthermore, after oral mitotane treatment (100 mg) in rats, 7.1% was excreted in the urine and 87.8% of mitotane was found in the feces within 8 days [55]. Thus, unchanged mitotane appears to be eliminated largely through biliary excretion. However, mitotane, DDA, and DDE can also undergo enterohepatic circulation; chyle could be enriched due to hepatic transformation [35]. The mitotane clearance and volume of distribution in the steady state were determined in 22 patients as 0.94 ± 0.37 L h-1 and 161 ± 68 L kg-1 of the lean body mass, respectively [29]. In addition, DDA was found in the urine of mice treated with mitotane (250 mg kg-1, p.o.) for a total of 96 h [24] as well as in the feces [56]. Although renal elimination also plays an important role in mitotane clearance, the rate is lower than that of biliary elimination. In a study of 19 patients receiving mitotane at 3–6 g day-1 over a period of 30–60 days, the half-life was found to be between 18 and 159 days after mitotane was withdrawn [26]. Long-term mitotane elimination observed was probably due to its accumulation in fat tissue, attributable to its lipophilic characteristics.