JPP
JOURNAL OF Pharmacy and Pharmacology
ROYAL PHARMACEUTICAL SOCIETY
Circannual variation of mitotane and its metabolites plasma levels in patients with adrenocortical carcinoma
Jessica Cusatoª,* ID, Silvia De Franciab,*, Sarah Allegraa ID, Simona Carrellab, Elisa Pirrob, Francesca Maria Piccioneb, Francesca De Martinob, Anna Ferrero“, Fulvia Claudia Daffarad, Massimo Terzolod, Alfredo Berrutie, Francesco Di Carlob, Marco Tampellini and Antonio D’Avolioª iD
aUnit of Infectious Diseases, Department of Medical Sciences, Amedeo di Savoia Hospital, University of Turin, Turin, Italy, bDepartment of Biological and Clinical Sciences, S. Luigi Gonzaga Hospital, University of Turin, Orbassano, TO, Italy, ‘Division of Oncology, Department of Oncology, “San Luigi” Hospital-Orbassano, University of Turin, Orbassano, TO, Italy, ªDivision of Internal Medicine, Department of Oncology, S. Luigi Gonzaga Hospital, University of Turin, Orbassano, TO, Italy and eMedical Oncology Unit, Department of Medical & Surgical Specialties, Radiological Sciences & Public Health, Spedali Civili Hospital, University of Brescia, Brescia, Italy
Keywords
adrenocortical carcinoma; DDD; gender medicine; HPLC; seasons; Therapeutic Drug Monitoring
Correspondence
Jessica Cusato, Unit of Infectious Diseases, Laboratory of Clinical Pharmacology and Pharmacogenetics, Department of Medical Sciences, University of Torino, ASL Città di Torino, Amedeo di Savoia Hospital, Corso Svizzera 164, Turin 10149, Italy.
E-mail: Jessica.cusato@yahoo.it
Received June 6, 2017 Accepted July 17, 2017
doi: 10.1111/jphp.12798
*Both authors equally contributed to the work.
Laboratory of Clinical Pharmacology and Pharmacogenetics: UNI EN ISO UNI EN ISO 9001:2008 and 13485:2012 (CE-IVD) Certi- fied Laboratory; Certificate No. IT-64386 Certification for: “DESIGN, DEVELOPMENT AND APPLICATION OF DETERMINATION METHODS FOR ANTI-INFECTIVE DRUGS. PHARMACOGENETIC ANALYSES.”
Abstract
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 treat- ment. 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 sub- stance (o,p’-DDD, o,p’-DDE and o,p’-DDA) plasma levels, in terms of acro- phases 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.
Introduction
Mitotane (dichlorodifenyldichloroethane, o,p’-DDD, com- mercially known as Lysodren®), an analogue of the DDT insecticide, has been used since the 60s for the treatment of adrenocortical carcinoma (ACC), a rare tumour character- ized by a dismal prognosis and by a 5-year survival rate <40%, after diagnosis.[1-3) Complete surgical removal of ACC is the most important outcome predictor.[1-3] How- ever, fully half of the patients, who underwent complete
surgery, are destined to relapse, often with distant metas- tases 49]; it is still unknown why they are destined to relapse, even at stage I-II of disease. The high recurrence rate of ACC has prompted to consider the use of an adju- vant therapy, after radical tumour resection; to this pur- pose, mitotane, alone or in combination with others antineoplastic agents, has been considered.[10] As conse- quence, even if its use as postoperative measure remains controversial, mitotane, recently, has been widely employed in daily clinical management of patients with ACC.[11]
This drug acts as an adrenocortical suppressant and as a steroid synthesis inhibitor. It has been reported that its two active metabolites, o,p-‘DDE (dichlorodiphenylethene) and o,p’-DDA (dichlorodiphenylacetate), cause toxicity through oxygen activation with superoxide formation or by covalent binding to specific proteins.[12] A range of drug efficacy has been well established in the past. In 1984, van Slooten et al.[13] defined a therapeutic range for mitotane: its antitumour efficacy was observed with plasma concen- trations >14 mg/l, whereas toxicities were associated with concentrations >20 mg/l.
Mitotane, o,p’-DDE and o,p’-DDA are lipophilic agents and tend to accumulate into fatty tissue. It is known that adult men have two kinds of adipose tissues: white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is a storage tissue; BAT is responsible for thermogenesis in neonatal and in adult life. Studies on Siberian hamster[14] demonstrated that the proportion of BAT depends on pho- toperiod. The longer dark period is associated with the greater melatonin (MLT) release; thus, in winter a great amount of MLT is secreted. MLT binds specific receptors and activates sympathetic nervous system, thus noradrenaline (NA) is released, and it determines BAT hypertrophy and WAT lipolysis. Saito et al., in 2009,[15] later confirmed this study in humans, demonstrating that BAT metabolic activity shows a seasonal rhythm actually depending on two factors: light/dark period and temperature. Furthermore, in the same paper, it was demonstrated that the proportion of metaboli- cally active BAT is inversely correlated with BMI, hypothesis better explained by Vijgen et al.[16] cold induced BAT activity decreases when BMI increases. Au-Yong et al.,[17] further- more, confirming previous findings, demonstrated that the percentage of metabolically active BAT is higher in winter and than in summer, when BAT is lower and lipolysis of WAT decreases.
It is known from literature, moreover, that body mass index (BMI) and body composition tend to change season- ally, with a reduction during summer.[18]
Response to mitotane treatment could be related to many different parameters; also its plasma levels could be subjected to seasonal variation.
For these reasons, aim of the study was to assess the potential presence of a seasonal trend of o,p’-DDD, o,p’- DDE and o,p’-DDA plasma levels, in patients treated with Lysodren® for at least 6 months continuously.
Materials and Methods
Patients
For this retrospective study, we enrolled a group of 86 ACC diagnosed patients, referred to Internal Medicine and Med- ical Oncology of St. Luigi Hospital (Orbassano, Turin,
Italy). Inclusion criteria were as follows: age 18 years or older, histologically confirmed diagnosis of ACC, complete tumour resection, availability of postoperative follow-up information including the results of imaging tests (com- puted tomography or MRI scans) and regular determina- tions of plasma mitotane concentration. Exclusion criteria were macroscopically incomplete resection, incomplete tumour staging, incomplete follow-up information or fol- low-up duration of <6 months and initiation of mitotane treatment longer than 6 months after surgery. For the anal- ysis, we selected 24 patients, 10 female patients and 14 male patients, characterized by maintaining the same dosage of Lysodren® for at least 6 months (1-4 g/die). A total of 62 patients were not eligible because mitotane therapy chan- ged, or it was interrupted for severe adverse events appear- ance. All patients received glucocorticoid replacement, whereas thyroxine and sex steroid hormone replacement was provided if deemed appropriate. Characteristics of selected patients group were reported in Tables 1 and 2. Patients underwent mitotane plasma level monitoring every 3 weeks.
Study protocol was approved by the local Ethics Com- mittee (sperimentazioni@sanluigi.piemonte.it). A written informed consent for the study was obtained from each study participant.
HPLC analysis
Each patient sample underwent o,p’-DDD, o,p’-DDE and o,p’-DDA plasma level determination. Patients blood sam- ples were collected in heparin tubes (BD Vacutainer® Bec- ton, Dickinson and Company, Franklin Lakes, New Jersey, USA) ~12 h after the dose administration of mitotane, just before the subsequent administration; plasma was sepa- rated by centrifugation (2000g, 4℃, 10 min) and then stored at -80℃ until analysis. Substances quantification was performed by a validated high-performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV).[19] Substances separation, after specific liquid extraction, was achieved on a RP-18 column. Internal stan- dard quantification was used, fitted with linear regression.
As summarized in Table 3, to evaluate o,p’-DDA plasma concentration, some parameters (extraction solution, col- umn, mobile phase, flow, UV nanometers and internal standard) of the mentioned method were changed.
Statistical analysis
Statistical analysis was performed with Statistica 6.0 (Stat- soft Inc., Tulsa, OK, USA). Data were expressed as median and range (age at diagnosis, duration of mitotane treat- ment, time to reach mitotane therapeutic range) or as mean ± standard error (mitotane grams administered and
| Patient ID | Age at diagnosis (years) | Treatment duration (months) | Mitotane dose (g/die) | Concomitant treatments | Surgery (no) | Chemotherapy |
|---|---|---|---|---|---|---|
| 15 | 58 | 12 | 1.5 | Rosuvastatin Simvastatin Fluvastatin | 1 | Yes |
| 16 | 41 | 8 | 2.5 | Ursodesossicolic acid | 2 | Yes |
| 17 | 29 | 10 | 1.5 | Estroprogestinic hormones | 1 | No |
| 18 | 43 | 14 | 4 | No | 1 | No |
| 19 | 32 | 15 | 2 | Pantoprazole Fluoxetine | 5 | Yes |
| 20 | 38 | 13 | 1 | Ursodesossicolic acid Pantoprazole Escitaliopram | 1 | Yes |
| 21 | 34 | 7 | 1 | No | 1 | No |
| 22 | 39 | 13 | 1.5 | No | 1 | Yes |
| 23 | 40 | 11 | 2 | No | 2 | No |
| 24 | 33 | 10 | 2 | Estroprogestinic hormones | 3 | Yes |
| Patient ID | Age at diagnosis (years) | Treatment duration (months) | Mitotane dose (g/die) | Concomitant treatments | Surgery (no) | Chemotherapy |
|---|---|---|---|---|---|---|
| 1 | 50 | 6 | 2.5 | No | 1 | Yes |
| 2 | 55 | 10 | 3 | No | 2 | Yes |
| 3 | 48 | 6 | 3 | No | 1 | No |
| 4 | 31 | 8 | 3 | No | 1 | No |
| 5 | 22 | 9 | 2 | Pantoprazole | 5 | Yes |
| 6 | 44 | 6 | 3 | No | 1 | Yes |
| 7 | 36 | 25 | 1.5 | Warfarin Rosuvastatin Fluvastatin | 1 | No |
| 8 | 46 | 9 | 3 | Rosuvastatin | 1 | Yes |
| 9 | 53 | 10 | 2 | No | 2 | No |
| 10 | 29 | 8 | 3 | No | 3 | Yes |
| 11 | 55 | 14 | 1.5 | Testosterone enanthate | 1 | No |
| 12 | 29 | 9 | 2.5 | No | 3 | Yes |
| 13 | 31 | 8 | 2 | Phenobarbital Topiramate | 2 | Yes |
| 14 | 35 | 7 | 4 | Lamotrigine Alendronate | 1 | Yes |
mitotane plasma levels reached). P < 0.05 was considered to represent statistical significance. Differences in drug administered dose and in mitotane plasma levels reached between genders were assessed using Wilcoxon paired test. Cosinor method has been employed for rhythm analysis: acrophase (the time at which the peak of the rhythm occurs), amplitude (the difference between the peak and the mean value) and mesor (the mean value around which the variable oscillates) were calculated.
In order to establish whether the obtained results were reliable or not, we compared each drug amplitude/mesor ratio to variability parameters of the chromatographic methods. Results were considered reliable if amplitude/ mesor ratio was higher than variability.
For o,p’-DDD and o,p’-DDE, we compared the ampli- tude/mesor ratio to the interday variability parameter because their plasma levels were determined in different days of analyses.
For o,p’-DDA, we compared the amplitude/mesor ratio to the intraday variability parameter, as o,p’-DDA concen- tration was determined in one day analysis.
Results
Median age at diagnosis for selected patients was 38 years (22-58). Concerning female patients, four of them showed the involvement of left adrenal gland and one a hepatic neurinoma; evaluating male group, two patients showed
| (a) HPLC-UV method for measuring o,p'-DDD and o,p'-DDE | (b) HPLC-UV method for measuring o,p'-DDA |
|---|---|
| · extraction: liquid/liquid (plasma/acetone) | · extraction: liquid/liquid (plasma/acetonitrile-methanol) |
| · column: RP-18 (5 mm, 250 × 4.6 mm) | · column: RP-18 (3 mm, 150 x 4.6 mm) |
| · mobile phase: H2O/MetOH/CH3CN (10 : 10 : 80) | · mobile phase: CH3CN/MetOH/H2O-MetOH-Et3N (40 : 20 : 40) |
| · flow: 1 ml/min | · flow: 0.5 ml/min |
| · UV: 218 nm | · UV: 235 nm |
| · Tº: 25°℃ | · Tº: 25℃ |
| · internal standard: DDT | · internal standard: nilotinib |
the involvement of left and two the right adrenal glands, one a thymoma and one a thyroid cancer. One of the enrolled patients had a metastatic disease and he underwent five surgeries.
Median duration of mitotane treatment, at the same dose, was 10 months (6-25); 16 patients are currently on mitotane therapy, eight died. During the time lapse of constant considered drug dose, 50% (five female patients and seven male patients) reached the drug therapeutic window (14-20 µg/ml). These patients, from starting therapy, reached efficacy range after a median time of 3 months (1-6). By Wilcoxon paired test, we observed a significant difference (P = 0.04) in terms of the adminis- tered drug dose between female patients (1.9 g ± 0.9) and male patients (2.6 g ± 0.7), whereas no significant difference (P = 0.78) was found in median mitotane plasma level between sex (females: 13.1 µg/ml ± 6.4; males: 13.4 µg/ml ± 6.2).
Considering BMI evaluation, most of the patients (70%) selected for the study resulted overweighted.
Regarding Cosinor analysis, we observed an amplitude/ mesor ratio and interday variability respectively of 8.3% and 4.8% for o,p’-DDD, for o,p’-DDE 28.5% and 11% and for o,p’-DDA 4.8% and 1.9%. All the obtained results were considered reliable, but, in o,p’-DDE case, we had many null values in plasma measurements.
Acrophases for the three substances occurred in different period of the year, as shown in Figure la-c: for o,p’-DDD, it occurred in August, whereas lower values were registered in winter months; for o,p’-DDE, acrophase was in March and lower values in summer months, whereas for o,p’- DDA, it was in April and lower values in autumn months.
Discussion
It is known that in humans, physiologic rhythms exist and regulate a great number of metabolic responses. These rhythms are genetically determined but, to mature, they interact with exogenous synchronizers. One of these syn- chronizers is the light/dark alternation, and the proportion of WAT and BAT seems to be regulated by the
photoperiod. As previously described, in winter, the pro- portion of BAT is higher, whereas WAT decreases.[14,15,17] Moreover, the proportion of metabolically active BAT seems to be inversely correlated with BMI, whereas WAT positively correlates. [14-1 [14-16]
As BMI and body composition tend to seasonally change[18] and mitotane is a lipophilic drug, we supposed that o,p’-DDD and its metabolites plasma concentrations could change during the year.
Our data suggested an evidence of a seasonal trend for o, p’-DDD (Figure la), with a peak in summer (August) and lower values registered in winter. O,p’-DDE (Figure 1b) showed an opposite trend, with a peak in Spring and lower values during summer. Finally, for o,p’-DDA (Figure 1c), even if very weak, trend was similar to that of o,p’-DDD, with acrophase in April and lower values in early autumn months. As already mentioned, all the results obtained were reliable, because in all cases ratio amplitude/mesor was higher than related method variability.
Evidence of seasonal trends for the three substances could be explained with the BAT and WAT variation over the year, depending on the photoperiod, demonstrated in the literature.[14,15,17] During summer, WAT higher pro- portion could cause an increase in o,p’-DDD storage in fatty tissue, with a potential slow and continuous drug release in plasma compartment. In fact, we observed that o, p’-DDD plasma levels during summer were higher. If we suppose that BAT, metabolically active compartment, could be involved in o,p’-DDD metabolites formation, as in sum- mer season it is reduced, we can also explain low levels of o,p’-DDE and o,p’-DDA in plasma (Figure 1b and 1c).
During the winter, on the contrary, the lower proportion of WAT could cause a lower o,p’-DDD storage in fatty tis- sue with a minor continuous release of the drug in plasma compartment. In fact, we observed that o,p’-DDD plasma levels during winter were lower and BAT, which is increased in this season, could explain medium-high levels of o,p’-DDE and o,p’-DDA in plasma (Figure 1b and 1c).
Our results not comply with the study conducted by Wolff et al.,[20] which showed a negative correlation between DDE and BMI, but in o,p’-DDE case, however, we had many null
o,p’-DDD CIRCANNUAL RHYTHM
o,p’- DDE CIRCANNUAL RHYTHM
30
(a)
4,5
8
(b)
4,0
25
o,p’-DDD concentration (µg/ml)
3,5
o,p’-DDE concentration (µg/ml)
20
3,0
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15
2,0
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10
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8
0,0
0
-0,5
0
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6
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14
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4
6
8
10
12
14
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Month
o,p’- DDA CIRCANNUAL RHYTHM
140
(c)
120
o,p’-DDA concentration (µg/ml)
100
80
60
40
8
8
0
000
8
000
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00
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000
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values in plasma measurement, which might have interfered with the final result. No clear indications are present in the literature, up to now, instead, about potential correlation between BMI and o,p’-DDD or o,p’-DDA.
Moreover, we could consider another factor, which is able to change its levels during the year, also in cancer patients: vitamin D.[21] This molecule regulates different biological processes, such as skeletal metabolism and cell growth and survival.[22] Higher vitamin D circulating levels were related to reduced risk to develop several diseases, including cancer.
Calcitriol is the active vitamin D form, and it modulates gene expression through its receptor vitamin D receptor (VDR). Vitamin D synthesis starts in skin, then cholecalcif- erol (vitamin D3) is hydroxylated to calcifediol in liver and in kidney converted to calcitriol by cytochromes and trans- ported in blood to target tissues by vitamin D binding protein.[24]
In-vitro studies on ACC human cells (H295R) assessed that calcitriol treatment (1 nM) inhibits 20% of cellular proliferation, together with steroids secretion reduction; a 10 nm dose increase leads to G1 phase stop cell cycle due to
reduced cyclin-dependent kinase 4 activity. This effect is not present through VDR gene silencing. Moreover, VDR expression was reduced in ACC compared to benign adrenocortical tumours. These results reveal a VDR protec- tive role against malignant adrenocortical cell growth. [25]
For these reasons, vitamin D could have a role in ACC mitotane-treated patients and it could be helpful to evalu- ate these molecule concentrations during the year correlat- ing them to mitotane levels, outcome and toxicity. Furthermore, in future, a genetic variation investigation in vitamin D pathway-related genes could be performed to clarify the involvement of heritability in this scenario. In fact, there are several pharmacogenetic studies showing the influence of polymorphisms in genes related to vitamin D pathway in affecting therapy outcome, toxicity and drug concentrations. [26-30]
Eventually, only the 50% of enrolled patients get to a therapeutic level, and this could be explained considering that several factors may influence drug bioavailability: intrinsic ones as age, sex, BMI and diet and extrinsic ones. [3]] In addition, we have to remember the importance of pharmacogenetics in mitotane therapy, as showed in our study.[32]
Conclusions
Considering the small sample size (24 patients), we could only suggest the possible presence of a seasonal trend. Mitotane is administered in dose escalation, and then pos- sibility to reach a larger population of patients, treated with the same dose for at least 6 months, is difficult. By the way, repetition of the analysis on a larger number of patients could help to establish a potential and real rhythm of mito- tane and its metabolites plasma levels in patients.
Even if in our limited sample size no differences were found in terms of mitotane plasma levels, a significant asso- ciation among genders and drug dosage has been observed. Considering that most of the patients were overweighted, preliminary observation could lead to higher drug dosage in male patients than female patients to reach mitotane effi- cacy range. It could be also possible that this difference, not related to BMI, could be attributed to sex hormone pattern, element that should be considered in clinical protocol drug setting. In conclusion, our results, even if related to a very small cohort of patients, could help physicians to set a real
individualized therapy different on the basis of the moment of the year chosen for drug administration.
Larger prospective studies, incorporating pharmacoge- netic markers (such as polymorphisms in vitamin D path- way genes), vitamin D serum levels, and comparative studies with other diseases are warranted. We had not eval- uated calcitriol plasma levels (considering it is a retrospec- tive study), which could be considered a limitation of this study, but we are working to add this evaluation to future studies.
Another important limit of our study consists in the pos- sible effect of the chemotherapy underwent by many of our patients, which may influence the natural biorhythms.
Further investigations including the estimation of the body fat composition and the proportion of BAT could specifically explain our results.
The finding for the present study may be helpful in developing personalized medicine, and the used method may also be applied in other pathologic populations to study their unique seasonality.
Declarations
Funding
This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors.
Conflict of interest
The Author(s) declare(s) that they have no conflicts of interest to disclose.
Acknowledgements
We thank CoQuaLab (www.coqualab.it) for its method- ological support and assistance in the preparation and exe- cution of the pharmacogenetic analysis.
Informed consent
Informed consent was obtained from all individual partici- pants included in the study.
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