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Bioorganic Chemistry
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BIO-ORGANIC CHEMISTRY
Two-dimensional chromatography for enantiomeric analysis of mitotane and its metabolite o,p’-DDA in patients with adrenocortical carcinoma indicates enantioselective metabolism
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Gabriela Stadler a,b,1, Alan de Almeida Veiga a,b,1, Claudia Rita Corso a, b, Camila Bach de Assis a,b, Beatriz de Toledo Nogueira a,b, Lucia Regina Rocha Martins ”, Beatriz Cruz Bonk , Flávia Lada Degaut Pontes , Bonald Cavalcante de Figueiredo a,b, Lauro Mera de Souza ª,
a Instituto de Pesquisa Pelé Pequeno Príncipe, Curitiba 80250-060, Brazil
b Faculdades Pequeno Príncipe, Curitiba 80230-020, Brazil
· Universidade Tecnológica Federal do Paraná, Curitiba 81280-340, Brazil
d Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba 81531-990, Brazil
ARTICLE INFO
Keywords: Two-dimensional chromatography Chiral chromatography Drug metabolism o,p’-DDD o,p’-dichlorodiphenyl acetic acid o,p’-dichlorodiphenyldichloroethane
ABSTRACT
Mitotane is a chiral drug used to treat adrenocortical carcinoma, being metabolized to the o,p’-dichlorodiphenyl acetic acid (o,p’-DDA), also a chiral compound. Despite of its therapeutic significance, the overall ratios and enantiomers have not been known. In this study, we analyzed the enantiomers of mitotane and o,p’-DDA in the plasma of patients by a newly developed chiral-phase method employed in two-dimensional chromatography. Important differences were observed in the ratio of (S)/(R)-mitotane, which varied substantially from 1:1.2 to 1:10 whereas the (S)/(R)-o,p’-DDA ratio was relatively conserved, at approximately 2:1. These findings provide evidence for the enantioselective metabolism and provide a method for further analyses of mitotane and me- tabolites, which can explain the variation in the therapeutic response.
1. Introduction
Adrenocortical carcinoma (ACC) is a rare malignant neoplasm of the adrenal gland cortex, with a worldwide incidence of 0.2 to 2.0 cases/ million inhabitants/year. There are two peaks of incidence, i.e., in children in the 1st decade (usually up to 5 years old) and in adults be- tween the 4th and 5th decade of life [1-3]. In southern Brazil, particu- larly in the state of Paraná, the incidence of ACC in children is 15 to 20 times higher than those in Europe and the United States [4-5]. Localized disease in pediatric ACC, irrespective of histopathological profile, re- quires complete resection of the tumor [2,6]. Advanced staging at diagnosis is a great challenge, with an overall 5-year survival rate of less than 40% and less than 10% in the presence of metastasis [1,7-9]. Currently, ACC cases not detected by neonatal screening combined with surveillance in Paraná are often diagnosed at a late stage, and more than 50% of patients at diagnosis have micro- or macroscopic spread beyond
the adrenal gland [10].
Adjuvant therapy with the drug mitotane [IUPAC name:1-chloro-2- [2,2-dichloro-1-(4-chlorophenyl)ethyl]benzene], analogous to the pesticide p,p’-DDT, has been recommended in late-stage ACC for more than five decades, including in cases of non-resectable and metastasized tumors and even after the presumed complete removal of the primary tumor at a late stage [11-12]. However, the objective response rate to mitotane therapy is less than 35%; therefore, it is frequently combined with chemotherapies (e.g., etoposide, doxorubicin, and cisplatin) to treat aggressive ACC forms [9,13-19]. The therapeutic effects of mito- tane are observed at a plasma concentration of 14-20 mg.L-1, reached through high daily doses, resulting in toxicity with frequent and intense side effects [12-13,19-21].
Mitotane undergoes enzymatic metabolism, resuting in two main metabolites that are found in the blood of patients, namely 1,1-(o,p’- dichlorodiphenyl)-2,2 dichloroethene (o,p’-DDE) and 1,1-(o,p’-
* Corresponding author at: Instituto de Pesquisa Pelé Pequeno Príncipe, Av. Silva Jardim, 1632, CEP: 80250-060, Curitiba, PR, Brazil.
E-mail addresses: lauro.souza@pelepequenoprincipe.org.br, lauro.bioq@gmail.com (L. Mera de Souza).
1 These authors have equal contributions to this work.
https://doi.org/10.1016/j.bioorg.2023.106835
dichlorodiphenyl) acetic acid (o,p’-DDA). The effects of o,p’-DDA and o, p’-DDE on ACC are still unclear; however, it is believed that they act in tumor inhibition. This inhibitory effect is associated with the ability of mitotane to bind to proteins after metabolic activation, considering that a highly reactive intermediate is produced by the enzymatic reactions that release o,p’-DDA [12,22-26]. Although the relationship between mitotane metabolism and its mechanism of action is not clearly estab- lished, it could explain the inter- and intra-patient variation in the concentrations of mitotane and its metabolites [26-28].
Although mitotane has a chemical structure similar to p,p’-DDT, the different configurations of the Cl elements attached to the aromatic rings (ortho and para’) of mitotane, result in an asymmetric carbon, yielding two enantiomers, (R) and (S), which are marketed in the racemic form. The main metabolites of mitotane are produced via - and ß-oxidation. The product of a-oxidation, o,p’-DDE, has a double bond in that earlier asymmetric carbon, resulting in the loss of chirality. However, B-oxidation gives the o,p’-DDA molecule, without compromising its chirality [24,29-31].
Racemic mixtures are not easily separated; thus, the effects of the individual enantiomers of mitotane have not been determined. More- over, although the ratio of mitotane to its metabolites has been inves- tigated [27-28], little is known about the ratio of enantiomers of mitotane in patients with ACC as well as the o,p’-DDA enantiomer ratio. In fact, it is not known whether o,p’-DDA, originating from mitotane metabolism in patients with ACC, exists as two enantiomers or if only one enantiomer is produced as a result of enzymatic selectivity.
Mitotane and metabolites can be easily analyzed using reversed- phase liquid chromatography (RP-LC) [27-28],however, enantiomeric resolution is obtained only using specific chiral columns. These columns are usually less versatile than RP columns and are frequently insufficient for analyses of complex matrices. Two-dimensional liquid chromatog- raphy (2D-LC) provides significant improvements in separation power and selectivity over those of RP-LC and is an interesting alternative for chiral analysis of complex matrices.
In this study, using the resolving power of the reversed phase in the first dimension in combination with the enantio-selectivity of the chiral phase in the second dimension, we were able to separate the enantio- mers of mitotane and o,p’-DDA from complex matrices. We applied this simple method to the plasma of pediatric patients with ACC, providing a basis for routine monitoring. Furthermore, we present, for the first time, the ratio of the enantiomers from mitotane and its metabolite, o,p’-DDA, in the plasma of children receiving mitotane therapy.
2. Materials and methods
2.1. Patients and ethical considerations
Plasma samples from six pediatric ACC patients with monitored levels of mitotane were analyzed. The patients included four males and two females, with ages ranging from 3 to 7 years. Patients received mitotane therapy for at least 3 months, with the drug managed by the medical staff. Blood samples were collected by peripheral puncture in heparinized tubes, and plasma was separated by centrifugation at 1,500 x g and stored at -20 ℃. Blank plasma samples were obtained from three anonymized healthy volunteers who did not undergo mitotane treatment. All experiments were in compliance with the ethical guide- lines for human studies, and this study was approved by the Center for Ethics and Research of Hospital Pequeno Príncipe under registration number 4.434.382, CAAE:40281420.8.0000.0097.
2.2. Reagents
The following solvents, all high-performance liquid chromatography (HPLC) grade, were used: acetonitrile, methanol, acetone, n-hexane (95%), isopropanol, n-propanol, and n-butanol. Type-1 ultrapure water (Milli-Q Millipore, Merck, Darmstadt, Germany) was used. Standards of
mitotane (racemic), o,p’-DDE, p,p’-DDD, p,p’-DDE, and p,p’-DDA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Racemic o,p’-DDA was purchased from ASCA GmbH (Berlin-Adlerschof, Germany).
2.3. Sample preparation
Quantification of mitotane, o,p’-DDE and o,p’-DDA was performed by single dimension reversed-phase liquid chromatography (RP-HPLC). Standards were prepared as stock solutions in methanol at 1 mg.mL-1. Calibration curves were obtained for low and high concentrations of mitotane and metabolites at 0.1, 1, 5, 10, 15, and 20 µg-mL-1, dissolved in a mixture of methanol/water (1:1, v/v). To calculate the lower limits of detection/quantification, standards were prepared at 100 ng.mL-1. The recovery rate was determined using blank plasma samples spiked with three concentrations of mitotane and its metabolites (1, 15, and 50 ug.mL-1). For internal standards, p,p’-DDE, p,p’-DDA, and p,p’-DDD were evaluated. The spiked blank plasma and patient plasma samples were stirred for 1 min and held at room temperature for 1 h. For RP- chromatography, an aliquot of 400 uL of each sample was transferred to microtubes and 800 uL of acetone was added. The samples were stirred vigorously for 2 min and then centrifuged at 13,000 rpm for 1 h. The soluble layers were transferred to chromatographic tubes and analyzed using RP-HPLC.
For enantiomeric analysis, the o,p’-DDA needed to be previously converted to ester-derivatives (as described below). After establishing the best condition, the plasma samples from patients were treated accordingly.
2.4. Preparation of esters of 2,4’-DDA
For the chiral analysis of o,p’-DDA, a derivatization step was necessary. Considering the presence of a carboxyl group, o,p’-DDA was esterified with different alcohols, such as methyl, isopropyl, n-propyl, and n-butyl alcohols. The acid-alcohol reagents were prepared by bubbling HCl gas, obtained from a mixture of NaCl (200 g) and H2SO4 (200 mL), until no more vapor was formed. The reactions were per- formed with 50 µg of o,p’-DDA dissolved in each acid-alcohol reagent at 80 ℃ for 2 h. After the reaction, the reagents were evaporated under a nitrogen stream, and the products were dissolved in MeOH and analyzed by gas chromatography-mass spectrometry (GC-MS), to confirm the reaction, by reversed-phase HPLC for purity evaluation, and by chiral phase HPLC for enantiomeric separation. The plasma samples, after being analysed by reversed-phase chromatography, were evaporated under nitrogen stream and similarly treated, in order to convert o,p’- DDA to the ester derivative.
2.5. GC-MS analysis
The GC-MS analysis was developed using Shimadzu equipment, model QP-2020, using an RTX-5MS capillary column (30 m × 0.25 mm i. d.) with 0.25 um of film. Helium (5.0) (analytical grade) was used as the carrier gas, set at a linear velocity of 45 cm-sec-1 (1.5 mL.mL-1). The temperature setup was as follows: injector at 280 ℃, interface and ion source at 280 ℃, column oven programmed from 80 ℃ to 280 ℃, with a heating rate of 20 ℃·min-1, and a total run time of 20 min. The analyses were carried out with 1 uL of each sample injected via an AOC-6000 auto-sampler (PAL System), with a split ratio of 1:20. Detection was performed using single-quadrupole MS (m/z 45-500) with electron ionization at 70 eV.
2.6. Liquid chromatography
HPLC was performed using the LC20A Prominence (Shimadzu, Kyoto JP), equipped with a degasser, autosampler, column oven, and fraction collector. Solvent delivery was provided by a quaternary pump, for the single dimensional analysis, or was combined with an isocratic pump
interfaced with two Rheodyne valves, each with six ports/two positions, placed inside the oven, for 2D configuration. Detection was performed using a photodiode array detector (PDA, 200-400 nm). Each dimension was individually optimized as follows.
For reversed-phase separation, the first dimension was carried out in Ascentis® Express columns, C8, 150 x 2.1 mm (L x I.D.) x 3 um (par- ticle size) or C18-PCP, 150 x 4.6 mm (L x I.D.) × 2.7 um (particle size), held constantly at 40 ℃. The mobile phase consisted of ultrapure water containing 0.05% (v/v) formic acid (solvent A) and acetonitrile con- taining 0.02% (v/v) formic acid (solvent B). For C8, the gradient was developed at 0.3 mL·min-1, initiated with 50% B, increasing to 100% in 8 min, held for 12 min, returned to 50% in 13 min, and held for 18 min for system re-equilibration. For C18-PCP, the gradient was developed at 0.5 mL·min-1, initiated at 40% B, increasing to 100% in 15 min, held for 20 min, and returned to the initial condition in 22 min, with 5 min of re- equilibration.
The chiral phase (as a single or the second dimension) was developed in Astec® Dimethylphenyl Cellulose (DMPC), with a 5 um particle size and 25 cm × 4.6 mm (L x I.D.), held at 40 ℃. An isocratic mobile phase composed of methanol-water (85:15 v/v) was employed in the chiral separation at a flow rate of 0.8 mL·min-1. Samples (10 uL) were injected directly from the autosampler to optimize chiral separation as a single- dimension analysis (as per the manufacturer’s recommendation, acetone must be avoided in the Astec® DMPC column).
2.7. Two-dimensional liquid chromatography
Heart-cutting 2D-LC was performed by trapping the peaks of interest in the interfacing Rheodyne valves, equipped with two 50 uL loops. The loops were connected to the two valves (valve 1 and valve 2), one at a time, receiving the solvent from both pumps. The effluent of the RP- column in the first dimension, receiving the solvent from the quater- nary pump, was connected to the entry port of valve 1, which distributes all of the column effluent through the first loop, connected to the waste, from valve 2. The solvent of the second dimension passed through the second loop, delivering it to the second column (chiral), which was connected to the detector.
The valves were programmed to simultaneously change their posi- tions by alternating the solvent in each loop. Therefore, the effluent from first dimension, trapped in the first loop, was directed to the second column and then to the detector. The second loop started receiving the effluent from the first dimension and diverted the effluent to the second column with the next valve transition (Fig. 1). Considering the pre- established retention times of the peaks of interest from the first
dimension, the enantiomeric compounds mitotane and o,p’-DDA (chemically modified to ester derivatives), along with non-chiral o,p’- DDE, could be transferred from the first dimension (reversed-phase) to the second dimension (chiral-phase) in the same chromatographic run.
2.8. Circular dichroism
The enantiomers of R/S-mitotane and o,p’-DDA-n-propyl ester standards were collected, individually, from the DMPC chiral column using the fraction collector. Chromatography was performed to confirm enantiomeric enrichment and by circular dichroism (CD), carried out on a Jasco J-815 spectropolarimeter (JASCO). CD-UV/Vis spectra were obtained from the isolated enantiomers prepared in methanol (500 ug.mL-1), and methanol was used as a blank for background correction. The analysis was performed using a 1 cm quartz cuvette at a wavelength range of 200-400 nm, with a scanning speed of 100 nm/min at 20 ℃. The data interval was set to 0.5 nm and bandwidth was set to 1.00 nm. Three sequential measurements were then performed.
3. Results
3.1. Reversed-phase separation of mitotane and metabolites
The standards of o,p’-DDA, mitotane, and o,p’-DDE were easily separated by RP chromatography. Therefore, the two RP columns were effective. On a C8 column, the retention times were as follows: o,p’-DDA at 7.15 min, mitotane at 11.75 min, and o,p’-DDE at 12.59 min. Using a C18 column, the retention times were as follows: o,p’-DDA at 11.58 min, mitotane at 17.76 min, and o,p’-DDE at 19.38 min. By this method, p,p’- DDA and p,p’-DDE were effectively separated from their isomers; however, p,p’-DDD was not separated from the peak of mitotane and thus was not a suitable internal standard.
For quantification, calibration curves were prepared for low and high concentrations, considering that the plasma concentrations of mitotane are usually lower than the therapeutic range; however, it is not un- common to find higher concentrations. Considering the dilution factor (3 x ) during plasma sample preparation, external calibration curves were over the range of 0.1 to 20 µg.mL-1 for each compound, resulting in an R2 ≥ 0.99.
The recovery rates of mitotane, o,p’-DDA, and o,p’-DDE were eval- uated at three concentrations. Blank plasma samples from three volun- teers were spiked with each standard at final concentrations of 1, 15, and 50 µg.mL-1 for low, medium, and high concentrations, respectively, since most real samples evaluated had concentrations within this range.
Position 1
Loop 1
P1 (Bin)
1D (RP)
2D (CH)
AS
PDA
Waste
P2 (Iso)
Loop 2
Position 2
Loop 1
P1 (Bin)
1D (RP)
2D (CH)
AS
PDA
Waste
P2 (Iso)
Loop 2
At lower concentrations, the recovery rate for each compound was 90% (88.5-92.3%). For the medium concentration (15 µg.mL-1), the recov- ery rates were 95.3% (93.7-97.2%) for o,p’-DDA, 101% (97.8-103%) for mitotane, and 96.6% (93.8-98.7%) for o,p’-DDE. At higher concen- trations (50 µg·mL-1), recovery rates were 100.8% (99.8-101.8%) for o, p’-DDA, 105.3% (104.5-106.6%) for mitotane, and 103% (93.5-109.2%) for o,p’-DDE. Under the analytical conditions used here, the lower limits of detection obtained with standards prepared at 100 ng.mL-1 were an injection volume of 1 µL at 230 nm for mitotane, 1 µL at 245 nm for o,p’-DDE, and 2 uL at 220 nm for o,p’-DDA, with a signal to noise (S/N) ratio of ~ 5:1. The low limits of quantification were 2 uL of mitotane and o,p’-DDE and 4 uL of o,p’-DDA, with S/N ≥ 10.
3.2. Derivatization of o,p’-DDA
The o,p’-DDA standard was subjected to chiral chromatography following the same method used for mitotane; however, in the first analyses, no peak was observed. This could be explained by the strong interaction of the carboxyl group from o,p’-DDA with the carbamate linkage present in the chiral stationary phase. To resolve this issue, formic acid (0.05%) was added to the mobile phase, resulting in the detection of a single peak for racemic o,p’-DDA. Different solvents and additives were tested for these enantiomers separation in the DMPC phase; however, none gave resolved enantiomer peaks.
As an alternative, blocking carboxylic acid as an ester derivative was hypothesized as a way to augment the selectivity of o,p’-DDA on the DMPC phase. For the first modification, MeOH-HCl was used as the reactant, yielding methyl ester derivatives (o,p’-DDA-Me). Nonetheless, o,p’-DDA-Me analyzed by chiral phase HPLC gave two poorly resolved peaks, even in different mobile phases. This represented a great advance when compared to the non-derivatized o,p’-DDA analysis. Therefore, alcohols with longer carbon chains, such as isopropyl, n-propyl, and n- butyl alcohols, were tested.
Esterification reactions were confirmed by GC-MS, which showed a single peak for each derivative. EI-MS gave ions consistent with the expected products at m/z 322 (o,p’-DDA-n-Prop) and m/z 336 (o,p’- DDA-But), with some fragments similar to mitotane [32-33], consistent with the expected derivatives (Fig. 1). The reactions were also evaluated by reversed-phase HPLC to check for the presence of nonvolatile
byproducts or incomplete reactions, since o,p’-DDA is not volatile, it should not appear in GC-MS analysis. For each derivative of o,p’-DDA, a single peak was observed with a higher retention time than that of the native compound owing to the higher nonpolar interaction of the esters on the RP columns.
3.3. Chiral analysis of mitotane and o,p’-DDA as ester derivatives
The absolute enantiomeric configuration was obtained from enriched samples and analyzed by CD. Although the chiral column was not the same, the elution profile of mitotane was the same obtained by Tanaka and coworkers [34], confirmed by CD spectra, with the first peak corresponding to the levorotatory configuration, confirming that it is the (S)-mitotane enantiomer. Previous data for the enantiomers of o,p’-DDA were not found; however, the CD spectra were similar to those of (S)/ (R)-mitotane, indicating that the first peak of o,p’-DDA-nProp was also the (S)-enantiomer (Fig. 2).
Although the monitoring of mitotane and metabolites has been explored as a prognostic or toxicology tool [25-28], few studies have focused on the value of enantiomeric analyses, beyond the mitotane standard. Ali & Aboul-Enein [35] used different polysaccharide-based chiral columns to analyze o,p’-DDT and o,p’-DDD (mitotane) from pesticide sources. They effectively separated o,p’-DDT; however, the columns and methods failed to separate enantiomers of o,p’-DDD, except for one method capable of partially separating these enantiomers, with a resolution factor (Rs) of 0.60. In that work, the stationary phase, tris-3,5-dimethylphenylcarbamate cellulose (DMPC), was ineffective at resolving the enantiomers of mitotane. Herein, using a similar chiral phase but a different mobile phase [i.e., methanol and water at a ratio of 85:15 (v/v)], we obtained good enantiomeric separation, sufficient for the complete separation of mitotane, with a good resolution of Rs = 3.304 (Fig. 3A).
The best results for o,p’-DDA enantiomer separation were achieved with derivatives of n-propyl (i.e. o,p’-DDA-n-Prop), followed by n-butyl (o,p’-DDA-nBut), with resolution factors of 1.177 and 1.128, respec- tively (Fig. 3C, D; Table 1). However, in this chiral analysis, a peak for o, p’-DDE was co-eluted with the peak of (S)-mitotane and, also, with o,p’- DDA-nBut (Fig. 3A,B,D), which could impair quantification (Table 1). For the chiral analysis of mitotane and o,p’-DDA from patient
R
0
R = Methyl - 294
R = Propyl - 322
R = Butyl - 336
CH
CI
CI
R
O
O
165
+
CI
C
L
CH
CH
R = Methyl - 259
R = Propyl - 287
R = Butyl - 301
CI
199
CI
CI
235
125
%
%
%
R = Methyl
R
= Propyl
R = Butyl
100
235
100
235
100-
235
75
165
237
75
75
165
237
237
50
50-
165
50-
199
25-
259
25
199
287
25-
199
207
301
113
139
207
294
281
115 133
207
289
322
115 133
281
336
0
180
0-
181
267
0
181
267
100
150
200
250
300
100
150
200
250
300
100
150
200
250
300
350
₥AU
ALL
A
Mitotane
(S)
250
(R)
20
B
o,p’-DDE
200
15
150
10
100
5
50
0
0
10,0
11,0
12,0
13,0
14,0
15,0
16,0
17,0
18,0
19,0
min
10,0
11,0
12,0
13,0
14,0
15,0
16,0
17,0
18,0
19,0
min
₥AUL
₥AU
20
C
(S) (R)
o,p’-DDA-nProp
30-
D
o,p-DDA-nBut
(S)
(R)
15
20
10
10
5-
0
0-
10,0
11,0
12,0
13,0
14,0
15,0
16,0
17,0
18,0
19,0
min
10,0
11,0
12,0
13,0
14,0
15,0
16,0
17,0
18,0
19,0
min
mAU
50
E
Plasma sample
40
30
20
10
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
min
| Analyte | tR | k1 | k2 | α | Rs |
|---|---|---|---|---|---|
| Mitotane | 16.86/18.67 | 3.586 | 4.398 | 1.226 | 3.304 |
| o,p'-DDA-Ipa | 11.26/11.58 | 2.255 | 2.354 | 1.041 | 0.778 |
| o,p'-DDA-nBut | 15.75/16.28 | 3.552 | 3.706 | 1.043 | 1.128 |
| o,p'-DDA-n-Prop | 13.38/13.89 | 2.866 | 3.014 | 1.051 | 1.177 |
plasma, a modification in sample preparation was necessary because, as recommended by the manufacturer (Sigma-Aldrich), the use of solvents such as acetone must be avoided in Astec® DMPC column, even for sample dissolution. Thus, the samples prepared for direct use in this chiral column were prepared in methanol instead of acetone, with a considerable increase in the matrix effect, compromising the analysis (Fig. 3E).
Despite the effective chiral separation of ester derivatives on DMPC column, the RP C8 column failed to separate the ester derivatives of o,p’- DDA, which co-eluted with mitotane or o,p’-DDE. In this method, o,p’- DDA-nProp overlapped with mitotane, whereas o,p’-DDA-nBut over- lapped with o,p’-DDE (Fig. 4A). Even after several gradient changes, o, p’-DDA-nProp was not well separated from mitotane. For the resolution of these overlapping peaks, different columns/methods were evaluated. C18-PCP was optimal among the tested columns, allowing the separa- tion of all components. Using the C18-PCP method, the non-derivatized o,p’-DDA was eluted at 11.58 min, mitotane at 17.76 min, o,p’-DDA-
nProp at 17.96 min, o,p’-DDA-nBut at 18.94, and o,p’-DDE at 19.38 min (Fig. 4B,C).
3.4. 2D-LC: reversed-phase x chiral phase
The matrix effect in an analysis of patient plasma along with over- lapping peaks (Fig. 3) limited the use of chiral phase chromatography. 2D-LC was then used to improve the selectivity of chromatography. Owing to the robustness of the reversed-phase columns over the DMPC column, two RP phases were chosen for the first dimension because they separate mitotane, o,p’-DDA, and o,p’-DDE with good selectivity, even in the presence of complex samples, with no matrix effect.
Both columns (Ascentis Express C8 and C18-PCP) were employed as the first dimension column. In this 2D method, the effluent from the first dimension was interfaced to second by two parallel Rheodyne valves (six ports x two positions) with simultaneous position switching (Figure 1). When the peaks of interest pass through the loops, the valves switch the effluent from the first dimension to the second. Thus, the peaks from the reversed-phase columns with previously established retention times were injected into the chiral phase, resulting in clean chromatograms without matrix or extractive solvent (acetone) interference.
The two o,p’-DDA derivatives (nProp and nBut) were also tested in the 2D-LC by simply adapting valve switching in accordance with their retention times in reversed-phase chromatography. Using the column C18-PCP, all peaks were separated in the first dimension (Fig. 4B,C) with good resolution. However, when the peaks were transferred to the sec- ond dimension, a loss of resolution was observed (Fig. 5A). Since the enantiomers of mitotane had notable resolution in the chiral phase single-dimension (Rs 3.304), the loss of resolution observed in 2D-LC did not affect separation. However, for o,p’-DDA-nProp and mainly for o,p’- DDA-nBut, this lost resolution in the second dimension could impair the
mAU
500
A
o,p’-DDA
Mitotane + o,p’-DDA-nProp
o,p’-DDE + o,p’-DDA-nBut
250
0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
min
mAU
50
B
Mitotane
25
o,p’-DDA
o,p’-DDA-nProp
o,p’-DDE
0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
min
mAU
40
C
o,p’-DDA
o,p’-DDE
30
Mitotane
o,p’-DDA-nBut
20
10
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
21.0
min
DMPC
DMPC
A
o,p’DDA-nBut
(S)
(R)
20.0
B
20.0
C18-PCP
19.0
C18-PCP
o,p’DDA-nProp (S) (R)
Mitotane
19.0
(R)
(S)
Mitotane
(S)
(R)
18.0
18.0
30.0
32.5
35.0
37.5
40.0
42.5
30.0
32.5
35.0
37.5
40.0
42.5
DMPC
8
DMPC
C
D
o,p’DDA-nBut
(R)+ o,p’-DDE
Mitotane
(S)
C8
o,p’DDA-nProp (S) (S) (R)
13.0
(R)
C8
13.0
12.0
Mitotane
(S)
(R)
12.0
22.5
25.0
27.5
30.0
32.5
35.0
25.0
30.0
35.0
accuracy of analyses (Fig. 5A,B).
On the Ascentis® Express C8 column, the peak of o,p’-DDA-nProp was eluted together with mitotane. However, by 2D-LC, this peak overlap was beneficial, as a single switch of the valves was enough to commute all enantiomers of o,p’-DDA and mitotane to the chiral col- umn, which was able to separate all of the enantiomers without losses in resolution (Fig. 5C). In contrast, with o,p’-DDA-nBut, two valve transi- tions were necessary, resulting in a loss of chromatographic resolution. Moreover, in this method, o,p’-DDA-nBut remained overlapped with o, p’-DDE in both dimensions (Fig. 5D). The loss of resolution observed in the two-dimensional analysis could be attributed to the excess solvent coming from the first column, which is inevitably injected into the chiral phase. Therefore, with only one transition, less solvent from the first column had minor effects on chiral chromatography.
3.5. Patient plasma analysis
The quantification of mitotane and metabolites in patient plasma samples, carried out by RP-HPLC, showed the concentration of o,p’-DDA was higher than those of mitotane and o,p’-DDE; however, the ratios varied. As an exception, in four independent analyses of patient “C,” levels of o,p’-DDA were lower than those of mitotane, at a ratio ranging from 1:1.4 to 1:4.2 (o,p’-DDA/mitotane). In all cases, the plasma con- centration of o,p’-DDE was lower than those of mitotane and o,p’-DDA (Table 2). These findings are in agreement with those of previous in- vestigations showing that the mitotane/metabolite ratio may be a useful prognostic parameter [27-28].
To our knowledge, all five molecules (i.e., (R) and (S)-mitotane, (R)- and (S)-o,p’-DDA, and o,p’-DDE) have not been comprehensively eval- uated or identified as a useful therapeutic parameter. In the patient samples, the ratio of (R)-mitotane was higher than that of (S)-mitotane, ranging from 1.2:1 to 10:1 (Fig. 6, Table 2). As determined, during the method development, the n-propyl esters of the o,p’-DDA enantiomers resulted in the best chiral resolution, therefore, o,p’-DDA-nProp de- rivatives were used for determining the ratio of (S)/(R)-o,p’-DDA in patient samples. Accordingly, we observed for the first time that the enantiomeric ratio varies, with a prevalence of (S)-o,p’-DDA-nProp over (R)-o,p’-DDA-nProp. Moreover, unlike the (S)/(R) mitotane ratio, the observed ratio for (S)/(R)-o,p’-DDA was relatively conserved in all pa- tients over time, with estimates close to 2:1 (i.e., 1.7:1 to 2.6:1). No direct correlation was observed for the enantiomer ratios; however, the sum of (S) enantiomers [i.e. _ (S)-mitotane + (S)-o,p’-DDA] and the sum of (R) enantiomers [≥(R)-mitotane + (R)-o,p’-DDA], with a few excep- tions, did not return to the racemic ratio (Table 2), which could indicate that the enantiomers undergo different rates of metabolism and/or
o,p’-DDA-nProp
Mitotane
(S)
(R)
(R)
(S)
25.0
27.5
30.0
32.5
elimination.
4. Discussion
Mitotane monitoring is essential to control drug levels in the blood of patients, ensuring safety and an improved therapeutic efficacy by avoiding sub- or supra-therapeutic concentrations. Moreover, mitotane metabolites have been identified as important prognostic markers for patients with ACC. Reversed-phase columns are employed extensively for therapeutic drug monitoring owing to their robustness and versa- tility. Here, different RP-columns were tested (C18 and C8 columns), all of which were able to separate mitotane and metabolites; however, o,p’- DDA required acidic mobile phases to augment interactions with the stationary phase and improve peak symmetry. Our analysis revealed important differences in the ratio of mitotane and its metabolites, identifying the importance of o,p’-DDA for toxicology and therapeutic monitoring, since it was found at higher concentrations than those of mitotane in most pediatric patients, in agreement with previous results for adult patients [26-28]. In a particular case (i.e., patient “B”), the ratio of o,p’-DDA to mitotane was up to 45:1, with a low plasma mito- tane concentration (0.9 µg.mL-1). This suggests that the metabolic rate
| Patient | o,p'-DDA Σ mg/ml | %RSD | (S)/(R) ratio | Mitotane Σ μg/ml | %RSD | (R)/(S) ratio | Σ (S) ug/mL | Σ (R) µg/mL | E ratio (S)/(R) | o,p'-DDE µg/mL | %RSD |
|---|---|---|---|---|---|---|---|---|---|---|---|
| A_1 | 10.6 | 1.88 | 2.1/1 | 6.6 | 0.63 | 4.2/1 | 8.5 | 8.7 | 0.98/1 | 1.4 | 0.91 |
| A_2 | 21.9 | 1.41 | 1.9/1 | 13.4 | 1.08 | 2.8/1 | 18.0 | 17.3 | 1.04/1 | 2.7 | 0.64 |
| B 1 | 40.5 | 1.08 | 1.8/1 | 0.9 | 0.82 | 1.2/1 | 26.6 | 14.8 | 1.80/1 | tr | – |
| B_2 | 28.0 | 1.35 | 2.1/1 | 1.4 | 1.35 | 2.2/1 | 19.4 | 10.0 | 1.95/1 | 0.2 | 2.38 |
| B 3 | 9.3 | 1.39 | 2.0/1 | 2.7 | 1.23 | 6.0/1 | 6.6 | 5.4 | 1.22/1 | 0.2 | 2.44 |
| B_4 | 22.7 | 3.69 | 2.0/1 | 6.1 | 1.21 | 5.1/1 | 16.1 | 12.6 | 1.28/1 | 0.9 | 1.31 |
| C_1 | 15.0 | 4.41 | 1.8/1 | 22.0 | 0.89 | 2.5/1 | 16.0 | 21.0 | 0.76/1 | 1.0 | 1.99 |
| C_2 | 12.5 | 1.48 | 1.7/1 | 18.0 | 0.99 | 2.5/1 | 13.0 | 17.5 | 0.74/1 | 1.1 | 1.25 |
| C_3 | 4.6 | 7.80 | 2.0/1 | 10.7 | 1.12 | 3.8/1 | 5.3 | 10.0 | 0.53/1 | 1.1 | 1.36 |
| C_4 | 2.5 | 1.80 | 2.3/1 | 10.4 | 0.48 | 6.2/1 | 2.3 | 9.3 | 0.25/1 | 1.8 | 0.68 |
| D_1 | 1.59 | 2.44 | 2.2/1 | 0.6 | 1.69 | 2.3/1 | 1.3 | 0.9 | 1.43/1 | tr | – |
| D_2 | 36.9 | 1.35 | 2.0/1 | 15.6 | 0.95 | 4.5/1 | 27.3 | 25.1 | 1.09/1 | 2.9 | 0.68 |
| E_1 | 9.2 | 1.22 | 2.2/1 | 3.3 | 0.76 | 3.0/1 | 8.2 | 5.9 | 1.39/1 | tr | – |
| E 2 | 4.4 | 3.69 | 2.1/1 | 6.8 | 0.84 | 9.8/1 | 3.6 | 7.7 | 0.47/1 | 0.5 | 1.14 |
| F 1 | 13.8 | 1.10 | 2.4/1 | 5.8 | 0.92 | 10.0/1 | 11.8 | 10.0 | 1.18/1 | tr | – |
| F_2 | 45.2 | 3.51 | 2.6/1 | 7.0 | 0.77 | 5.2/1 | 33.9 | 18.2 | 1.86/1 | tr | – |
differed from those of other patients, probably leading to an increased elimination rate and consequently requiring higher daily doses of mitotane with an increased risk of toxicity.
A novel 2D-LC method was developed using two different reversed- phase columns in the first dimension, allowing the separation of mito- tane, o,p’-DDA, and o,p’-DDE with good selectivity, even in the presence of complex samples (i.e., blood plasma) with no matrix interference. Nevertheless, in the 2D analysis, the C8 column had a lower internal volume (2.1 mm i.d.) than those of other RP-columns evaluated here. This allowed for a lower flow rate and, consequently, the use of short interface loops (50 uL) because longer loops (e.g., 100 or 200 uL) pro- moted the loss of resolution in the second dimension. However, the C8 column promoted an overlap of peaks from mitotane and o,p’-DDA- nProp, providing an easy way to commute them from the first to second dimensions. The chiral column used here (i.e., DMPC) had a limited range of solvents, flow rates, and back pressures and was therefore inappropriate for complex matrix analyses. To overcome this difficulty, we used a chiral column in the second dimension, taking advantage of the higher separation capacity of the reversed-phase used in the first dimension. Therefore, the selectivity of the chiral column was improved as a second dimension because the matrix effect was reduced substan- tially and was retained in the first-dimension column. Thus, the (R)-and (S)-mitotane showed good separation rates.
Mitotane is used in patients with ACC in the racemic form, thus equal amounts of (R)- and (S)-mitotane are administered. Nevertheless, our analyses demonstrated that enantiomer ratios vary. In particular, (R)- mitotane was the major enantiomer present in the plasma, while (S)-o, p’-DDA was the most abundant enantiomer in the plasma, suggesting that (S)-mitotane is converted to (S)-o,p’-DDA at a higher rate than the conversion of (R)-mitotane to (R)-o,p’-DDA. This indicates that enan- tioselective metabolism results in the differential accumulation/elimi- nation of enantiomers, as reported by Shen et al., [36] who observed different elimination ratios in human placentas exposed to enantiomeric pollutants, including o,p’-DDD and o,p’-DDT.
Further investigations should confirm the therapeutic value of this enantioselective metabolism. Whereas the (S)/(R)-o,p’-DDA ratio was consistently near 2:1, the ratio of (R)/(S)-mitotane was much more variable [ranging from 1.2:1 to 10:1], and this could be responsible for the interpatient differences in the plasma concentration of mitotane. Cantillana et al. [37] also observed variation in the ratio of (R)/(S)- mitotane in minipigs administered a single dose of racemic mitotane (30 mg·kg-1). Analyses of plasma and adipose tissue samples showed vari- ation in distribution profiles, with the predominance of (S)-mitotane in two minipigs and (R)-mitotane in three minipigs. In our analysis, how- ever, the variation in the ratio of (R)/(S)-mitotane did not influence the dominance of the (R)-enantiomer, which was the main enantiomer in all samples.
We observed a general predominance of (S)-enantiomers [that is, (S)- mitotane + (S)-o,p’-DDA] over (R)-enantiomers, with one exception (patient “C”), characterized by the dominance of (R)-enantiomers and the metabolite o,p’-DDA at lower concentrations than mitotane, in all the four independent plasma samples. In another patient (“E”), based on two independent plasma samples, the first ES/R ratio was 1.39:1 and o, p’-DDA was more prevalent than mitotane (2.78:1). Interestingly, at the second dosage, the ES/R ratio was reversed (0.47:1) and the concen- tration of o,p’-DDA was lower than that of mitotane (0.65:1). Therefore, there may be a correlation between the metabolic synthesis of o,p’-DDA and stereoselective metabolism, as lower amounts of (S)-enantiomers were accompanied by lower amounts of o,p’-DDA; this remains to be better investigated.
Mitotane metabolism involves a-hydroxylation, which yields the end product o,p’-DDE, and ß-hydroxylation, which yields the end product o, p’-DDA [29]. Metabolization has been described in dogs; it occurs mainly in the mitochondria of adrenal cells via CYPs involved in ste- roidogenesis, and results in adrenolytic effects by covalent binding to macromolecules [22]. If adrenal glands are important mitotane-
metabolizing sites in humans, as they are in dogs, the tumor mass and physiology may influence the metabolism rate, which may, in part, explain the interpatient differences. Mitotane is known to inhibit rate- limiting enzymes in steroidogenesis, such as CYP11A1, CYP21A2, CYP17A1, hydroxy-8-5-steroid dehydrogenase 3 ß- and steroid 8-isom- erase type 2 and type 1 [38-39]. These enzymes are responsible for the secretion of aldosterone, cortisol, dehydroepiandrosterone, testos- terone, and estradiol by adrenal cells. Thus, the inhibition of these en- zymes reduces hormone secretion and ACC symptoms. Moreover, in vitro investigations, with adrenal cell NCI-H295, have demonstrated that mitotane also targeted the adrenal enzyme Sterol-O-Acyl Transferase 1 (SOAT1), whose inhibition avoids the esterification of cholesterol, leading to a toxic lipid accumulation, with a blocking effect on the cholesterol synthesis. Since cholesterol is the precursor for many steroid hormones, the impaired synthesis and the depletion of cholesteryl esters contribute to preventing steroidogenesis, resulting in the adrenolytic effect [40].
Interestingly, the effects of (S)-mitotane on dehydroepiandrosterone and cortisol hormone levels are slightly weaker than those of (R)- mitotane or the racemic mixture [41], suggesting different rates of metabolism via adrenal CYPs in the mitotane enantiomers. Chiral drugs can undergo different pharmacokinetic and pharmacodynamic effects owing to the presence of enantiomers [42]. In addition, the enantiomers of mitotane and the main metabolite o,p’-DDA, along with the non- chiral o,p’-DDE, could interact with drugs metabolized by CYP en- zymes, since CYPs (e.g., CYP3A4, CYP2B, CYP17A1, CYP11A1, and CYP19A1) are up- or downregulated in the presence of these compounds [43-45]. This affects pharmacokinetic and drug-drug interactions and could interfere with mitotane enantiomers and metabolite ratios, as observed in the plasma of patients undergoing mitotane treatment.
5. Conclusions
Therapeutic drug monitoring is essential to ensure patient safety and improve outcomes. Here, we developed a two-dimensional liquid chromatography method with an expanded analytical capacity for mitotane and its metabolites. Applying this method, we found that mitotane undergoes chiral-oriented metabolism, which may have an important impact on patients with ACC. Thus, monitoring the enantio- mers of mitotane and o,p’-DDA, its main metabolite, is now possible, and further investigations will demonstrate the pharmacological properties and relationship between this chiral metabolism and therapeutic out- comes in ACC.
CRediT authorship contribution statement
Gabriela Stadler: Methodology, Validation, Formal analysis, Investigation, Writing - original draft. Alan de Almeida Veiga: Meth- odology, Validation, Formal analysis, Investigation, Writing - original draft. Claudia Rita Corso: Writing - original draft, Methodology, Investigation. Camila Bach de Assis: Methodology, Investigation. Beatriz de Toledo Nogueira: Methodology, Investigation. Lucia Regina Rocha Martins: Methodology, Investigation. Beatriz Cruz Bonk: Methodology, Investigation. Flávia Lada Degaut Pontes: Methodology, Investigation. Bonald Cavalcante de Figueiredo: Writing - original draft, Supervision. Lauro Mera de Souza: Concep- tualization, Methodology, Formal analysis, Resources, Supervision, Writing - review & editing, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data availability
No data was used for the research described in the article.
Acknowledgments
This study was supported by the Instituto de Pesquisa Pelé Pequeno Príncipe (Associação Hospitalar de Proteção à Infância Dr. Raul Car- neiro). We thanks to the Brazilian Agencies Coordenação de Aperfei- çoamento de Pessoal de Nível Superior (CAPES) (Financial code 001, Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (Processo 310169/2018-6, Brazil) and Fundação Araucária - PPSUS, for the support.
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