Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma
Matthias Kroiss ** , Dietmar Plonnét, Sabine Kendl*, Diana Schirmer$, Cristina L. Ronchi™, Andreas Schirbel ** , Martina Zink*, Constantin Lapa ** , Hartwig Klinkers, Martin Fassnacht ** 1, Werner Heinz® and Silviu Sbiera™
*Endocrine and Diabetes Unit, Dept. of Internal Medicine I, University Hospital Würzburg, Würzburg, Germany
$ Infectiology Unit, Dept. of Internal Medicine II, University Hospital Würzburg, Germany
* Comprehensive Cancer Center Mainfranken, University of Würzburg, Germany
# Medical Care Centre of Human Genetics Ulm, Division of Laboratory Medicine, Ulm, Germany
** Department of Nuclear Medicine, University Hospital Würzburg, Würzburg, Germany
1 Clinical Chemistry and Laboratory Medicine, University Hospital Würzburg, Germany
Running title: Lipoprotein binding of mitotane
Key words: adrenal cancer, adrenocortical carcinoma, pharmacokinetics, drug metabolism, therapeutic drug monitoring
Word count (excluding abstract, tables and references): 4092
Correspondence:
Dr. Matthias Kroiss
Department of Internal Medicine I
University Hospital Würzburg
21 22 23 24 25 26 Oberdürrbacher Str. 6 97080 Würzburg, Germany;
27 28 Tel: +49-931-201-39740
Fax: +49-931-201-6039740
E-mail: Kroiss_M@ukw.de
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33 Abstract
34 35 Objective: Oral mitotane (o,p’-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). Serum mitotane concentrations >14mg/l are aimed for improved efficacy 36 but not timely achieved in about half of patients. Here we aimed at a better understanding of 37 intestinal absorption and lipoprotein association of mitotane and metabolites o,p’-DDA and 38 o,p’-DDE.
39 Design: Lipoproteins were isolated by ultracentrifugation from chyle of a 29-years old patient 40 41 and serum from additional 14 ACC patients treated with mitotane. High-performance liquid chromatography was applied for quantification of mitotane and metabolites. We assessed 42 43 NCI-H295 cell viability, cortisol production and expression of endoplasmic reticulum stress (ER-stress) marker genes to study the functional consequences of mitotane binding to lipoproteins.
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Results: Chyle of the index patient contained 197mg/ml mitotane, 53mg/ml o,p’-DDA and 51mg/l o,p’-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3) and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction. Mitotane
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content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p’- DDE was similarly distributed but 87.9±4.2% of o,p’-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti- 52 proliferative and anti-hormonal effects on NCI-H295 cells and reduced ER-stress marker gene 53 expression.
Conclusion: Mitotane absorption involves chylomicron binding. High concentrations of o,p’-
54 55 DDA and o,p’-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority 56 of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane 57 suggesting that lipoprotein-free mitotane is the therapeutically active fraction.
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Introduction
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Adrenocortical carcinoma (ACC) is a rare tumor with dismal prognosis (see 1,2 for recent reviews). Mitotane (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichlorethane, o,p’-DDD) has 62 been clinically used for treatment of ACC for decades3 either as monotherapy or in 5. 63 combination with cytotoxic chemotherapies4,5. Only recently our laboratory identified sterol- 64 O-acyl-transferase 1 as a key target of mitotane. We have shown that SOAT1-inhibition by 65 mitotane leads to intracellular accumulation of toxic lipids which results in activation of the endoplasmic reticulum stress response which results in reduced steroidogenesis and apoptosis6,7 of adrenocortical carcinoma cells.
66 67 68 Mitotane treatment is hampered by the requirement of therapeutic drug monitoring and
69 unfavorable pharmacokinetic properties8, 9
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Several studies reported an association of mitotane serum concentrations above 14mg/l with treatment response10-14. Since serum concentrations >20mg/l are associated with more frequent and more severe adverse effects15, serum concentrations between 14 and 20mg/l are aimed at16, 17. However, even with high doses of mitotane for three months, concentrations >14mg/l are achieved in only half of patients18, 19 and are never reached in some patients. The
72 73 74 75 reasons for this are unknown.
76 The long time interval required for reaching effective mitotane serum concentrations is 77 directly related to the excessively long elimination half-live of mitotane which approaches up to 6 months20. Alkyl oxidation of mitotane leads to formation of the inactive metabolite21 78
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o,p’-dichlorodiphenylacetic acid (o,p’-DDA)21, which is present in blood in up to ten fold higher concentrations compared to the parent compound, and is excreted in urine22. In
81 contrast, the second metabolite o,p’-dichlorodiphenyl-dichloroethene (o,p’-DDE) is much less 82 abundant23.
Mitotane has been described earlier to be associated with lipoproteins in serum24,25 and it has
83 84 been suggested that mitotane binding to lipoprotein particles may contribute to the apparent
85 tissue specificity for the adrenal cortex. Since adrenocortical cells require large amounts of cholesterol for steroidogenesis, it appears conceivable that lipoprotein binding might favor entry of mitotane into adrenocortical cells similar to a Trojan horse. Recently, lipoprotein association of mitotane has been studied in more detail26 and lipoprotein-free mitotane reported to confer cytotoxicity in cell culture disproving this hypothesis. It is also known that mitotane treatment increases serum LDL-cholesterol which has been attributed to increased hepatic cholesterol synthesis27, 28
92 Here we report a translational study starting with the analysis of mitotane in chyle of a 29- year old mitotane-treated ACC patient to gain insights into mitotane absorption in the intestine. Subsequently we challenged data published previously by analyzing association of mitotane and metabolites with serum lipoproteins26 in 14 additional patients. Cell culture assays were used to study the functional consequences of the association of mitotane with chylomicrons and serum lipoproteins.
Subjects and methods
Chemicals
Mitotane for in vitro treatment was from ISP Chemical Products (Columbus, OH, USA), o,p’- DDD, o,p’-DDE and p,p’-DDE for HPLC were from Sigma (PESTANAL, Taufkirchen, Germany), all buffers and solvents from Merck (Darmstadt, Germany) unless otherwise stated.
Iodixanol was supplied as 60% w/v solution (OptiPrep™M) by Axis-Shield PLC (Dundee, UK). OptiSeal™M tubes (3.2ml) were supplied by Beckman Coulter (Krefeld, Germany).
Triacylglycerol, Cholesterol, LDL-Cholesterol, HDL-Cholesterol assays, calibrators and serum lipid control level I and level II were purchased from Roche (Mannheim, Germany). All lipid measurements were performed on the Cobas C501 from Roche.
Synthesis of DDA
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o,p’-DDA was synthesized as described in Supplementary Data.
112 HPLC
Stock solutions of o,p’-DDD (250ug/ml), o,p’-DDA (500µg/ml), o,p’-DDE (50µg/ml) and p,p’-DDE (250µg/ml, internal standard), respectively were prepared in acetonitrile (AcN). Working solutions of o,p’-DDD (25µg/ml), o,p’-DDA (50µg/ml) and o,p’-DDE (5µg/ml) were prepared in can and stored at -20°℃. Seven different calibration samples were prepared by accurately spiking 250ul of 5% BSA (in H2O) with stock and working solutions to realize calibration ranges of o,p’-DDD 0.5-40µg/ml, o,p’-DDA 1.25- 100µg/ml and of o,p’-DDE 0.25-20 µg/ml. A volume of 25 ul p,p’-DDE was added to 250ul aliquots of calibration samples or serum and iodixanol gradient fractions, mixed with 250ul of 0,1N sodium acetate pH 4.6. 5 ul of ascites were used to minimize matrix interferences. Analyte extraction was performed by adding twice 3ml each of tert-butylmethylether for 5 min, followed by centrifugation at 5,000xg for 5min. The organic layer was transferred into glass tubes and evaporated to dryness (40℃) under a gentle stream of nitrogen. The residue was reconstituted with 125ul of AcN and diluted with 125ul H2O-0.1% trifluoroacetic acid (TFA) adjusted to pH 3.0 with Triethylamine (TEA). A volume of 10ul of this solution was injected into the HPLC-system.
Quantitative analysis was performed on a Hitachi LaChrom Ultra HPLC system (VWR, Darmstadt, Germany) equipped with a L-2160 solvent pump, a L-2455U UV-VIS photodiode array detector, a L-2200 autosampler and a L-2350 column oven. Chromatographic separation of analytes was carried out on ASCENTIS Express C18 column (100 x 2.1mm/2.7um; SUPELCO) protected by a C18 guard column (4×2mm ID; Phenomenex, Aschaffenburg, Germany) using a linear gradient A/B 50%-0-5min-90%B-+90%B/3.5min-+50%B in 0.5min→6.5min Re-equilibration. The mobile phase A consisted 0.1% trifluoroacetic acid (TFA) adjusted to pH 3.0 with Triethylamine (TEA) and acetonitrile (70:30, v/v solvent A;
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10:90, v/v solvent B). The flow rate was 0.2ml/min, the column was maintained at 40℃.
136 137 Detection was performed at 230nm.
138 Retention times for o,p’-DDD, o,p’-DDA, o,p’-DDE and p,p`-DDE (IS) were 8.5, 4.2, 9.9 and 10.8 min, respectively (Fig. S1).
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Patients
A total of 15 ACC patients treated with mitotane including the index case (10 with palliative 143 intention and 5 as adjuvant treatment) were included in this study. The study was approved by the ethics committee of the University of Würzburg (No. 93/02 and 88/11) and written informed consent was obtained from all patients. Anonymized serum samples from routine laboratory analyses of healthy individuals without mitotane treatment were used to prepare lipoprotein pools not containing mitotane.
Density Gradient Ultracentrifugation of Lipoproteins
Serum samples of the 14 ACC patients were collected during mitotane treatment and stored at 4°℃ until density gradient ultracentrifugation.
The procedure for the separation of lipoprotein subclasses using iodixanol originally described by Graham et al.29,30 was adapted as follows. 1.0ml of serum, 1.24ml of HBS buffer (10 mM HEPES; 0.8% NaCl; pH7.4) and 0.54ml of OptiPrep™M (60% iodixanol) were transferred into a Beckman (Krefeld, Germany) OptiSeal™M tube (3.2ml) and thoroughly mixed by gentle, repeated over-head rotation. Finally, the mixture was carefully overlayed with distilled water to fill the tube. The tubes were housed in a Beckman TLN100 rotor and centrifuged at 100,000rpm (350,000xg) for 3:00h at 15℃ with acceleration program 9 and
deceleration program 9 in a Beckman Optima-E-max benchtop ultracentrifuge.
Gradients were collected using a Beckman gradient unloader, which pierces the tube bottom, a Watson-Marlow 520S/R precision peristaltic pump and a Gilson (Villiers, France) fraction
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162 collector. 20 fractions each 0.16ml were collected by continuously pumping the gradient from 163 164 165 166 167 168 the bottom of the tube to the fraction collector numbering the fractions from top (1) to bottom (20). The fractions of the gradient were pooled to obtain the following lipoprotein fractions: VLDL (fractions 1-2); IDL (fractions 3-4); LDL1 (fractions 5-8); LDL2 (fractions 9-11); LDL3 (fractions 12-13); HDL (fractions 14-17); lipoprotein-depleted serum (fractions 18-20). In each of the lipoprotein fractions triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol were measured on the Cobas c501 (Roche, Basel, Switzerland) with the reagent 169 kits from Roche. To isolate chylomicrons, ascites was mixed with HBS and centrifuged for 10 min at 100,000xg.
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As controls, 14 anonymous samples from individuals without mitotane treatment were fractionated in the same manner and pooled as described.
Cell culture
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The adherent variant of the ACC cell line NCI-H295 (NCI-H295R) was cultured as described31.
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Cell-based assays
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Viability testing using WST1 reagent was performed according to manufacturer’s protocol (Roche). To assess the efficacy of mitotane in chyle, NCI-H295 cells have been incubated with medium containing increasing concentrations of mitotane dissolved in ethanol or the equivalent quantity of mitotane from chyle from the index patient. Quantification of mitotane in chyle was performed by HPLC as described above. To assess the impact of lipoproteins on mitotane efficacy, equal quantities of lipids (cholesterol+triglycerides) from the VLDL, LDL2, and HDL2 fractions of pooled control serum samples were added to serum-free cell culture medium and either diluent, 25uM or 50uM mitotane was added. The viability of the control cells cultivated in serum-free and medium containing fetal calf serum was very similar
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(100±8.3 vs 91.34±4.4%). Cortisol was measured in the supernatant with an Immulite2000 analyzer (Siemens Healthcare Diagnostics). Quantitative real-time PCR was performed as described7.
Statistics
Patients were divided into high and low lipid groups according to the median of their total lipid (cholesterol+triglyceride) serum concentration. Statistical analyses were made using 192 Prism 6.0 (GraphPad, La Jolla, CA, USA). Statistical differences between groups were calculated using either Fisher’s t-test or ANOVA non-parametric test (Kruskal-Wallis) depending on the number of data sets. Correlation analyses were carried out using the
193 194 195 nonparametric Spearman r correlation test. All error bars show standard deviation (SD). Box 196 plots indicate mean values and quartiles and the whiskers represent the minimal and maximal values.
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Results
Case description
A 29-year old female ACC patient was admitted to the hospital for disease staging after 202 chemotherapy with gemcitabine, capecitabine and mitotane (Table 1, index case)32. The patient complained about abdominal discomfort and abdominal swelling. An 18FDG-PET-CT revealed abundant ascites (Fig. 1A-B). Paracentesis was performed and 4.1 l of opaque ascites removed. Cytology showed abundant mast cells and lymphocytes without detection of tumor cells. Chemistry demonstrated 48550ug/ml triglycerides consistent with the clinical diagnosis of chylous ascites.
In the absence of detectable peritoneal carcinosis and gross liver metastases, we suspected misplaced pedicle screw implants to be causative of chylous ascites keeping in mind the close proximity of Th11 screws to central abdominal lymph vessels. Vertebra L1 had been replaced and spondylodesis of Th11-L3 performed due to ACC metastasis to L1 (Fig. 1A-D). The chylomicron fraction isolated by ultracentrifugation contained 179840µg/ml triglycerides and 54400ug/ml cholesterol (Fig. 1E).
Mitotane was discontinued and the patient placed on a fat-reduced diet with medium-chain fatty acid supplementation. This resulted in nearly complete remission of ascites until the next tumor evaluation three months thereafter.
Mitotane and its metabolites are abundant in chylomicrons
We employed high performance liquid chromatography to quantify mitotane and its metabolites o,p’-DDA and o,p’-DDE in chyle. Strikingly, we detected 197mg/l mitotane, 53mg/l o,p’-DDA and 51mg/l o,p’-DDE in the ascites specimen (Fig. 1F). At the time of
217 218 219 220 221 paracentesis the serum level of mitotane in this patient was 5.5 mg/l.
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222 Mitotane bound to chylomicrons has reduced activity in NCI-H295 cells
To determine the biological activity of chylomicron-associated mitotane, we made use of the NCI-H295 ACC cell line. Equal amounts of chylomicron-bound mitotane from the index patient and mitotane diluted in ethanol were added to cell culture medium and cell viability was assessed by WST-1 assay. We found the EC50 after 24h to be 15.4uM for free mitotane and >100uM for chylomicron bound mitotane (Fig. 1G). Accordingly, free mitotane but not chylomicron associated mitotane led to inhibition of cortisol synthesis by more than 4 fold (from 1.45 to 0.35-fold compared to baseline).
Endoplasmic reticulum (ER) stress, the underlying molecular effector mechanism of both impaired steroidogenesis and apoptosis, was strongly activated by free mitotane. ER-stress marker C/EBP homologous protein (CHOP) mRNA was induced 19.2±3.2-fold, 23.0±1.3- fold and 14.8±1.9-fold at 25uM, 50uM and 100uM, respectively. At variance, chylomicron associated mitotane did not induce any changes of CHOP mRNA expression (Fig. 1J). Similarly, activation of XBP1-mRNA splicing indicative of ER-stress was observed with free mitotane (4.8±0.1-fold, 9.3±0.1-fold and 61.5±8.7-fold with 25, 50 and 100uM mitotane, respectively) but not with mitotane bound to chylomicrons (p<0.001).
The majority of circulating mitotane is bound to lipoproteins
We next investigated the distribution of mitotane and its metabolites in serum lipoproteins in 14 ACC patients treated with mitotane (see Table 1 for patient characteristics) by subjecting serum samples to LipoDens® density gradient ultracentrifugation. Patients were classified as high (>12.3mg/l) vs. low mitotane (<12.3mg/l) based on the median serum value and no significant difference in total triglycerides (1180±663µg/ml vs. 1560±1094ug/ml, p=0.5, Fig. 2A) and total cholesterol (2571±650µg/ml vs. 2731±816ug/ml, p=0.9, Fig. 2B) was observed.
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21.7±21.4% of total mitotane (Fig. 2C) was found in the VLDL fraction, 1.9±0.8% in IDL, and 8.9±5.5% in LDL1, 18.9±9.6% in LDL2, 10.1±4.0% in LDL3and 26.3±13.0% in HDL2 lipoprotein fractions. Only 12.3±5.5% were present in the lipoprotein-depleted fraction. In contrast o,p’-DDA (Fig. 2D) was mostly present in the HDL2 fraction (21.9±6.9%) and in the lipoprotein-depleted fraction (68.1±8.8%). o,p’-DDE (Fig. 2E) distribution was similar to mitotane (VLDL: 16.4±14.3%, IDL: 2.1±1.6%, LDL1: 7.9±4.2%, LDL2: 14.2±5.5%, LDL3: 9.3±2.9%, HDL2: 21.2±8.6%; lipoprotein depleted: 28.7±6.7%). We next determined concentrations of mitotane and lipids in individual lipoprotein classes and examined the impact of lipid composition on mitotane binding to individual lipoproteins. Mitotane content of lipoproteins showed a positive correlation with both cholesterol (r=0.77, 95%CI of regression curve=0.67-0.84, Fig. 2F) and triglyceride content (r=0.59, 95%CI=0.45-0.71, Fig. 2G). Best correlation was obtained for the sum value of triglycerides and cholesterol (r=0.83, 95%CI=0.75-0.88, Fig. 2H). Accordingly, relative abundance of mitotane in cholesterol and triglyceride rich VLDL particles was higher in the group of patients with high total cholesterol and triglyceride serum concentrations (32.0±25.0% vs. 11.4±11.1, p<0.05, Fig. 2 C, low lipid group: blue symbols; high lipid group: red symbols).
A moderate correlation was found for total mitotane and lipoprotein free mitotane (Fig. 2I) (r=0.68, 95%CI=0.22-0.89). Total serum lipids inversely correlated with lipoprotein free mitotane (r= - 0.33, 95%CI =- 0.74-0.26, Fig. 2J).
Mitotane efficacy can be reduced by co-incubation with lipoproteins
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To examine the impact of lipoproteins on mitotane efficacy, NCI-H295 cells were exposed to 25uM and 50uM mitotane and increasing quantities of lipids from isolated mitotane free human lipoproteins (Fig. 3). As expected, mitotane treatment in diluent significantly reduced viability of NCI-H295 cells after 6h treatment to 60.2+4.3% and 47.0±6.6% for 25uM and 50uM, respectively (Fig. 3A). In contrast, addition of VLDL particles rescued cell viability to
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91.0±5.2% and 86.8±6.8% for 25uM and 50uM of baseline (p<0.05 each). Similarly, addition of LDL2 particles rescued cell viability to 89.5±7.9% and 82.9±6.5% for 25uM and 50uM of baseline (p<0.05 each). Of note, HDL2 alone increased cell viability compared to other lipoproteins and cell culture medium to 136.5±3.3% (compared to solvent control, p<0.05), but addition of mitotane suppressed cell viability even more strongly than co-incubation with VLDL and LDL2 particles. Similarly, mitotane alone but not mitotane co-incubated with lipoproteins inhibited cortisol secretion in the supernatant (viability 25uM mitotane: 7.0±0.3% and 50uM mitotane 8.9±0.7% vs an average viability of 68.9±5.5% and 40.6±4.4%, respectively, p<0.001, after lipoprotein addition)(Fig. 3B). Cortisol production in the presence of lipoproteins was generally higher as the lipoproteins represent an external source of substrate cholesterol for steroidogenesis. The role of HDL, LDL and VLDL in promoting adrenal steroidogenesis has been quite well established33-36
We next studied the impact of added lipoproteins on mitotane-induced ER-stress gene expression and found CHOP expression after co-incubation with mitotane to be reduced from 12.2±0.2-fold (25uM mitotane) to 4.9±0.5-fold (VLDL added), 2.3±0.4-fold (LDL2 added), and 2.3+0.1-fold (HDL2 added). XBP1 mRNA splicing was completely blunted after co- treatment with lipoproteins (25uM mitotane: 8.2±1.2-fold, VLDL added: 0.4±0.02-fold, LDL2 added: 0.4±0.04-fold, HDL2 added: 1.1±0.04-fold).
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Discussion
Mitotane is routinely administered orally for adjuvant and palliative treatment of ACC. The physicochemical properties of mitotane are characterized by a high degree of lipophilicity. It is current clinical practice to advise patients to take oral mitotane together with fat containing food such as whole-milk yoghurt in order to improve resorption and increase the likelihood of achieving therapeutic drug concentrations. In this study, we exploited the availability of chyle in an ACC patient treated with mitotane to study association of mitotane and its metabolites with lipoproteins.
To unambiguously prove that the ascites removed from the index patient contained chyle, we performed ultracentrifugation to isolate chylomicrons. The high content of triglycerides and cholesterol in the flotating fraction after ultracentrifugation confirmed the diagnosis of chylous ascites. The precise cause of ascites could not decisively be clarified. However it is likely that the physical activity of the patient (horse riding) and the known misplacement of pedicle screws in the Th11 vertebra led to injury of large abdominal lymph vessels. During follow-up in this patient, near complete remission was achieved by conservative treatment.
We found chyle to contain large amounts of mitotane indicating that mitotane absorption from the intestinal lumen largely occurs as a by-load of nutrient lipids and more specifically long- chain triglycerides. We were surprised to also detect in the chyle significant amounts of both o,p’-DDA and o,p’-DDE, the two best described mitotane metabolites. o,p’-DDA concentrations are known to be up to ten-fold higher than the parent substance in serum whereas o,p’-DDE is less abundant13. Rat liver microsomes have been shown to be capable of mitotane transformation to the inactive metabolite o,p’-DDA37 21. Circumstantial evidence of mitotane transformation in the liver of humans is suggested by its strong inducing effect on hepatic cytochrome P450 enzymes38-41. To our knowledge, intestinal metabolization of mitotane has not yet been described and the origin of o,p’-DDE in serum is not precisely
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314 315 316 317 known. Therefore, our finding of o,p’-DDA and o,p’-DDE in chyle suggests that mitotane biotransformation may occur already during resorption in the intestine. The mechanisms underlying the differences in relative abundance of o,p’-DDA and o,p’-DDE in serum vs. chyle can not be clarified from this data. On the one hand, beyond intestinal mitotane 318 metabolism, hepatic and potentially extra-hepatic metabolism of mitotane may contribute to 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 the overall high abundance of o,p’-DDA, since the amount of o,p’-DDA recovered from the chyle was only around a quarter of that of mitotane and not ten fold higher as most often found in the serum of mitotane treated patients. On the other hand, o,p’-DDE levels were similar to those of o,p’-DDA in chyle while in patients’ serum it is 10 times less then that of mitotane. This suggests that mitotane metabolization in the gut may be directed more towards production of o,p’-DDE. On the other hand, o,p’-DDE might be further metabolized in organs such as the liver, resulting in only trace amounts present in the serum. As a third alternative, the relatively small proportion of circulating mitotane vs. o,p’-DDA may be attributable in part to diffusion into lipid-rich tissues. We cannot exclude at this point that o,p’-DDA and o,p’-DDE undergo an enterohepatic circulation and hence chyle would be enriched in o,p’- DDA resulting from hepatic transformation. However, no significant quantities of o,p’-DDA have been reported in feces22, 42. Conversely, intestinal microsomes are highly active in xenobiotic transformation and CYP3A-mediated metabolism is prevalent in intestinal mucosa43, 44. Therefore, intestinal exposure to mitotane may lead to enzyme induction and metabolic transformation already during absorption. Inhibition of intestinal (and hepatic) CYP3A enzymes may therefore shorten the time interval to reach therapeutic serum levels of mitotane45. There are already inhibitors of CYP3A4 used in the clinic like ritonavir or cobicistat, which are pharmacokinetic enhancers of protease inhibitors46,47. Our observation is limited by the availability of only one suitable case. Therefore, animal studies - preferably in non-rodents48 - are required to confirm our finding.
Mitotane association with lipoproteins was reported earlier24 and mitotane-induced increase of serum lipids is frequent49.
In our study, serum lipids were similar between patients with high and low mitotane concentrations precluding selection bias. We found the vast majority of mitotane to be bound to lipoproteins with only 12.3±5.5% lipoprotein-free mitotane. Distribution of mitotane between serum lipoproteins was variable and depended on the lipid status of the patient. A narrow correlation was observed between lipoprotein associated mitotane and total cholesterol+triglyceride concentration in the VLDL fraction. A similar distribution was seen for o,p’-DDE. In contrast, o,p’-DDA was preferably detected in HDL2 and lipoprotein- depleted fraction. Hence, lipoprotein binding most likely is a result of the different lipophilicity of the parent compound and metabolites rather than specific binding to lipoprotein constituents (so mitotane and o,p’-DDE might just be having a higher affinity for lipids while o,p’-DDA is not lipophilic).
This is in good agreement with data by Gebhardt et al24 but not completely identical to the very recent data by Hescot et al26. In the latter paper, serum lipids were not measured and therefore it could be that relatively low overall lipid and especially triglyceride levels in the study population may have led to relative high lipoprotein-free mitotane levels. Our patient samples cover a range of physiological and elevated lipid concentrations potentially explaining this difference. Moreover care was applied in our study to avoid freeze-thaw cycles which are known to potentially impair lipoprotein integrity. It is unclear whether this was the case in the work by Hescot et al. Of note, we found free mitotane to correlate with total serum mitotane and to inversely correlate with total cholesterol+triglyceride concentrations suggesting that lipoproteins constitute a circulating pool of mitotane in the serum. From a pharmacological perspective, our combined clinical data can be interpreted in several ways: One possibility is that mitotane bound to chylomicrons or their remnants enters
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hepatocytes and is incorporated in triglyceride rich VLDL and cholesterol-rich LDL and HDL particles. Alternatively, mitotane may be exchanged between lipoproteins passively which is likely given the close correlation of mitotane and lipid content of lipoproteins. We cannot exclude however that shuttling proteins contribute to exchange of mitotane and its metabolites between the different lipoproteins.
Human adrenocortical cells mainly rely on LDL cholesterol for steroid synthesis but HDL- cholesterol is a relevant source of cholesterol as well50. Therefore, it is tempting to speculate that mitotane enters adrenocortical cells using HDL and/or LDL particles as a Trojan horse. However our in vitro data employing the NCI-H295 model cell line contradict this hypothesis. Chylomicron associated mitotane failed to impair viability and reduce steroidogenesis even at high concentrations. Given the correlation of mitotane and combined cholesterol and triglyceride concentration, we decided to add lipoproteins to defined concentrations of mitotane and assess the consequences at gene expression level and in terms of viability and steroidogenesis. By normalizing for lipid content, we found that VLDL and LDL particles alleviated the effect of mitotane on cell viability. HDL particles alone increased viability. HDL particles are known to be taken up by adrenocortical cells through the scavenger receptor B150 and promote cell growth and proliferation by activation of the PI3 kinase/Akt pathway51. However, a proportional decrease of viability was seen when mitotane was added. The notion of blunted response to mitotane in the presence of lipoproteins was even more clear when steroid hormone production and transcriptional up-regulation of ER-stress markers CHOP and XBP1 mRNA splicing, two early steps during mitotane action, were assessed7. Together these data provide compelling evidence that lipoprotein associated mitotane is less active in reducing cell viability compared to unbound mitotane and likely functions as a buffer or sink which may also prevent mitotane efficacy in vivo in some cases.
364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387
388 389 390 391 392 393 394 395 396 397 The data presented here have significant clinical consequences. First, patients with a disposition towards low serum lipids may be unable to achieve total serum concentrations of mitotane deemed effective. Conversely, patients with severe hypertriglyceridemia - as sometimes observed during mitotane treatment for unknown reasons - are likely to have overall higher mitotane serum concentrations with albeit lower lipoprotein free mitotane. This is consistent with the clinical experience that excessively high mitotane blood concentrations are tolerated in hyperlipidemia without significant side effects. Efficacy of mitotane in these patients has not yet been studied systematically. Second, our data together with the study by Hescot et al.26 put into question the current threshold of 14mg/l total mitotane in serum that has been demonstrated to be associated with superior treatment response. It may improve 398 patient care to instead quantify lipoprotein-free mitotane in order to better balance toxicity and efficacy.
Clinically, two obvious consequences remain to be investigated: We cannot, at present, finally argue for or against a particular diet for patients taking mitotane. Increased dietary lipids may be expected to increase total serum lipids but fail to increase actual lipoprotein-free mitotane concentration. A pharmacokinetic study of lipoprotein-free mitotane concentration in a fasting state vs. taken with a meal could solve this issue. Pharmacologic lowering of triglycerides and cholesterol may be another approach to increase the levels of lipoprotein-free mitotane. Though, we have reported that - at low mitotane concentrations - hydroxymethyl-glutaryl- CoA (HMG-CoA) reductase inhibitors counteract cytotoxic effects of mitotane on adrenal cortex cells by lowering concentrations of free intracellular cholesterol7 which induces lipotoxic ER-stress. Moreover, it remains to be demonstrated that administration of lipid- lowering drugs indeed increases lipoprotein-free mitotane.
In conclusion, we found that the intestine likely contributes to metabolic transformation of mitotane and incorporation into chylomicrones. We confirm previous data indicating that
399 400 401 402 403 404 405 406 407 408 409 410 411 412
lipoprotein-free mitotane is the active form of the drug. Lipoproteins decrease the bioavailability of active mitotane. A better understanding of the impact of food and co- administration of lipid-lowering drugs on lipoprotein-free mitotane concentrations is necessary to improve patient care. We additionally propose a prospective clinical trial to study disease response in relation to total and lipoprotein free mitotane concentrations.
413 414 415 416 417 418
Declaration of interests: M.F. received a lecture fee from HRA Pharma and has been a co- investigator of an HRA-Pharma sponsored clinical trial on the pharmacokinetics of mitotane.
Funding: This publication was supported by grants of the Deutsche Forschungsgemeinschaft 423 (grant KR4371/1-1 to M.K., FA 466/3-1 to M.F.) and a fellowship of the Comprehensive Cancer Center Mainfranken to M.K
Acknowledgment: We are grateful to Michaela Haaf for maintaining the ACC database.
419 420 421 422
424 425 426 427
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Figure Legends
Figure 1: Analysis of the index patient
Coronal (A), axial (B,C) and sagittal (D) slices of computed tomography (CT) displayed in both soft tissue (A, B) and bone (C, D) windows are given. The 29-year-old patient was referred for re-staging. Note important quantities of ascites in the abdomen (A, B; arrows). Misplaced pedicle screws in close proximity to the chyle cistern were the likely cause of the chylous ascites (C, D; arrowheads). No intravenous contrast agent was administered due to chronic kidney disease caused by nephrectomy and previous platinum based chemotherapy 5. Ultracentrifugation (E) proved ascites to contain chylomicrons (arrows) which float on buffer. (F) High performance chomatography of chyle provided evidence of o,p’-DDA (retention time 4.2 min), mitotane (8.5 min, and o,p’-DDE (9.9 min). p,p’-DDE (10.8 min) served as internal standard. Viability of NCI-H295 cells (G) was significantly less reduced when equal amounts of mitotane associated with chylomicrons (closed symbols) were used compared with mitotane dissolved in ethanol (open symbols). Cortisol secretion of NCI-H295 cells (H) exposed to equal amounts of mitotane associated with chylomicrons (closed symbols) was less reduced compared to mitotane dissolved in ethanol (open symbols). Expression of endoplasmic reticulum stress marker gene CHOP (I) and XBP1 mRNA splicing (J) was not increased in NCI-H295 cells exposed to mitotane associated with chylomicrons (closed symbols) but strongly increased in cells exposed to equal concentrations of mitotane in ethanol (open symbols). * p<0.05, *** p<0.001.
591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611
Table 1: Patient characteristics
The patient with chylous ascites is termed “index case”.
612 613 614
Serum trigylcerides (A) and serum cholesterol (B) did not differ significantly between patients with low (left) and high (right) mitotane serum concentrations. Lipoprotein binding of mitotane (C), o,p’-DDA (D), o,p’-DDE (E) in patients with high serum lipids (triglycerides and cholesterol, red) and low serum lipids (blue) reveals differential association of mitotane and o,p’-DDE with lipoproteins whereas o,p’-DDA is mostly present in lipoprotein-depleted and HDL2 fractions. Lipoprotein association of mitotane correlates with cholesterol (F) and triglycerides (G) in the respective fraction. Best correlation is obtained with combined cholesterol and triglycerides (H). Lipoprotein free mitotane concentration correlates with total mitotane serum concentration (I) and negatively correlates with serum cholesterol and triglyceride concentration (J).
615 616 617 618 619
620 621 622 623 624 625 626
Figure 3: In vitro efficacy of lipoprotein associated and lipoprotein-free mitotane
Normal mitotane-free lipoprotein fractions (VLDL, LDL2 and HDL2) were added to 25uM and 50uM mitotane and viability (A), cortisol secretion (B), CHOP mRNA expression (C) and XBP1 mRNA splicing in NCI-H295 cells measured. Addition of VLDL (open square) and LDL2 (black triangle) increased viability compared to equal concentrations of mitotane in ethanol. Addition of HDL2 (open circle) increased viability compared to solvent control but addition of mitotane decreased viability proportionally more than LDL2 and VLDL.
Similarly, inhibition of cortisol secretion by mitotane was alleviated through addition of lipoproteins (B). CHOP expression (C) likewise reduces by lipoproteins compared to lipoprotein free mitotane. The decrease of CHOP expression at 50uM lipoprotein-free mitotane compared to 25uM has been observed previously and may be associated with feed- back inactivation through downstream events. XBP1 mRNA splicing, an early event of ER- stress, is completely abolished when lipoproteins are added. * p<0.05, ** p<0.01, *** p<0.001
627 628 629 630 631 632 633 634 635 636 637 638 639 640
Figure S1: Chromatograms of serum of mitotane treated ACC patient (PID 15)
Elution profiles demonstrate retention times of mitotane, o,p’-DDA, o,p’-DDE and internal standard p,p’-DDE in serum of PID 15.
641 642 643 644
| PID | sex | age (years) | ENSAT stage | treatment intention | serum mitotane (mg/l) | serum triglycerides (µg/ml) | serum cholesterol (µg/ml) | sum serum cholesterol+triglycerides (µg/ml) |
|---|---|---|---|---|---|---|---|---|
| index case | F | 29.1 | 4 | palliative | 5.1 | 1230 | 820 | 2050 |
| 19 | M | 54.7 | 2 | adjuvant | 12.1 | 1160 | 3493 | 4653 |
| 20 | F | 75.9 | 3 | adjuvant | 20.1 | 1213 | 4197 | 5410 |
| 22 | M | 44.0 | 4 | palliative | 16.6 | 802 | 3096 | 3898 |
| 23 | F | 37.1 | 4 | palliative | 6.6 | 678 | 1862 | 2540 |
| 31 | M | 46.4 | 4 | palliative | 7.7 | 891 | 3174 | 4065 |
| 32 | M | 52.7 | 2 | adjuvant | 12.5 | 1618 | 1877 | 3495 |
| 34 | F | 50.4 | 4 | palliative | 25.1 | 739 | 3003 | 3742 |
| 38 | M | 69.7 | 4 | palliative | 8.1 | 2498 | 2040 | 4538 |
| 40 | M | 54.5 | 4 | palliative | 14.3 | 2024 | 2310 | 4334 |
| 42 | M | 63.5 | 4 | palliative | 20.2 | 3779 | 2755 | 6534 |
| 43 | M | 63.1 | 4 | palliative | 17.0 | 743 | 1876 | 2619 |
| 46 | M | 57.3 | 2 | adjuvant | 2.3 | 531 | 2512 | 3043 |
| 52 | M | 54.8 | 3 | adjuvant | 3.4 | 1504 | 2959 | 4463 |
| 55 | M | 50.6 | 4 | palliative | 5.2 | 994 | 1954 | 2948 |
| median | 1160 | 2512 | 3898 | |||||
| normal range | 740-1720 | 1300-2200 |
A
B
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.
F
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Coronal (A), axial (B,C) and sagittal (D) slices of computed tomography (CT) displayed in both soft tissue (A, B) and bone (C, D) windows are given. The 29-year-old patient was referred for re-staging. Note important quantities of ascites in the abdomen (A, B; arrows). Misplaced pedicle screws in close proximity to the chyle cistern were the likely cause of the chylous ascites (C, D; arrowheads). No intravenous contrast agent was administered due to chronic kidney disease caused by nephrectomy and previous platinum based chemotherapy 5. Ultracentrifugation (E) proved ascites to contain chylomicrons (arrows) which float on buffer. (F) High performance chomatography of chyle provided evidence of o,p’-DDA (retention time 4.2 min), mitotane (8.5 min, and o,p’-DDE (9.9 min). p,p’-DDE (10.8 min) served as internal standard. Viability of NCI-H295 cells (G) was significantly less reduced when equal amounts of mitotane associated with chylomicrons (closed symbols) were used compared with mitotane dissolved in ethanol (open symbols). Cortisol secretion of NCI-H295 cells (H) exposed to equal amounts of mitotane associated with chylomicrons (closed symbols) was less reduced compared to mitotane dissolved in ethanol (open symbols). Expression of
endoplasmic reticulum stress marker gene CHOP (I) and XBP1 mRNA splicing (J) was not increased in NCI- H295 cells exposed to mitotane associated with chylomicrons (closed symbols) but strongly increased in cells exposed to equal concentrations of mitotane in ethanol (open symbols). * p<0.05, *** p<0.001. 126x201mm (600 x 600 DPI)
A
5000
p=0.5
B
5000
p=0.9
4000
4000
µg/ml triglycerides
pg/ml cholesterol
3000
3000
2000
2000
1000
1000
0
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mitotane low (n=7)
mitotane high (n=7)
mitotane low (n=7)
mitotane high (n=7)
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