Development of Mitotane Lipid Nanocarriers and Enantiomers: Two-in-One Solution to Efficiently Treat Adreno-Cortical Carcinoma
F. Menaa*, and B. Menaa*,2
1Fluorotronics, Inc, Department of Bio-Medical and Pharmaceutical Sciences, 2453 Cades Way, Bldg C, San Diego, CA 92081, USA; 2 Fluorotronics, Inc, Department of Chemistry and Nanotechnology, 2453 Cades Way, Bldg C, San Diego, CA 92081, USA
Abstract: Adrenocortical carcinoma (ACC) is a rare but aggressive malignancy with a poor prognosis. Treatment options for advanced ACC are limited. Indeed, radical tumor resection can lead to local or metastatic recurrence, and mitotane (Lysodren®), the only recog- nized adrenolytic drug, offers modest response rates, notably due to some of its physico-chemical and pharmacological properties (i.e. hydrophobicity, low bioavailability). Meantime, high cumulative doses of Lysodren® usually cause systemic toxicities. To reduce adverse health effects, the search of safe and efficient mitotane nano-formulations as well as the full characterization and testing of its enanti- omers can represent valuable therapeutic options. Interestingly, recent investigations showed that solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) could considerably improve the efficacy of mitotane (i.e. enhanced solubility and bioavailability, progressive release of the loaded drug into blood and targeted tissues) as well as its safety (i.e. lower toxicity, higher biocompatibility). These two nano-carriers for mitotane delivery and targeting are of particular interest over other polymeric particles (i.e. low-cost, effi- cient and simple scaling to an industrial production level following green methods). Besides, emerging studies suggested that the S-(-)- mitotane is more potent than the R-(+)-mitotane for ACC treatment. Therefore, the production of pure and active S-(-)-mitotane might of- fer synergic or additive benefits for ACC patients when combined to solid lipid-based nanocarriers. In this review, we first provide an updated overview of the ACC disease before emphasizing on the promising mitotane drug nano-systems, as well as on the separation, purification and production of single mitotane enantiomer using state-of-art chromatographic-based methods.
Keywords: Adreno-cortical carcinoma, cancer endocrinology, chiral separation, endocrine cancer chemotherapy, lysodren®, medicinal chem- istry, mitotane enantiomers, mitotane pharmacology, mitotane synthesis, nanostructured lipid carriers, nanomedicine, simulated moving bed chromatography, solid lipid nanoparticles, supercritical fluid chromatography, translational medicine.
1. ADRENO-CORTICAL CARCINOMA: AN UPDATE
1.1. Epidemiology and Etiology
In contrast to benign adreno-cortical adenomas (ACA) that oc- cur in at least 3% of the population aged over 50 years, adreno- cortical carcinoma (ACC) is a rare endocrine malignancy [1-3]. Indeed, the worldwide incidence of this orphan disease is estimated to be around 0.5-2 cases per million/year and its prevalence about 4-12 cases per million population [1-3]. Exceptionally, in some regions of the world such as southern Brazil, the annual incidence in the children population - under 15 years old - is about 3.4-4.2 cases per million, which is considerable comparatively to the worldwide incidence [4]. A bimodal age distribution has been ob- served in ACC patients with peaks in childhood, before the age of 5, and in the fourth to fifth decades of life [5, 6]. Moreover, women are slightly more predisposed to the disease than men (ratio 1.5) [2, 7,8].
The majority of ACCs are sporadic neoplasms of undetermined etiology while familial predisposition can occur. Interestingly, so- matic mutations in genes predisposing to some syndromes associ- ated with the increased susceptibility of cancer (e.g. Li-Fraumeni, Beckwith-Wiedemann), have also been identified in either benign or malignant sporadic adreno-cortical tumors (ACTs) [9]. Thereby, inactivating mutations at the 17p13 locus including the TP53 - pro- tein considered as the “guardian of the genome” - [10], as well as alterations of the 11p15 locus leading to IGF-II (type II Insulin Growth Factor) over-expression and adrenal cancer cell prolifera- tion, were frequently observed [11].
1.2. Prognosis, Diagnosis and Therapy
ACC is characterized by a poor prognosis and a high risk of re- currence post-therapy. Indeed, the recurrence is about 49% after
adjuvant chemotherapy and up to 85% after surgery without adju- vant treatment [12, 13]. In about 40% of the cases, the disease re- lapse is manifested by the development of metastatic disease to the lungs, liver or bone at the diagnosis or within 6-24 months of surgi- cal resection [7, 8, 12-14]. Moreover, the unsatisfactory overall 5- year survival ranges between 23% and 60% [2, 6, 8, 15]. Three main prognostic parameters are significantly associated with a shorter patient’s survival: (i) older age at diagnosis; (ii) tumor stage at presentation (i.e. stage III aka involvement of local lymph nodes and, stage IV aka local organ invasion or distant metastases), which can slightly differs according to the staging system used (i.e. MacFarlane-Sullivan [16] or European Network for the Study of Adrenal Tumors (ENSAT) [17]); (iii) hypersecretion of cortisol, a major adrenal steroid hormone [18].
At present, early diagnosis of this aggressive malignancy is mainly monitored by cortisol levels, which are too high in 60% of patients - especially in children (about 90%) - frequently leading to Cushing’s syndrome with or without virilization [1, 2, 5].
Complete surgical resection of the tumor (i.e. ipsilateral adrenalectomy with or without nephrectomy and/or splenectomy) is the only therapy that has consistently shown to prolong patient’s survival, particularly if disease is detected at early stages (I and II) but this, usually concerns less than 60% of the patients [1, 19, 20]. Indeed, median survival in patients with unresectable tumors or incomplete tumor resection is usually less than 12 months (about 3 to 9 months) [14, 21, 22] whereas, after complete resection, the median survival is generally improved (13 to 28 months) as shown in retrospective studies [19, 23-25]. Radiotherapy is only indicated as palliative treatment for patients with bone (and brain) metastases [1], and international prospective randomized studies are still insuf- ficient to evaluate its benefit in the treatment of unresectable dis- ease [26]. Finally, chemotherapy mainly consists of using mitotane, the only Food and Drug Administration (FDA)-approved drug against ACC, and can be administrated as follows: (i) alone; (ii) as combined regimens (e.g. mitotane plus etoposide-doxorubicin- cisplatin (EDP/M)) preferably in patients with incomplete, not pos- sible or not successful tumor resection; (iii) as adjuvant in patients with high risk of recurrence at presentation or at relapse [1, 20, 27].
*Address correspondence to this author at the Fluorotronics, Inc, Department of Bio- Medical and Pharmaceutical Sciences, 2453 Cades Way, Bldg C, San Diego, CA 92081, USA; Tel/Fax: +1 (858) 274-2728; E-mail: dr.fmenaa@gmail.com
*Both authors have equally contributed to this work.
Interestingly, adjuvant treatment (i.e. chemotherapy and/or radio- therapy after surgery) might significantly decrease the disease re- currence after surgery and, increase the overall survival of the pa- tients [12, 13, 18, 28-31]. Nevertheless, the potential benefits of adjuvant treatment have not been confirmed in some other studies [32-34], most likely because of incomplete surgery and variable drug metabolism [1, 35, 36]. Besides, current available systemic therapies provided incomplete efficient responses (<50%) in cases of advanced ACC and, remain severely limited mainly because of the rarity of ACC disease that had hampered the ability to undertake international randomized clinical studies to identify the most effec- tive first- and second-line cytotoxic regimens [37]. Consequently, in spite of its relative efficacy, mitotane drug therapy remains the cornerstone, mainly in metastatic stage [38, 39].
Hopefully, the two most recent international randomized clini- cal studies, FIRM-ACT (First International Randomized trial in locally advanced and Metastatic Adrenocortical Carcinoma Treat- ment) and, ADIUVO (an international prospective, randomized, open-label, and controlled phase III trial for patients with ACC after radical resection), endorsed by the ENSAT [40], will show interest- ing data. Briefly, FIRM-ACT aims to assess the efficacy of mito- tane combined to other drugs (e.g. EDP/M) as first line treatment versus Streptozotocin plus mitotane (Sz/M), while ADIUVO con- sist to evaluate the efficacy of mitotane as adjuvant treatment ver- sus observation in patients with ACC at low-intermediate risk of recurrence after radical resection [27, 37]. However, until results from all randomized clinical trials become available, healthcare professionals will be challenged by an uncertainty.
Alternatively, the better understanding of the molecular patho- genesis of ACC, such as IGF signaling pathway, might allow the design of promising therapeutic targets [20, 41].
Eventually, the rapid emergence of the nanotechnology and state-of-art chromatography systems, shall significantly contribute to the development of mitotane chiral nano-formulations, which might present greater therapeutic features than the free mitotane drug formulation (i.e. lower toxicity, significant efficacy to lower the disease progression, capability to enhance patient’s survival and patient’s quality of life). However, the development of generic mi- totane formulations shall be avoided due to the Narrow Therapeutic Index (NTI), and inadequate glucocorticoids administration must be prevented in order to limit the risk of adverse effects.
2. MITOTANE: SYNTHESIS, STRUCTURE AND DRUG PROPERTIES
2.1. Synthesis Route
The synthesis route of mitotane (o,p’-DDD aka 1-(2- chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane, or 1-chloro- 2-[2,2-dichloro-1(4-chlorophenyl)ethyl]benzene, or 1,1-(o,p’- dichlorodiphenyl)-2,2-dichoroethane, or 1,1-dichloro-2-[o-chloro phenyl]-2-[p-chlorophenyl]ethane) has been more recently re- viewed [42]. The manufacturing process is simply carried out in 5 steps Fig. (1) - three of them include chemical reactions and the other two correspond to recrystallizations -, and gas chromatogra- phy coupled to mass spectroscopy (GC-MS) is generally sufficient to characterize mitotane during this process or in the final product. Thereby, the classical synthesis route starts with the diazotisation of 2-chloroaniline (I) with NaNO2 and HBr in H2O that furnishes 2- chlorobenzenediazonium bromide (II), which is then brominated with CuBr and HBr to afford 1-bromo-2-chlorobenzene (III) [43]. 1-bromo-2-chlorobenzene (III) is treated with Mg in ether, and the obtained Grignard reagent is condensed with dichloroacetaldehyde (IV) in ether, yielding 2,2-dichloro-1-(2-chlorophenyl)ethanol (V), which finally is condensed with chlorobenzene (VI) in the presence of H2SO4 to provide mitotane [44].
Cl
Br
Cl
Cl
N
H2N
N+
Br
NaNO2
CuBr
HBr
HBr
(III)
(I)
(II)
Mg
Et2O
Cl
H
Cl
(IV)
O
1
Cl
Cl
Cl
Cl
Cl
Cl
Cl
(VI)
H2SO4
HO
Cl
Mitotane
(V)
2.2. Structure and Physical-Chemical Properties
This oral antineoplastic agent is best known by its trivial name, o,p’-DDD. Its systematic chemical name, according to IUPAC (In- ternational Union of Pure and Applied Chemistry) nomenclature, is 1-chloro-2-[2,2-dichloro-1(4chlorophenyl)ethyl]benzene [45]. The 2D and 3D chemical structures of mitotane (C14H10C14) are shown in Fig. (2).
Cl
Cl
(a)
Cl
Cl
(b)
According to the US Pharmacopeia (USP) [46], Lysodren® must present the major following chemical-physical and pharma- ceutical features: (i) a monoisotopic mass /molecular weight (MW) of about 318-320 Da; (ii) a melting point ranging between 75-81℃; (iii) a dosing of 500 mg of the active substance mitotane; (iv) a white granular solid composed of clear colorless crystals; (v) a tasteless and slight pleasant aromatic odor; (vi) a low solubility in water, an acceptable solubility in either ethanol, ether, hexane, iso- octane, carbon tetrachloride, fixed oils or fats; (vii) inactive ingre- dients represented by Avicel (matrix of microcrystalline cellulose), Polyethylene Glycol (PEG) 3350, colloidal silicon dioxide, and corn starch; (viii) an optimal stability when stored at 25°℃ (77ºF), with excursions permitted to 15°C-30°C (59ºF-86°F); (ix) absence of chromophores that absorb at wavelengths >290 nm, to avoid direct photolysis by sunlight; (x) an estimated usual half-life of 90 days, taking into consideration the possible atmosphere-degradation by a reaction involving photochemically-produced hydroxyl radi- cals.
These properties underlining the effect of the particle size and physical form on the dissolution, hence the bioavailability of the active substance - consequently need to be tightly controlled to ensure the clinical safety and efficacy of the medicinal product. [46].
2.3. Biological Effects and Pharmacological Properties
Mitotane (Lysodren®), developed in 1960, is an isomer of DDD (dichloro-diphenyl-dichloro-ethane), a derivative of the pesticide DDT (dichloro-diphenyl-trichloro-ethane) which was shown to produce adrenal atrophy in dogs in 1948 and, represents up-to-date the only Food and Drug Administration (FDA)-approved drug for the treatment of ACC [47, 48]. Mitotane acts both as an inhibitor of steroidogenesis and an adrenolytic agent. Mechanistically, it inhib- its directly 11ß-hydroxylase, and cholesterol side-chain cleavage (SCC) in the mitochondria of steroidogenic cells together with an- tagonizing chemotherapy drug efflux, therefore blocking cortisol synthesis and reducing multidrug resistance (MDR), respectively [49]. Interestingly, mitotane has been shown to induce a p53- independent irreversible G2-arrested in cultured adrenocortical cell lines when combined to radiotherapy, as well as an increase of the radiotherapy cell growth inhibitory effect [50]. Furthermore, recent findings established a critical role of IGF signaling in ACC patho- physiology and provide rationale for use of targeted IGF-1R (type I Insulin-like Growth Factor Receptor) antagonists - especially when combined with mitotane - to treat ACC in future clinical trials [51].
Pharmacological analysis of oral Lysodren® in humans showed that about 40% only is absorbed, and approximately 10% of the administered dose is recovered in the urine as a water-soluble me- tabolite [52]. A variable amount of metabolite (1%-17%) is ex- creted in the bile within 24 hours and, because of its lipophilicity, the balance is apparently stored in the tissues (e.g. mainly adipose, liver, brain and adrenal tissues) [52]. Peak plasma Lysodren® con- centrations occur 3-5 hours after a single oral dose of the drug and distribution of the drug between plasma and tissues is completed within 12 hours [52]. Consequently, cumulative high dose admini- stration of mitotane is often required (up to 4-6 g/day during 3-5 months) which, subsequently, can lead to higher toxicity events [52, 53]. Following discontinuation of Lysodren®, the plasma terminal half-life has ranged from 18 to 159 days, but can even last longer in some tissues (e.g. storing tissues such as fat ones) [52]. It is not known whether Lysodren® or its metabolites are able to cross the placenta or distribute into milk. The NTI of mitotane anti-tumor activity is achieved at the plasma concentration of 14 mg/L [52, 54- 56], and significant side effects have been noticed in more than 80% of all patients particularly when systemic levels of mitotane were greater than 20 mg/L [1, 57]. The adverse effects include the gastro-intestinal system (e.g. nausea, vomiting, diarrhea) or the
central nervous system (e.g. lethargy, ataxia, depression), which can be reversible after cessation of mitotane [1, 57-60]. According to the World Health Organization (WHO) criteria, the overall response rate in 72 patients was 49%, including five patients with complete response [27]. This inter-patient variability, low response and con- siderable drug toxicity might be explained by many factors and mechanisms (e.g. genetics, epigenetics, ability of human tumor cells to efflux the drug or to metabolize it) and, underlines the im- portance of personalized medicine for mitotane dose titration as well as close clinical supervision.
In fact, mitotane metabolism is being studied for almost four decades to better understand its pharmacokinetics and pharmacody- namics and so, its molecular activity, which would help in carrying out the treatment [61-70]. Several approaches, using chromatogra- phy and/or spectrometry, have been developed to quantitatively determine mitotane and its metabolites in body fluids (e.g. serum, plasma, and urine) as well as in feces, and associate them with clinical outcomes [61, 66-70]. Among the major mitotane metabo- lites, we can cite o,p’-DDE (i.e. 1,1-(o,p’-dichlorodiphenyl)-2,2 dichloroethane aka 1,1-dichloro-2-[p-chlorophenyl]-2-[o-chloro phenyl]ethane) [61], and o,p’-DDA (1,1-(o,p’-dichlorodiphenyl) acetic acid) [62] which has been identified through a proposed route involving the adrenal mitochondrial cytochrome P450-catalyzed hydroxylation of mitotane at the B-carbon [63, 64]. Subsequent dehydrochlorination of the hydroxylated product forms the corresponding acyl chloride that, in the presence of water, formed the acidic metabolite, o,p’-DDA, although it could alternatively bind to tissue nucleophiles [65]. Interestingly, the synthesis route of B-3H-mitotane has been reported few years ago for use in a assay for mitotane metabolism [65], and consisted in the reduction of 1- (2-chlorophenyl)-1-(4- chlorophenyl)-2,2,2-trichloroethane (o,p’- DDT) by an aluminium-Hg2Cl2 couple in the presence of tritied water (3H20). Thereby, the determination of the 3H+, released to the aqueous media after organic solvent extraction, constituted a spe- cific, faster (about 2-3 hours), and more sensitive assay for mitotane metabolic activation mediated by -hydroxylation than 14C- mitotane-high-performance liquid chromatography (HPLC) [65]. Initial experiments in rats using 4C-labeled mitotane along with thin-layer chromatography (TLC), gas-liquid chromatography (GLC) and MS, reported that most of metabolites (87.8%) was concentrated in the feces (e.g. o,p’-DDA and its hydroxy-derivates such as 4-hydroxy-, 3-hydroxy-, and 3,4-dihydroxy-substituted o,p’- DDA, as well as o,p’-DDE) [66]. Interestingly, it was shown using GC-MS/Selected Ion Monitoring (SIM) that o,p’-DDA is present in the plasma at a concentration about 10 times higher than the levels of o,p’-DDD (mitotane) and o,p’-DDE [67]. This finding was later confirmed by other approaches using HPLC separation [61, 68]. The clinical significance of such high plasmatic o,p’-DDA levels is not established yet. Nevertheless, a relatively recent study that ex- plored the relationship between the plasma levels of mitotane and its metabolites, o,p’-DDA and o,p’-DDE determined by HPLC, with the efficacy of mitotane therapy during a long-term follow-up of pediatric and adult patients with adrenal cancer, suggested that plasmatic o,p’-DDE concentrations could be more closely related to clinical improvement or remission than the mitotane levels [69]. Indeed, higher o,p’-DDE and o,p’-DDE/o,p’-DDD seemed to be associated with a good/favorable prognosis during the prolonged mitotane therapy [69], and so might consitute interesting/important factors in clinical practice.
3. MITOTANE: PROMISING EMERGENT FORMULA- TIONS
3.1. Mitotane Nano-Formulations: From Concept to Develop- ment
Mitotane is a hydrophobic drug (i.e. displaying low solubility in water) with relative high toxicities and low therapeutic responses.
Consequently, the search of safe and efficient mitotane formula- tions can represent valuable therapeutic options. Among emerging technologies (i.e. self-microemulsifying drug delivery system (SMEDDS) for mitotane [71]), nanotechnology using nano-carriers for drug delivery could offer tremendous possibilities to improve efficacy and significantly decrease the side effects associated with drug hydrophobicity which, in turn can compromise the drug bioavailability, safety and efficacy (i.e. disease targeting). Indeed, the pharmacological activity of a drug molecule depends on its ability to dissolve and interact with its biological target, either through dissolution and adsorption, or through dissolution and re- ceptor interaction. The low bioavailability that characterizes hydro- phobic drugs, such mitotane, is usually attributed to the dissolution kinetic profile.
The novel strategies to effectively deliver these drugs include nanosizing approaches based on the production of: (i) drug nanoc- rystals dispersed in an aqueous surfactant solution; (ii) drug loading nanoparticles; (iii) lipid-based nanocarriers aka solid-lipid nanopar- ticles. To select the best approach, there are however some critical considerations to take into account such as the: (i) physicochemical properties of the drug; (ii) possibility to scale-up the production process; (iii) toxicological considerations of the use of solvents and co-solvents; (iv) selection of an environmentally sustainable meth- odology; (v) development of a more patient-friendly dosage form; (vi) relevant drug administration route(s). Interestingly, the two promising and emerging approaches consist in using either solid lipid nanoparticles (SLN) or nanostructured lipid carriers (NLC), to possibly enhance the therapeutic effects of the current mitotane free formulation. Indeed, those lipid-based nano-formulations are ex- pected to (i) improve gastrointestinal tract (GIT) absorption of the drug; (ii) allow progressive release of the drug into blood and spe- cific tissues; (iii) increase bioavailability of the drug notably by preventing chemical and enzymatic degradation of the loaded drug; (iv) enhance solubility of the drug; (v) lower toxicity problems than other polymeric nanoparticles because of biodegradable, biocom- patible and physiological lipids generally used [72, 73].
Solid lipid nanoparticles (SLN) were developed at the begin- ning of the 1990s as an alternative carrier system to emulsions, liposomes and polymeric nanoparticles, permitting potential phar- maceutical applications [74, 75]. SLN can be simply obtained by exchanging the liquid lipid (oil) of the emulsions by a lipid able to solidify at room as well as at body temperature. The two basic pro- duction methods for SLN are: (i) high pressure homogenization [76]; (ii) the micro-emulsion [77]. There are basically three differ- ent SLN models for the incorporation of active ingredients Fig. (3), represented by the: (i) homogeneous matrix model; (ii) drug- enriched shell model; (iii) drug-enriched core model [78]. The re- sulting structure depends on the formulation composition (i.e. lipid, active compound, and surfactant) as well as on the production con- ditions (hot versus cold homogenization). For instance, a homoge-
neous matrix would be mainly obtained when applying cold ho- mogenization method and when incorporating very lipophilic drugs (i.e. mitotane) in SLN with the hot homogenization method [79]. The scale production can be performed using specific piston-gap homogenizers, depending on the desired quantity. Thereby, for lab scale production, the Micron lab 60 system (10 Kg/20 minutes) can be sufficient while, for much higher scale production, a system such Rannie 118 (up to 2000 Kg/hour) is required [80]. Interestingly, incorporation of active compounds into the solid matrix of SLN can protect them against degradation [81]. Nevertheless, for sensitive drugs such mitotane, the choice of basic lipids as well as small par- ticles with largest interface lipid/ surfactant to water area, is pri- mordial to ensure the highest stability of the compound [82]. The effect of formulation parameters on the burst release of active com- pounds from SLN was usually optimal when: (i) producing at high- est temperatures, probably because of the induced solubility; (ii) highest amount of surfactant were used in the formulation; (iii) applying the hot homogenization method [74, 79]. SLN particles also possess an adhesive effect or “occlusion” that increases with: (i) decreasing particle size; (ii) low melting lipids; (iii) high crystal- line particles [83]. Eventually, the SLN formulations are, at some extend, interesting for mitotane production as they can present the following advantages: (i) protection of this labile molecule against environmental degradation during storage or oral administration (i.e. air, light, elevated temperatures, enzymes, chemicals etc); (ii) increase of the bio-availability of this lipophilic molecule; (iii) bio- compatibility of these biodegradable and physiological lipids; (iv) possibility to be scaled to an industrial production level at low cost and in a relative simple way; (v) no requirement for using organic solvents; (vi) no need of employing a higher concentration of sur- factants than the one normally used in marketed products, avoiding the potential necessity to perform a tolerability study for the excipi- ent [74, 79, 84]. Conversely, SLN can also present major disadvan- tages for mitotane production due to: (i) high water content of SLN dispersions; (ii) probable low drug-loading capacity because of the formation of a perfect lipid crystal; (iii) probable drug expulsion during storage [75, 85-87].
Nanostructured lipid carriers (NLC) are a novel type of lipid nanoparticles with solid matrix. NLC present quite similar features to the `old’ SLN system but, some improvements to minimize or avoid some problems associated with SLN have been realized [75, 85-89]. The concept of NLC consists to mix spatially different lip- ids (i.e. blending solid lipids with liquid lipids (i.e. oils) that can become solid at body temperature), allowing: (i) an imperfect nanostructure to accommodate the drug; (ii) increase of the pay- load for active compound, subsequently avoiding the expulsion of the compound. There are three different types of NLC Fig. (4) known as: (i) imperfect; (ii) amorphous; (iii) multiple [86].
Recently, preliminary studies have demonstrated the feasibility to develop lipid-based nanocarriers to enhance both the drug solu-
Homogeneous Matrix
Drug-enriched Shell
Drug-enriched Core
NLC - Imperfect Crystal
Drug incorporated into a mix of different lipids
SLN - Perfect Crystal
Amorphous lipid
Drug expulsion
Oil nano-compartments in solid lipid (fat)
bility and bioavailability of mitotane [90, 91]. Thereby, the loading of mitotane into SLN and NLC formulations were recently prepared by high shear homogenization followed by hot high pressure ho- mogenization [90]. SLN and NLC were then evaluated by particle size determination (PSD), zeta potential calculation, polydispersity index, encapsulation efficiency, and loading capacity. Hydrophilic coating of SLN with chitosan or benzalkonium chloride was effec- tive in changing zeta potential from negative to positive values [90]. While the authors showed that mitotane was efficiently loaded into SLN or NLC, becoming potential delivery systems for improv- ing mitotane loading capacity and controlled drug release, several issues are unanswered yet and, shall be then further investigated. such: (i) how to assess the quality assurance and quality control (QA/QC) of the final nano-product intended to market ?; (ii) what about the pharmacokinetics, bio-distribution, bioavailability, effi- cacy and safety of such mitotane nano-lipid formulations in vivo ?; (iii) what is the best route for mitotane nano-drug administration? Indeed, oral route could be considered to be the most preferred route for drug administration due to greater convenience, less pain, high patient compliance, reduced risk of cross-infection, and needle stick injuries [72]. Nevertheless, oral drugs - including oral nano- drugs - may still present a major risk of degradation by pancreatic lipases.
3.2. Mitotane Enantiomers: Another Valuable Therapeutic Option
In addition to the search of safe and efficient mitotane nanoma- terial based-formulations, recent investigations suggest that the characterization and comparative testing of mitotane enantiomers can lead to a valuable therapeutic option for patients with ACC.
Chiral separation, also called chiral resolution, is a procedure used to separate the two isomers of a racemic compound. Indeed, the reformulation in single enantiomeric form of a chiral drug (i.e. racemate) has gained considerable attention in the pharmaceutical industry as well as in clinical analysis [92]. The principal reason is that enantiomers (i.e. stereo-isomers that are mirror images of each other and so, spatially non-superposables) often display different pharmacological activities as well as pharmacokinetic and pharma-
codynamic properties, which can be potentially beneficial for health [93].
The integration of modern technologies for extensive charac- terization and development of pure and active but less toxic mito- tane enantiomer might then offer synergic or additive beneficial effects when combined to lipid-based nanocarriers. Indeed, mito- tane is a chiral drug marketed in the racemic form, and recent in vivo studies that aimed to evaluate pharmacological effects due to molecule chirality, indicated that one of the mitotane enantiomers (S-(-)-mitotane) is more potent (about 4 times) in cancer treatment than the other enantiomer (R-(+)-mitotane), which would justify its purification [94-97].
Several techniques and methods derived from the chromatogra- phy system have been widely used for enantiomeric separation (e.g. HPLC, GC), but supercritical fluid systems (e.g. supercritical fluid extraction (SFE), supercritical fluid chromatography (SFC)) [98- 107] as well as the multicolumn continuous preparative chromatog- raphy (e.g. Varicol simulating moving bed (SMB) chromatography system) [108] are now considered as the gold standard methods for screening, separation, qualitative and quantitative characterization and, development of chiral compounds such mitotane.
In general, pure enantiomers can be obtained by asymmetric synthesis and racemic resolution, the latter being less complex and capable of producing both compounds, essential for further investi- gations (i.e. biological and clinical testing). The racemic resolution includes: (i) enzymatic separation and formation of diastereomers which can be separated by crystallization or chromatography (indi- rect separation); (ii) direct chromatographic separation using the chiral stationary phase (CSP) [96, 109-111]. Globally, CSP-based chromatographic methods allow separation - through differences in elution retention times -, isolation and analysis of both enantiomers, with high optical purity and in a relatively short time, permitting the development of novel effective drug formulations [110, 112, 113]. The mechanism involved in CSP-based chromatographic methods to separate enantiomers is based in the interaction enantiomer- selective CSP. A CSP consists of an achiral matrix such as porous silica gel in which a type of ligand, as chiral selector, is chemically or physically attached (e.g. small molecules of MW< 3 KDa such
as exchange CSPs, complex (Pirkle-type) CSPs, crown ether CSPs, cyclodextrin CSPs and macrocyclic glycopeptide CSPs, or large molecules such as synthetic chiral polymers and naturally occurring chiral structures) [114-117]. A”three-point binding model” de- signed by Dalgliesh in 1952 explains that at least three different molecular interactions (i.e. steric-, hydrogen-, or polar-) must occur between the enantiomer and the CSP to allow chiral recognition and thus, contribute efficiently to resolve the enantiomeric separation [118, 119]. Three physical properties of silica gel are also important to be considered: (i) the pore size; (ii) the particle diameter; (iii) the surface area. Indeed, the column efficiency increases when the diameter of particle and pore size is smaller, in other words when the surface area of silica gel is greater [120]. Finally, separation of enantiomers by CSP-based chromatography is conditioned by their adsorption which is determined by their specific thermodynamic properties (i.e. Gibbs free energy (AGº) that depends on enthalpy (AHº) and entropy (ASº) terms, and which can cause either an exer- gonic (AG<0) or an endergonic (AG>0) reaction) [121-123]. Thereby, it was noted that the more (energetically) unstable diastereomeric binding complex (chiral selector-enantiomer) is first eluted [122].
Supercritical fluid extraction (SFE) is a selective and conven- ient technique for enantiomeric analysis [105-107] with a number of advantages over HPLC and GC [98]. Further, separation by SFE using carbon dioxide (CO2) is a green, rapid, productive method allowing solubility of the free enantiomeric mixture in the super- critical CO2, subsequently eliminating problems associated with method of separation of racemates via partial or total diastereomeric salt (complex) formation and crystallization from a proper solvent [93, 106, 107, 124, 125].
Supercritical fluid chromatography (SFC) that uses su- per/subcritical fluid CO2 and polar organic modifiers as mobile phase (e.g. alcohols such methanol or a less polar solvent) as well as packed columns as stationary phases, represents a golden choice to separate stereo-isomers (e.g. (E)-(Z) isomers) owing to its speed, efficiency, cost effectiveness [124, 126-129]. SFC has some signifi- cant advantages over standard HPLC methods [98, 130] such as: (i) more flexibility in solvent selection; (ii) less pressure drop across the columns; (iii) faster column equilibration; (iv) faster method development; (v) higher efficiency separations and significantly less generation of hazardous waste; (vi) improving selectivity and solubility; (vii) direct mixing of polar modifiers with CO2. The major advantage that SFC holds over GC is the ability to separate thermally labile compounds [98, 130]. The use of SFC for the sepa- ration of enantiomers has been one of its most successful applica- tions due to the inherent advantages of using liquid CO2 in the mo- bile phase, resulting from its high diffusivity and low viscosity [131]. SFC coupled with other analytical tools such MS (i.e. SFC- MS) has had a great advance in drug discovery process and produc- tivity due to increase in reliability and robustness of the combina- tory systems [132]. In addition, with SFC-MS, enantiomeric excess can be determined with much lower detection limits than ultraviolet (UV) and, much shorter analysis times compared to normal- phase/reversed-phase liquid chromatography [133]. Eventually, mitotane enantioseparation was possible in a semi-preparative scale and in controlled conditions (i.e. constant temperature and pressure) when a chiral stationary phase of supercritical fluid CO2 and opti- mal concentration of organic modifiers (i.e. methanol, ethanol or 2- propanol) as mobile phase, were used [97].
Besides, multicolumn chromatographic processes like simulated moving bed (SMB) chromatography is often applied for separating binary mixtures, like racemates, and diastereomers, as they increase throughput, purity, and yield compared to batch chromatography [134-138]. Interestingly, supercritical fluid-SMB processes under pressure gradient mode offered productivity improvement over the isocratic mode of operation [138]. Nevertheless, several theoretical
and experimental results in the literature indicate that, due to the new degree of freedom, the Varicol preparative chromatographic system represents a more favorable and innovative chromatographic technique than conventional SMB because of: (i) higher productiv- ity; (ii) lesser eluent consumption; (iii) smaller number of shorter columns; (iv) operation of a continuous unit by the advance of feed (inlet) and withdraw (outlet) streams of the adsorbent bed, in an asynchronous switching positions mode, to produce two outlet streams. Thereby, the more adsorbed component is the main com- pound in the extract stream while the less adsorbed component predominates in the raffinate stream [135, 136, 139]. A recent pio- neered study has reported the semi-preparative separation of pure mitotane enantiomers by a continuous Varicol unit operated on a scale of 30 g/day [108]. The enriched enantiomer streams were obtained using chiral chromatographic columns (1.0 cm inner di- ameter, 10 cm length) packed with amylose tris(3,5- dimethylphenylcarbamate) - supported on a matrix of silica - as the stationary phase (6.0 g/column) and acetonitrile-isopropanol mix- tures (1:3 v/v) as mobile phases. The enantiomeric purities obtained were 97.0% for S-(-)-mitotane and 96.8% for R-(+)-mitotane in the raffinate and extract streams, respectively. The unit provided satis- factory productivities of 1.14 Kg raffinate per day and Kg adsorbent for S-(-)-mitotane and, 0.68 Kg extract per day and Kg adsorbent for R-(+)-mitotane.
The chemical structures corresponding to the mitotane enanti- omers are depicted in Fig. (5).
CI
CI
(a)
(b)
Cl
H
Cl
H
Cl
Cl
Cl
Cl
CONCLUSIONS AND PERSPECTIVES
Adrenocortical carcinoma (ACC) is a rare but aggressive ma- lignancy with a poor prognosis. Owing to some of its physical- chemical and pharmacological properties (e.g. hydrophobicity, lability, and subsequent very low systemic bioavailability), mito- tane (Lysodren®), the only FDA-approved adrenolytic drug offers modest response rates in ACC cancer patients while causing sig- nificant health side-effects when chronic doses are employed. Thus, the management of ACC patients, particularly those with advanced ACC, requires multidisciplinary and innovative approaches to over- come these current therapeutic limitations. In this manuscript, we highlighted a possible “two-in-one” solution to efficiently treat patients with ACC, based on the recent and emerging investigations that suggest a favorable use of: (i) nanostructured lipid carriers (NLC) to load mitotane with greater features compared to other polymeric particles (e.g. in terms of safety, efficacy and cost of production), and (ii) S-(-)-mitotane, considered to be more potent than R-(+)-mitotane (Lysodren®) in cancer patients. Therefore, pure active S-(-)-mitotane loaded into NLC might offer better clinical results than S-(-)-mitotane as a free drug and, synergic or additive beneficial health effects for a larg number of ACC patients com- paratively to the use of R-(+)-mitotane loaded in the same experi- mental conditions into NLC. Eventually, more studies (e.g. in vivo, clinical, epidemiological ones) are needed to assess the pharmacol- ogical, physical and chemical properties as well as the risk/benefits ratio of such possible novel mitotane drug formulations (e.g. trig- gered release, long-term stability, safety/toxicity, efficacy) for the patients before tempting a large scale production that can be greatly conducted using “green” methods.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of interest.
[13]
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Abder Menaa, MD, for the critical review of this manuscript and his pertinent suggestions.
ABBREVIATIONS
ACC = Adreno-Cortical Carcinoma
CSP = Chiral Stationary Phase
EDP/M = Etoposide-Doxorubicin-Cisplatin/Mitotane
GC = Gas Chromatography
HPLC = High-Performance Liquid Chromatography
[18]
MS = Mass Spectroscopy
MW = Molecular Weight
[19]
NLC NTI
= Nanostructured Lipid Carriers
= Narrow Therapeutic Index
[20]
SFC
= Supercritical Fluid Chromatography
[21]
SFE
= Supercritical Fluid Extraction
SLN
= Solid Lipid Nanoparticles
SMB = Simulated Moving Bed
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