Taylor & Francis Taylor & Francis Group
H55Pc 1863-3450
PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY
informa healthcare
Mitotane liposomes for potential treatment of adrenal cortical carcinoma: ex vivo intestinal permeation and in vivo bioavailability
P. Zancanella, D. M. L. Oliveira, B.H. de Oliveira, T.D. Woiski, C.C. Pinto, M.H.A. Santana, E.B. Souto & P. Severino
To cite this article: P. Zancanella, D. M. L. Oliveira, B.H. de Oliveira, T.D. Woiski, C.C. Pinto, M.H.A. Santana, E.B. Souto & P. Severino (2020): Mitotane liposomes for potential treatment of adrenal cortical carcinoma: ex vivo intestinal permeation and in vivo bioavailability, Pharmaceutical Development and Technology, DOI: 10.1080/10837450.2020.1762645
To link to this article: https://doi.org/10.1080/10837450.2020.1762645
Accepted author version posted online: 28 Apr 2020.
Submit your article to this journal
Article views: 8
Q
View related articles
View Crossmark data ☒ CrossMark
Check for updates
Mitotane liposomes for potential treatment of adrenal cortical carcinoma: ex vivo intestinal permeation and in vivo bioavailability
Zancanella, P.1, Oliveira, D. M. L.2, de Oliveira, B.H.1, Woiski, T.D.3, Pinto, C.C.4, Santana, M.H.A.5, Souto, E.B.6,7*, Severino, P.2,5,8*
1 Department of Chemical, Federal University of Paraná, 81531-990, Curitiba-PR, Brazil
2 Biotechnology Industrial Program, Laboratory of Nanotechnology and Nanomedicine (LNMed), University of Tiradentes, Av. Murilo Dantas, 300, 49010-390 Aracaju, Sergipe, Brazil
3 Research Institute “Pelé Pequeno Príncipe”, 80-250-200 Curitiba, Paraná, Brazil
4 Institute of Technology and Research (ITP), Av. Murilo Dantas, 300, 49010-390 Aracaju, Sergipe, Brazil
School of Chemical Engineering, University of Campinas, 13082-852 Campinas, São Paulo, Brazil
6 Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal
CEB - Centre of Biological Engineering, University of Minho, Campus de Gualtar 4710-057 Braga, Portugal
8 3 Tiradentes Institute, 150 Mt Vernon St, Dorchester, MA 02125, USA
*Corresponding authors:
Patrícia Severino, Program in Industrial Biotechnology, Laboratory of Nanotechnology and Nanomedicine (LNMED), Institute of Technology and Research (ITP), Tiradentes University (UNIT) - Aracaju, Sergipe, Av. Murilo Dantas, 300, Farolândia, Aracaju Sergipe, CEP 49.032- 490, Rel .: +55 (79) 3218-2190 (R-2599), Fax: +55 (19) 999988910
Eliana B. Souto, Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra (FFUC), Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal, Tel .: +351 239 488 400; Fax: +351 239 488 503; E-mail: ebsouto@ff.uc.pt
Ačacada ManusČi E-mail: patricia_severino@itp.org.br
Abstract
The adrenal cortical carcinoma (ACC) treatment, for which mitotane (o,p’-DDD) is the drug of choice, still remains a challenge both because of the well-known solubility problems of the drug, and its serious side effects. Mitotane is currently administered as oral tablets. The loading of mitotane into nanocarriers has been suggested as a way to circumvent the low solubility of the drug and its limited oral bioavailability. In this work, we have developed liposomes containing mitotane to enhance its intestinal absorption and oral bioavailability. Liposomes were produced by spray-drying of a mixture of phospholipids and the developed formulation was optimized by studying the degree of crystallinity, spray-drying conditions, phospholipid/mitotane ratio, and influence of mannitol in the hydrating ethanolic solution. An optimal liposomal formulation was produced with a phospholipid:mitotane combination (3.34:1), exhibiting a mean hydrodynamic diameter around 1 um and spherical shape. The produced mitotane liposomes were re-suspended by hydrating the spray-dried powders in a stirred tank, and tested their intestinal permeability (ex vivo) and relative bioavailability (in vivo), against a free drug solution (with or without Trigliceril®CM). Our results support the conclusion that the loading of mitotane in liposomes enhanced its intestinal absorption and relative bioavailability.
Keywords: mitotane, liposomes, adrenal cortical carcinoma, spray-drying, intestinal absorption, bioavailability.
Accepted® de de los angeles sol
1. Introduction
Adrenal cortical carcinoma (ACC) is an extremely rare, highly aggressive cancer, with very poor prognosis (Lee et al. 2016; Stigliano et al. 2017). The survival rate of patients with advanced disease stages (III/IV) is below 10% (Barlaskar and Hammer 2007), while stages I/II disease patients have a higher survival rate (Asare et al. 2014). The treatment of first choice, - and potentially curative -, is the surgery. At stage III/IV, curative surgery is not recommended (Ohashi et al. 2016), and mitotane is the main chemotherapeutic choice, associated or not to additional antineoplastic drugs, as cisplatin, doxorubicin and etoposide (Xu and Zhu 2013). The number of cases reported in Southern Brazil is about 10 times higher than worldwide, The cases among Brazilian children under 15 years old are associated to a genetic mutation of Tp53 (R337H) (Pianovski et al. 2006).
Mitotane (1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane) is approved by Food and Drug Administration (FDA) for the treatment of ACC. The drug is very poorly-water soluble and shows low oral bioavailability (Biopharmaceutical Classification System BCS IV (Amidon et al. 1995)). The daily needs of high doses (up to 10 g) require daily administration of several pills while the persistent adverse side reactions compromise the quality of life of patients and their therapeutic compliance (Maiter et al. 2016).
The oral administration of mitotane associated with foodstuff (e.g. chocolate, oils, milk or other emulsions) increase its plasma levels compared to the oral intake of conventional tablets (Zancanella et al. 2006). These findings substantiate the need to develop new mitotane formulations in order to improve its solubility, intestinal permeation, and thus its oral bioavailability. Attempts to formulate mitotane in lipid nanocarriers, e.g. oil-in-water (o/w) microemulsions (Attivi D. et al. 2010; Lin et al. 2011), and in lipid nanoparticles have already been published (Grando et al. 2013; Severino Patrícia et al. 2013). Self-microemulsifying drug delivery systems (SMEDDS) containing mitotane were developed and their intestinal permeation evaluated in vivo (Attivi David et al. 2010). The bioavailability tested in rabbits was 3.4 times greater for mitotane-loaded SMEDDS than the commercial product (Lysodren®).
Other lipid nanocarriers extensively employed in drug delivery are the liposomes (Souto 2009; Severino P. et al. 2012; Clares et al. 2014). The main advantages of liposomes are related to their structure, which mimics cell membranes. In addition, these carriers show high biocompatibility and improved oral bioavailability for several drugs (Ali et al. 2012; Teixeira et al. 2017; Souto et al. 2019). Then, the loading of drugs in liposomes can increase their oral absorption, improve therapeutic efficacy, and ultimately reduce the toxicity of the treatment (Makwana and Tandel
2012). Successful liposomal formulations available on the market are those containing doxorubicin (Petersen et al. 2016) and amphotericin B (Li et al. 2016).
Hantel et al. (Hantel et al. 2014) reported the therapeutic efficacy of polychemotherapy regimens based on mitotane-loaded liposomes associated with etoposide, doxorubicin, cisplatin and paclitaxel in an ACC in a preclinical rodent model. Superior anti-tumoral effects were observed when combining different drugs in a liposomal formulation, both during short and long-term exposure. Jung et al. also described the testing of combined regimens of etoposide, doxorubicin, cisplatin, mitotane in liposomal formulations in patients with advance adrenocortical carcinoma (Jung et al. 2016).
The use of one single chemotherapeutic drug, formulated in drug delivery systems with capacity to increase drug bioavailability, will certainly reduce the risk of adverse side effects when compared to polychemotherapeutic regimens. In this work, we propose the development of a new mitotane-liposomal formulation by spray-drying to be resuspended prior to oral administration. Spray-drying technique has been referred as a scalable method in the bulk preparation of a dried lipid mixture for secondary production of multi-lamellar liposomes (Alves and Santana 2004). Production of liposomes are carried out in aqueous medium, where pluri- or uni-lamellar vesicles are formed. The operational variables such as nozzle diameter, lipid concentration, flow rate and temperature of the feed solution, concentration of mannitol as a core material, were found to affect the crystallinity of the obtained liposome powders. Amorphous powders resulting from the spray-drying contribute to increase the stability of loaded drugs, and to enhance the shelf-life of the formulations (Alves and Santana 2004). The aim of this work has been the study of the effect of mannitol on the stability of liposomes produced by spray-drying, and their capacity to load mitotane to improve its intestinal absorption and oral bioavailability. The ex vivo intestinal permeation/absorption and in vivo bioavailability of mitotane-loaded liposomes have also evaluated.
2. Material and methods
2.1. Materials
Hydrogenated soybean phospholipid S (PC-3) was donated by Lipoid (Newark, USA). Mannitol, sulfuric acid and ethanol were supplied by Synth (Diadema, Brazil). Mitotane (racemic mixture) was bought to Yick-Vic Chemicals & Pharmaceuticals (Hong Kong). Hepes/NaCl buffer, sucrose and Tris-HCl were acquired from Sigma (São Paulo, Brazil). TC-199, composed of NaCl, KCI, CaCl2, Na2HPO4 was obtained from Neon (São Paulo, Brazil), and Trigliceril®CM from Danone Nutricia (Campinas, SP, Brazil). Double distilled water was used after filtration in a Millipore system (home supplied).
2.2. Methods
2.2.1. Production and characterization of dried liposomes
Dried liposomes were prepared in a spray-dryer (B-190, Büchi, Switzerland). Initially, feeding liquid dispersions composed of lipids, mitotane and mannitol in ethanol were prepared and pumped into the drying chamber. During pumping, the dispersions were kept heated at 50℃ (TE-0853, Tecnal, Brazil) and under stirring. The composition of the lipid dispersions, and the respective feed flow rates, is summarized in Table 1. The dispersions were atomized into the drying chamber at a temperature of 90+5℃ using a spray nozzle of 1 mm in diameter, and dried in a co-current air flow. The spray-dryer particles were collected in a reservoir attached to a cyclone and stored in a refrigerator prior to their characterization and further use. Three group categories of formulations (Group A, Group B and Group C) were studied, in a range of operational conditions previously defined by Alves and Santana (Alves and Santana 2004). Liposomes of different lipid-to-mitotane (L/M) ratios were produced and the effect of mannitol tested. These spray-dried formulations were further evaluated for their ability to load mitotane.
[Please insert Table 1 about here]
2.2.2. Mass production yield (Recovery)
The mass production yield (%R, Recovery) of the dried particles produced by spray-drying was determined using the equation %R=(Mf/Mi)×100, where My stands for the final mass obtained after spray-drying and Mi for the initial mass weighed to prepare the solution of supply.
2.2.3. Wide angle X-ray diffraction (WAXD)
Polymorphism and crystalline properties of the lipids were studied by WAXD using a diffractometer X-ray (Philips, model X ipert, Pennsylvania, USA), with a copper anode. WAXD measurements were taken from 10° to 35° (20) in 0.015° steps (1 s per step) (Alves and Santana 2004; Edwards and Baeumner 2006).
2.2.4. Scanning Electronic Microscopy (SEM)
The morphology of the dried particles was assessed by SEM (LEO 440i, Leica, EUA) operated at 5 - 6 kV. The samples were coated with gold prior to examination (Alves and Santana 2004; Edwards and Baeumner 2006).
2.2.5. Production of liposomes by hydration and characterization
Liposome formulations were prepared with hydrogenated soy phosphatidylcholine, obtained from the purification of soy lecithin (molar mass of 790 g/mol), which is approved by the FDA for oral and parenteral use. Liposomes were produced by mechanical stirring, in Hepes/NaCl (10 mM/120 mM) buffer solution. Spray-dried samples were put in a tank kept at 65℃, and a sufficient volume of buffer solution was added to obtain a semi-solid mixture (lamellar phase less than 30% of the total volume). This mixture was kept under mechanical stirring (TE-039-1, Tecnal, Brazil) at 100 rpm, for 15 min, using an anchor impeller for homogenization. In the second phase, an excess of buffer solution was added and the dispersion was kept under mechanical stirring for 1 h at 4000 rpm using a blade impeller.
2.2.6. Mean diameter and size distribution
The mean hydrodynamic diameter of liposomes was determined by Dynamic Light Scattering (DLS) (i.e. Photon Correlation Spectroscopy (PCS), Malvern Autosizer 4700, Malvern, UK). For the measurements, the temperature was kept at 25℃ and the laser was set at 633 nm wavelength (Edwards and Baeumner 2006).
2.2.7. Phospholipid content in the spray-dried powders
The concentration of phospholipids in the dried liposomes was estimated by determining the phosphate content, using the method described by Chen and Toribara (Chen et al. 1956). A calibration curve was prepared from a phospholipid standard solution. Each point of the calibration curve was evaluated in triplicate. Briefly, liposomes were added with H2SO4 10 M and heated at 280℃ for 20 minutes, followed by cooling down for 10 minutes. H2O2 was then added and heated again at 280℃ for additional 30 minutes. Finally, deionized water, ammonium molybdate, L-ascorbic acid were added, the mixture homogenized by vortexing, and kept at 100℃ for 7 minutes. After cooling, the reading was done in a spectrophotometer (FEMTO 800XI, São Paulo, Brazil) at 2=830 nm.
2.2.8. Percentage of mitotane recovery and total mitotane content in the powders
To determine the mitotane percentage of recovery (%R) and total mitotane content in the dried powders, a discontinuous sucrose gradient was prepared (15% and 50% sucrose in 5 mM Tris- HCI pH 7.4) (Copland et al. 2000; Nguyen et al. 2002; Daghastanli et al. 2004). Samples of liposomes without drug (control) and loaded with mitotane (100 uL) were placed on the top of gradient and centrifuged (5415, Eppendorf, Germany) at 14.000 x g for 3 h, operated at ambient temperature. The fractions were extracted with hexane (1:1 V/V), based on the need for quantification of mitotane, and homogenized for 60 seconds (Vortex-Genie 2, Model G560, EUA). This procedure was repeated thrice. Hexane was evaporated and the fractions were re- suspended in acetone, and mitotane concentration quantitatively determined by a HPLC system (Varian, Inc (3120 Hansen Way, Palo Alto, CA 94304) with a quaternary pump (model 9012Q), diode array detector (model 9065), and automatic sampler (model AI200)). Chromatography was performed in a Nucleosil C-18 column (4.6 x 250 mm, 5 um particles, 50 Å pores, injection volume 20 uL) and using 85% methanol in 15% water as mobile phase, which was fed at 1
mL/min. The ultraviolet detector was set at 239 nm. Each analysis was run for 7 min (Zancanella et al. 2006). The %R was then determined as the ratio between the weight of loaded mitotane (total mitotane content in liposomes) and the initial weight of mitotane added to the formulation, i.e .:
Initial mass (g) of mitotane weighted for the production (Mi) × 100 %R = Mass (g) of mitotane loaded in spray - dried liposomes (Mf)
2.2.9. Intestinal permeability
The intestinal permeation of mitotane was evaluated using the everted gut sac model. Wistar rats were anesthetized by intraperitoneal injection of sodium pentobarbital and a duodenal segment of the intestine was immediately dissected and flushed with TC-199. The solution was kept at 1ºC. The intestinal duodenal segment was gently everted with the aid of a flexible cotton swab with its extremity protected by a fine fabric. One end of the segment was clamped, filled with fresh TC199 medium and sealed with a second clamp in order to obtain a closed sac. Then, the everted sacs were placed in flasks containing the formulations in TC199 medium with the addition of 10 mM glucose. The solutions were kept oxygenated (O2:CO2= 95:5) and incubated at 37°C. The everted sacs were collected by removing the sacs from the flasks, and then were externally washed with fresh TC199 medium.
To set-up the in vivo experiment, animals were split into three groups, namely, MIT-S (group treated with a formulation produced by adding mitotane powder to TC-90 solution without Trigliceril®CM), MIT-T (group treated with a formulation produced by adding mitotane powder to TC-90 solution containing Trigliceril®CM) and MIT-L (group treated with liposomal formulation), and treated with a volume corresponding to 30 mg/kg of mitotane. Each formulation was administered to the animal once a day, by oral gavage, for 28 days. The duodenal segments were incubated separately for times ranging from 5 to 120 minutes. Mitotane was determined in three compartments, namely: (i) the internal duodenum medium (permeated drug), (ii) the duodenum segment (drug retained in the intestinal mucosa), and (iii) the external environment (non-permeated drug). For each time, three animals were used (n = 3). As a control sample (blank), incubation was conducted for 120 minutes without drug (da Silva et al. 2009).
Samples were analyzed by HPLC, as described above (Zancanella et al. 2006). The recorded data were statistically analyzed by ANOVA, randomized models and randomized blocks, and by Tukey test, using the statistical package Statistica 6.0 (StatSoft). Statistical significance was set
at p <0.05. The in vivo experiment was approved by the Local Ethics Committee, performed and supervised by Cat-C FELASA certified personnel.
2.2.10. Relative mitotane bioavailability
Wistar rats (350-450 g) were split in three groups and treated, respectively, with MIT-S (mitotane solution without Trigliceril®CM), MIT-T (mitotane solution with Trigliceril®CM) and MIT-L (liposomal formulation). The administered mitotane dose was 30 mg/kg. Each formulation was administered to the animal once a day, by oral gavage, for 28 days. Approximately 1 mL of blood was collected with heparin anticoagulant, via tail of each animal, on day 0, 7, 14, and 21 of the experiment. Plasma was separated by centrifugation and kept frozen until the time of analysis. Aliquots of 120 uL of plasma were added with 180 uL of acetone, mixed and centrifuged. The supernatant was separated and concentrated near dryness with the aid of nitrogen gas flow. The residue was re-suspended in 1 mL hexane and applied onto columns for solid phase extraction (Manirakiza et al. 2000; Diez et al. 2006; Rodrigues et al. 2007). After extraction, samples were evaporated to dryness with the aid of nitrogen flow. Samples were then heated at 45℃, re-suspended with 1 mL hexane and analysed using a previously validated gas chromatography assay coupled with an electron capture detection (GC- EC) as described by Hermansoon et al. (Hermansson et al. 2008). Briefly, a Varian 3400 Gas Chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with a CP-SIL 8CB column (25 m x 0,15 mm x 0,12 um) and automatic injector was used, starting at 80℃ (1 min) with increment rates of 20℃/min up to 300℃ (kept for 10 min). Injector was kept at 260℃ and detector at 360℃. The recorded data were analyzed by ANOVA, using the statistical package Statistica 6.0 (StatSoft). Statistical significance was set at p <0.05. The in vivo experiment was approved by the Local Ethics Committee, performed and supervised by Cat-C FELASA certified personnel.
3. Results and discussion
The qualitative and quantitative composition of the developed liposomes (Table 1), and respective operational conditions, has been based on previously published data (Alves and Santana 2004; de Paula Rigoletto et al. 2012). After spray-drying of the bulk mitotane, no individualized particles of the drug have been recovered because of the cyclone efficiency and the very low density of the obtained spray-dried powders. When incorporating phospholipid in
the formulation, a significant increase of mitotane retention in the cyclone was observed. This has been attributed to the creation of a pasty material when homogenized, increasing the adhesion to the cyclone. The percentage of recovery of liposomes produced by spray-drying (%R) is shown in Table 2.
[Please insert Table 2 about here]
While depending on the cyclone efficiency, relatively low yields are usually obtained when spray-drying small amounts of samples. An increase of %R is usually expected when larger batches are produced and when using cyclones with greater efficiency. For drug-loaded liposomes, the production yield varied between 6.45% (F11) and 50.58% (F4), while the control group B (F5) presented the highest yield (58.43%). The loss of phospholipids was quantitatively determined for all the formulations.
WAXD analysis was used to evaluate the degree of crystallinity of bulk materials and liposomes, and should consider the presence and shape of the intensity peaks and their base width (Severino P. et al. 2011). The bulk raw materials were evaluated to monitor the changes of the crystallinity before and after the production process of liposomes. Figure 1 shows the diffractograms recorded for mitotane (1A), mannitol (1B) and hydrogenated soy phosphatidylcholine (1C) as bulk materials. For mitotane and mannitol, a high degree of crystallinity was detected by the presence of the peaks in the diffraction patterns, in comparison to a less crystalline profile (only one peak) of the phospholipid (1C). Liposomes clearly depicted more amorphous profiles (Figure 1 - D, E, F) in comparison to the bulk counterparts (Figure 1 - A, B, C), while the presence of mitotane and mannitol did not influence the shape of the peaks (i.e. a similar profile was recorded among all formulations within the 3 group categories). According to Kikuchi et al., the more amorphous the structure is, the faster it is hydrated when adding the aqueous solution for resuspension (Kukuchi et al. 1991). The hydration of our samples was indeed almost instantaneous, minimizing the time required for resuspension of the dried powders. This property is desirable in dried particles for pharmaceutical use due to the potential increase on the drug’s bioavailability; the ratio of polymorph solubility has however been shown to be typically less than 2 (Pudipeddi and Serajuddin 2005). The production of powders from drugs (e.g. spray- drying, lyophilization) is a common procedure to improve drug stability over storage time. As liposomes also exhibit limited shelf-life in aqueous dispersion, freeze-drying the formulations is usually required. Freeze-drying or lyophilization is however more time-consuming than spray- drying which also produces dried powders. The obtained dried powders should be as less
crystalline as possible to increase their solubility upon resuspension. The degree of crystallinity of the obtained powders is dependent on the production conditions i.e. nozzle diameter, lipid concentration, flow rate and temperature of the feed solution, and the concentration of mannitol. The presence of mannitol in the solid structures increases the hydration efficiency attributed to the increased surface area of the lipids. However, this effect depends on the distribution of mannitol in the total phospholipid matrix. Amorphous powders are usually preferred for oral drug delivery requiring their resuspension in an aqueous solution prior to administration; this increases the drug solubility and thus its bioavailability, opening the opportunity to reduce the dosage required for the therapeutic effect and thus the risk of adverse side effects. The bioavailability of the drug is therefore very much dependent on the polymorphism and degree of crystallinity of the drug delivery system, which translates the dependency between the rate of dissolution in vivo and the rate of absorption. The maximum plasma concentration (Cmax) and the time required to achieve it (Tmax) are the main parameters that have an impact in vivo. This fact reflects the consequences of the polymorphism on the solubility since the most stable form (lower free energy) has lower solubility. In most cases, this results in lower dissolution rate and, consequently, lower rate of absorption.
[Please insert Figure 1 about here] [Please insert Figure 2 about here]
Figure 2 shows the SEM results of the formulations produced as described in Table 1. Figures 2B, 2E, and 2H have similar crystalline patterns, which resemble the bulk phospholipid. Comparing the control groups (Figure 2 - B, E, H), the addition of mannitol caused the loss of the spherical shape of liposomes. Figure 2B shows the spherical shape of liposomes with uniform structural arrangement for pure phospholipid (Control Group A), with a diameter of 2.33 ± 0.91 um as determined by SEM. Figure 2E, for the formulation containing phospholipid with 36 mM mannitol, the presence of a film background was observed. Figure 2H, for the formulation containing phospholipid with 92 mM mannitol, no individual vesicles were observed, while the predominant lipid film was kept. Figure 2C (phospholipid/mitotane 1:1) and Figure 2D (phospholipid/mitotane 2:1) show that the addition of mitotane changed the vesicles’ morphology, where no homogeneous liposomes were observed. This result is in agreement with the increased polydispersity obtained when loading liposomes with mitotane, in particular for the L/M ratio 1:1 (Table 3). Figure 2E (Control Group B) shows few vesicles and the presence of a lipid film. In Figures 2F and 2G (phospholipid/mitotane 1:1 and phospholipid/mitotane 2:1) no
homogeneous vesicles were produced but the predominant lipid film was observed. In the presence of mitotane, no formation of vesicles was seen but only the typical lipid film. In Figure 2H (Control Group C) no vesicles were recorded. In Figure 2L (phospholipid/mitotane 2:1) vesicles are shown, while in Figure 2I (phospholipid/mitotane 1:0.25) and Figure 2J (phospholipid/mitotane 1:1) only a lipid film was kept.
The mean size diameter of liposomes was determined by dynamic light scattering and correlated with the percentage of mitotane recovery (%R) determined as the ratio between the mass of mitotane before and after spray-drying. Results are summarized in Tables 2 and 3. Among Group A, formulation 4 (L/M 2:1) showed the smallest hydrodynamic diameter but the highest %R of mitotane from spray-dried liposomes. Among Group B, the decrease of the hydrodynamic diameter of liposomes was followed by the increase of the %R of mitotane, for which the affinity of mitotane to phospholipids is attributed to the high lipophilic character of the drug. Group C formulations showed the highest diameter due to higher phospholipid content. Formulation 10 showed the presence of aggregates, noticed by particles with diameter higher than 3 um (i.e. 3.656±0.107 um). Particles larger than 1 um exhibited a trend towards precipitation.
[Please insert Table 3 about here]
The mean size of colloidal carriers (e.g. liposomes, nanoparticles) is known to have impact on the drug’s bioavailability and pharmacokinetics, as it influences clearance/phagocytosis via mononuclear system (at liver, spleen, bone marrow, among other tissues), responsible for taking the particles out of the circulation, releasing the drug over time (FDA 2008). The lipids, as excipients, can be digested and dispersed in the gastrointestinal tract, releasing the drug.
PLGA (poly-lactic acid and poly-glycolic acid 50:50 w/w) has been previously used to produce particles of different sizes (100 nm, 500 nm, 1 um and 10 um), to evaluate the effect of the mean particle size on the oral uptake of loaded bovine albumin by the gastrointestinal tract (Desai et al. 1996). In the study reported by Frohlich and Roblegg, the uptake efficiency was quantitatively analyzed by an in vitro assay. The 100 nm diameter particles underwent the highest gut uptake when compared to the other sized populations. In the duodenum, the number of particles/mm2 was 2.7 x 109 and 1.3 x 105, respectively, for 100 nm and 1 um of particle size diameters. In the ileum, the number of particles/mm2 was 4.4 x 109 and 6.5 x 105 for 100 nm and 1 um of particle size diameters (Frohlich and Roblegg 2012). The mean hydrodynamic diameter of our mitotane- loaded liposomes, produced by spray-drying, was within the range of 1 um. While having limited application for parenteral route, the size of our liposomes was not an exclusion criterion
for oral administration. Considering the limited long-term physicochemical stability of liposomes in water (e.g. risk of oxidation, drug leakage, vesicles aggregation and/or disruption), a case is made about the use of free-flowing liposome powders obtained by spray-drying. The advantages of using liposomes in oral drug delivery rely on the improvement of intestinal permeation and increased bioavailability by changes in the coefficient partition, which is dependent on the concentration of the phospholipids in the formulation. Biocompatibility, drug protection from the gastrointestinal tract and from pre-systemic metabolism, increased residence time of the drug in the site of absorption, increased drug diffusion through mucosal and epithelial layers, with enhanced bioavailability, are advantages of the oral administration of liposomes (Daeihamed et al. 2017; Teixeira et al. 2017; Pashirova et al. 2018).
The phospholipid concentration in the dried liposomes was estimated by the measurement of the phosphate content. The organic material was exposed to acid digestion and the generated molybdenum blue was quantified spectrophotometrically (2=830 nm). The analytical curve (5 points concentration) was obtained with a correlation coefficient of 0.9979. Table 4 shows the results of the quantitative analysis of phospholipids, before and after spray-drying. Each formulation was analyzed in triplicate. When comparing the results shown in Table 4 with the percentage of recovery of liposomes (Table 2), F4 was shown to have the highest yield of all mitotane-loaded formulations with ca. 50% of the phospholipid concentration after spray-drying. On the other hand, while F10 showed the highest phospholipid concentration after spray-drying (ca. 100%), its percentage of recovery of liposomes was 15.38%.
[Please insert Table 4 about here]
Table 5 shows the indirect determination of phospholipid/mitotane ratios. The mitotane molar concentration in the formulations was determined considering the initial mass taken for spray- drying, and the phospholipid molar concentration. Table 5 shows the results obtained for the determination of phospholipid/mitotane (L/M) ratios for F4 (Group A), F6 and F7 (Group B). As the mass of mannitol was not considered, the indirect L/M ratios recorded for groups B and C are an estimation. F4 was chosen for further ex vivo and in vivo experiments.
[Please insert Table 5 about here]
Mitotane-loaded liposomes of F4 and F2 (control) were separated from the free fraction by differential migration, depending on the density, layered with different sucrose concentrations.
The association efficiency of mitotane within liposomes was determined by separating the non- loaded drug by differential migration using sucrose gradient and quantifying the layers by HPLC. The sucrose gradient was separated in two portions, each of them analyzed by DLS and mitotane content determination by HPLC (Table 6). The obtained results clearly demonstrate that mitotane was incorporated within the lipid bilayers of liposomes during spray-drying, while after hydrating the dried liposomes, the drug was mostly encapsulated inside the vesicles. The formulation of highly hydrophobic drugs in liposomes by conventional methods usually leads to low loadings within the vesicles. The addition of the drug to the lipid mixture before spray- drying was shown to be a suitable loading process of drugs in liposomes, with the additional advantages of being a scalable process and offering improved physicochemical stability of the colloidal carriers over storage (Misra et al. 2009; Yin et al. 2014).
[Please insert Table 6 about here]
To evaluate the drug intestinal permeation, the intestinal segments were immediately removed from mice with maintenance of optimal biological activity for up to 2 hours by the use of TC 199 as cell culture medium. The tissue was kept at 37℃ to simulate the body temperature, and under constant stirring, which simulates the bowel movements during peristalsis. This system is used to follow the uptake of liposomes, proteins, and macromolecules, described for the study of oral delivery systems (Barthe L et al. 1998; He et al. 1998; Barthe Laurence et al. 1999).
The purpose of the test was to compare the mitotane permeation rate among the different formulations, namely, two conventional drug solutions (MIT-S, mitotane solution without Trigliceril®CM; MIT-T, mitotane solution with Trigliceril®C) and the optimized liposomal formulation developed in this work (MIT-L, F4 - L/M 2:1). Trigliceril®CM has been selected as oil medium because its use is a common practice in hospitals to improve the oral bioavailability of mitotane (Moolenaar et al. 1981). The calibration curve for the determination of mitotane in TC 199 medium was obtained from seven points (5 µg/mL; 25 µg/mL; 50 µg/mL; 500 µg/mL; 1 mg/mL; 5 mg/ml; 10 mg/mL), analyzed in triplicate, with a correlation coefficient of 0.9998.
For the ex vivo experiments, a volume of F4 corresponding to 6.516 mg of mitotane was added to the incubation medium. The amount of mitotane was determined by HPLC and the results are shown in Figure 3, where 3A stands for the amount of drug permeated in duodenum segment, 3B for the amount of drug retained in the tissue and 3C for the amount of drug not permeated in the tissue. After 120 minutes, the percentage of mitotane determined in the internal medium was 3.8%, 10.0% and 44.8%, respectively, for MIT-S, MIT-T and MIT-L (F4). These results
translate changes in the intestinal permeation induced by the liposomal formulation, attributed to the octanol-water partition. Notwithstanding is the dose of mitotane, which is about five times lower in liposomes than in conventional mitotane solutions used as reference.
[Please insert Figure 3 about here]
Statistically significant differences (p < 0.05) were recorded between the rates of permeated mitotane (measured in the internal medium, 3A) from MIT-L, in comparison to the standard solutions (MIT-S and MIT-T), which means that improved permeation rates were obtained with F4. Figure 3B shows the percentage of the total mass of mitotane in bowel segments obtained after extraction using hexane. About 26.9% of mitotane was recovered from intestinal segments. Mitotane concentrations of 9.3% and 26.9% were quantified in intestinal segment when using, respectively, free drug and conventional formulation. The amount of drug quantified in the internal medium was 3.2% and 5.8%, respectively, from free drug and conventional formulation. In the same experimental conditions, when using the liposomal formulation in the intestinal mucosa, the amount of drug absorbed varied between 10.7% and 25.9%. When mitotane is delivered as conventional formulation (MIT-T), the drug has greater affinity for the intestinal mucosa than the aqueous gastrointestinal fluids, being thus retained in the tissue. On the other hand, when delivered in form of liposomes (MIT-L), the vesicles adhere to the intestinal mucosa, being the drug taken-up in higher extent which contributes to the enhanced drug bioavailability (p <0.05).
Figure 3C shows the percentage of mitotane quantified in the external intestinal medium, which translates the non-permeated mitotane, illustrating the low water solubility of this drug. The variation of the percentage of mitotane over time was attributed to the non-homogeneous distribution of mitotane in aqueous medium. Mitotane is a class IV of the Biopharmaceutical Classification System (BCS) which means it shows low solubility and low bioavailability. This justifies the insolubility of the free drug in TCC medium. Adding the oily fatty acid Trigliceril® CM, it will promote the solubility of the drug in the buffer solution. The liposomal formulation had ~ 1000 nm size, characterizing a colloidal system consisting of concentric bilayers, comprising an internal aqueous compartment favoring the homogenization of the formulation in the buffer solution. In addition, liposomes exhibited high affinity for biological membranes.
While the concentration of mitotane in the liposomal formulation is about five times lower than the tested standard drug solutions (MIT-S and MIT-T), the amount of mitotane quantified by HPLC in the external intestinal medium over time was more homogeneous when treated with
MIT-L than with MIT-S and MIT-T. Indeed, the variations of mitotane concentration over the course of the experiment (Figure 3C) clearly translate that liposomes offer a more constant release, while in conventional formulations the low solubility of mitotane in the aqueous medium contributes to a non-homogeneous variation of the percentage of mitotane.
The use of the everted intestinal approach for the ex vivo permeability of drugs loaded in colloidal carriers offers the advantage of being simple, fast, reproducible and of low cost. However, it is also recognized that the cellular heterogeneity, and the fact that the luminal and basolateral membranes are studied together, compromises the significance of the results. Measures may be affected by the backflow of the drug and/or by the absorption via paracellular pathways. On the other hand, the animal model does not reflect the real in vivo situation in humans, as the physiological parameters, such as transit time or presence of food, and also the influence of irrigation and nervous system, are also not taken into consideration in the assay (Barthe Laurence et al. 1999). To demonstrate the specificity of the cholesterol absorption, Westover et al. marked cholesterol and its synthetic enantiomer (Westover et al. 2006), and formulations were administered to hamsters by oral gavage following the measurements by mass spectrometry of the intestinal mucosa and in tests recovery in feces. The results suggested that cholesterol absorption is structurally specific and appears to be mediated by cellular proteins that have enantiomeric specificity (Westover et al. 2006).
To study the effect of the enantiomers on the intestinal permeation of mitotane, the internal medium samples were analyzed by HPLC with chiral column of y-cyclodextrin for each tested formulation containing all mitotane as racemic mixture obtained after 120 minutes of incubation the intestinal permeation. In the analyzed samples, the enantiomers (R)-(+) and (S)-(-) of mitotane were identified in equal proportions, suggesting no chiral specificity in permeation of the intestinal membrane to mitotane.
According to the literature, when taken-up by the same administration route, it is expected that the pharmacokinetics of a drug-loaded in a liposomal formulation follows the same pathway as the kinetics of the non-loaded drug. For this reason, the pharmacokinetic study should include a comparative evaluation of liposomal and non-liposomal formulations for the establishment of a dose. As mitotane has a very narrow therapeutic window, which means that levels of toxicity can be reached easily and fast with the risk of animal’s death, for the in vivo studies, a dosage of 30 mg/kg of drug was administered to rats by oral gavage. Mitotane-loaded formulations were administered for 21 days to evaluate the plasma concentration in blood samples collected on day 0, 7, 14 and 21, for the assessment of mitotane bioavailability.
For the determination of mitotane concentration in the plasma of rats, plasma proteins were first precipitated by vortexing 120 uL rat plasma with 180 µL acetone, followed by centrifugation, and drying of supernatant under nitrogen stream. Briefly, approximately 1 mL of blood was collected with heparin as anticoagulant, from the tail of each animal on day 0, 7, 14, and 21. The plasma was separated by centrifugation and stored frozen until the time of analysis. The plasma samples were thawed at room temperature and prepared with acetone. After mixed and centrifugation, the supernatant was concentrated to dryness, the residue was re-suspended with 1 mL hexane and used for solid phase extraction. The eluate from the solid phase extraction was evaporated with nitrogen stream, the residue was re-suspended with 1 mL hexane and analyzed by GC-EC.
Figure 4 shows the plasma concentrations of mitotane, throughout the 21 days of treatment, for standard (MIT-S and MIT-T) and liposomal (MIT-L) formulations. It should be noted that the dose of available mitotane in liposomes (2.117 mg) is about five times lower than the dose administered to the animals treated with the standard solutions which received 10 mg of free mitotane daily. The mitotane concentration in plasma was shown to be lower when animals were treated with MIT-L (4.6 ng/ml), in comparison to the standard solutions of MIT-S (6.4 ng/ml) and MIT-T (5.3 ng/ml) (p<0.05).
[Please insert Figure 4 about here]
Figure 5 shows that mitotane plasma (expressed as a percentage of the total administered) for the liposomal formulation, from 14 days of chronic treatment, is higher than the concentration for obtained with the conventional formulations (p <0.05). These results corroborate those obtained with the everted intestinal segment, in which MIT-L offered slower permeation rates, compared to MIT-S and MIT-T
[Please insert Figure 5 about here]
In the evaluation of plasma concentrations, a significant difference between the behaviors of the animals was observed depending on the treatment. The animals receiving MIT-L were less irritable, more docile, and less resistant at the time of administration of the formulation. Such behavioral profile is attributed to the lower incidence of adverse side effects which could be associated with a higher dose of mitotane. Indeed, the main purpose of loading the drug in liposomes is to achieve a higher bioavailability with the minimum of administered dose, which is
translated by keeping the plasma concentrations within the therapeutic range (i.e. absence of concentration peaks and with lower incidence of adverse reactions).
4. Conclusions
To the best of our knowledge, this work represents the first in vivo study, reporting the development and administration of a resuspended liposomal formulation for the oral delivery of mitotane. We have achieved ca. 100% of association efficiency of mitotane in liposomes after re- hydrating the amorphous Lipid:Mitotane (2:1) formulation produced by spray-drying. We have demonstrated that mitotane was incorporated within the lipid bilayers of liposomes during spray- drying, while after hydrating the dried liposomes, the drug was mostly encapsulated inside the vesicles. The amorphous status of spray-dried liposomes was confirmed by WAXS analysis which demonstrate that mannitol did not influence the crystallinity profile of liposomes, while the changes in their morphology after loading them with mitotane was confirmed by SEM. The mean hydrodynamic diameter was within the range of 1 um, which is considered suitable for oral administration. To evaluate the intestinal permeation of mitonane, ex vivo everted intestinal studies were carried out, comparing the mitotane-loaded liposomes with standard solution of the drug i.e. MIT-S (mitotane solution without Trigliceril®CM) and MIT-T (mitotane solution with Trigliceril®CM). When mitotane was delivered as conventional formulation (MIT-T), the drug has greater affinity to the intestinal mucosa than the aqueous gastrointestinal fluids, being therefore retained in the tissue. On the other hand, when delivered in form of liposomes (MIT-L) the vesicles adhere to the intestinal mucosa, being the drug up taken in higher extent which contributes to the enhanced drug bioavailability. The amount of mitotane quantified in intestinal segment when using free mitotane (without Trigliceril®CM) was low as 9.3%, in comparison to 26.9% and 25.9% for mitotane solution with Trigliceril®CM and liposomal formulations, respectively. The in vivo studies demonstrated that liposomes were able to control mitotane concentration in plasma, which was shown to be lower (4.6 ng/ml), in comparison to the standard solutions of free drug (6.4 ng/ml) and mitotane solution with Trigliceril®CM (5.3 ng/ml). These results justify the effect of mitotane plasma concentration on the behavior of animals, translating less side effects with the lowest drug concentration.
Acknowledgements
The authors wish to acknowledge the sponsorship of the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP - Processo 20801-2 21219-5), to Fundação de Apoio a Pesquisa e à Inovação Tecnológica do Estado de Sergipe (Fapitec) and to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, 2014-6, 2014- 5). This work was also financed through the project M-ERA-NET-0004/2015-PAIRED and UIDB/04469/2020 (strategic fund), from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/MEC) through national funds, co-financed by FEDER, under the Partnership Agreement PT2020.
References
Ali MH, Moghaddam B, Kirby DJ, Mohammed AR, Perrie Y. 2012. The role of lipid geometry in designing liposomes for the solubilisation of poorly water soluble drugs. International journal of pharmaceutics. Eng.
Alves GP, Santana MH. 2004. Phospholipid Dry Powders Produced by Spray Drying Processing: structural, thermodynamic and physical properties. Powder Technology. 145(2):139-148.
Amidon G, Lennernãs H, Shah VP, Crison JR. 1995. A theoretical basis for a biopharmaceutical drug classification: the correlation of in vivo drug product dissolution and in vivo bioavailability. Pharm Res. 12:414-420.
Asare EA, Wang TS, Winchester DP, Mallin K, Kebebew E, Sturgeon C. 2014. A novel staging system for adrenocortical carcinoma better predicts survival in patients with stage I/II disease. Surgery. 156(6):1378-1385; discussion 1385-1376.
Attivi D, Ajana I, Astier A, Demore B, Gibaud S. 2010. Development of microemulsion of mitotane for improvement of oral bioavailability. Drug development and industrial pharmacy. 36(4):421-427.
Attivi D, Ajana I, Astier A, Demoré B, Gibaud S. 2010. Development of microemulsion of mitotane for improvement of oral bioavailability. Drug development and industrial pharmacy. 36(4):421-427.
Barlaskar FM, Hammer GD. 2007. The molecular genetics of adrenocortical carcinoma. Rev Endocr Metab Disord. 8(4):343-348. eng.
Barthe L, Bessouet M, Woodley J, Houin G. 1998. The improved everted gut sac: a simple method to study intestinal P-glycoprotein. International journal of pharmaceutics. 173(1):255- 258.
Barthe L, Woodley J, Houin G. 1999. Gastrointestinal absorption of drugs: methods and studies. Fundamental & clinical pharmacology. 13(2):154-168.
Chen PS, Toribara TY, Warner H. 1956. Microdetermination of Phosphorus. Analytical Chemistry. 28(11):1756-1758.
Clares B, Calpena AC, Parra A, Abrego G, Alvarado H, Fangueiro JF, Souto EB. 2014. Nanoemulsions (NEs), liposomes (LPs) and solid lipid nanoparticles (SLNs) for retinyl palmitate: effect on skin permeation. Int J Pharm. 473(1-2):591-598.
BLUECALE p
Copland MJ, Rades T, Davies NM. 2000. Hydration of lipid films with an aqueous solution of Quil A: a simple method for the preparation of immune-stimulating complexes. International journal of pharmaceutics. 196(2):135-139. eng.
da Silva CF, Severino P, Martins F, Chaud MV, Santana MHA. 2009. The intestinal permeation of didanosine from granules containing microspheres using the everted gut sac model. Journal of microencapsulation. 26(6):523-528.
Daeihamed M, Dadashzadeh S, Haeri A, Akhlaghi MF. 2017. Potential of Liposomes for Enhancement of Oral Drug Absorption. Current Drug Delivery. 14(2):289-303.
Daghastanli KR, Ferreira RB, Thedei G, Jr., Maggio B, Ciancaglini P. 2004. Lipid composition- dependent incorporation of multiple membrane proteins into liposomes. Colloids and surfaces B, Biointerfaces. 36(3-4):127-137. eng.
de Paula Rigoletto T, Silva CL, Santana MHA, Rosada RS, de la Torre LG. 2012. Effects of extrusion, lipid concentration and purity on physico-chemical and biological properties of cationic liposomes for gene vaccine applications. Journal of Microencapsulation. 29(8):759-769.
Desai MP, Labhasetwar V, Amidon GL, Levy RJ. 1996. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharmaceutical research. 13(12):1838-1845. eng.
Diez C, Traag W, Zommer P, Marinero P, Atienza J. 2006. Comparison of an acetonitrile extraction/partitioning and “dispersive solid-phase extraction” method with classical multi- residue methods for the extraction of herbicide residues in barley samples. Journal of Chromatography A. 1131(1):11-23.
Edwards KA, Baeumner AJ. 2006. Analysis of liposomes. Talanta. 68:1432-1441.
mFDA. 2008. http://www.fda.gov/ohrms/dockets/ac/01/slides/3763s2_10_KUMI.PPT. Access 09/23/2015.
Frohlich E, Roblegg E. 2012. Models for oral uptake of nanoparticles in consumer products. Toxicology. 291(1-3):10-17.
Grando CRC, Guimarães CA, Mercuri LP, Matos JdR, Santana MHA. 2013. Preparation and Characterization of Solid Lipid Nanoparticles Loaded with Racemic Mitotane. Journal of Colloid Science and Biotechnology. 2(2):140-145.
Hantel C, Jung S, Mussack T, Reincke M, Beuschlein F. 2014. Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocrine-related cancer. 21(3):383-394.
He P, Davis SS, Illum L. 1998. In vitro evaluation of the mucoadhesive properties of chitosan microspheres. International journal of pharmaceutics. 166(1):75-88.
Hermansson V, Cantillana T, Hovander L, Bergman A, Ljungvall K, Magnusson U, Törneke K, Brandt I. 2008. Pharmacokinetics of the adrenocorticolytic compounds 3-methylsulphonyl-DDE and o,p’-DDD (mitotane) in Minipigs. Cancer chemotherapy and pharmacology. 61(2):267-274. eng.
Jung S, Nagy Z, Fassnacht M, Zambetti G, Weiss M, Reincke M, Igaz P, Beuschlein F, Hantel C. 2016. Preclinical progress and first translational steps for a liposomal chemotherapy protocol against adrenocortical carcinoma. Endocrine-related cancer. 23(10):825-837. eng.
Kukuchi H, Yamauchi H, Hirota S. 1991. A Spray-Drying Method for Mass Production of Liposomes. Chemical & Pharmaceutical Bulletin. 39(6).
Lee D, Yanamadala V, Shankar GM, Shin JH. 2016. Metastatic adrenal cortical carcinoma to T12 vertebrae. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia. 27:166-169.
Li S-S, Tang X-Y, Zhang S-G, Ni S-L, Yang N-B, Lu M-Q. 2016. Voriconazole combined with low-dose amphotericin B liposome for treatment of cryptococcal meningitis. Infectious Diseases. 1-3.
a
Lin YH, Chen YS, Wu TC, Chen LJ. 2011. Enhancement of dissolution rate of mitotane and warfarin prepared by using microemulsion systems. Colloids and surfaces B, Biointerfaces. 85(2):366-372.
Maiter D, Bex M, Vroonen L, T’Sjoen G, Gil T, Banh C, Chadarevian R. 2016. Efficacy and safety of mitotane in the treatment of adrenocortical carcinoma: A retrospective study in 34 Belgian patients. Annales d’Endocrinologie. 77(5):578-585.
Makwana K, Tandel H. 2012. Design and characterization of anionic PEGylated liposomal formulation loaded with paclitax for ovarian cancer. J Pharm Bioallied Sci. 4(Suppl 1):S17-18. eng.
Manirakiza P, Covaci A, Schepens P. 2000. Single step clean-up and GC-MS quantification of organochlorine pesticide residues in spice powder. Chromatographia. 52(11-12):787-790.
Misra A, Jinturkar K, Patel D, Lalani J, Chougule M. 2009. Recent advances in liposomal dry powder formulations: preparation and evaluation. Expert Opinion on Drug Delivery. 6(1):71-89.
Moolenaar A, Van Slooten H, Van Seters A, Smeenk D. 1981. Blood levels of o, p’-DDD following administration in various vehicles after a single dose and during long-term treatment. Cancer chemotherapy and pharmacology. 7(1):51-54.
Nguyen XT, Pabarue HA, Geyer RR, Shroyer LA, Estey LA, Parilo MS, Wilson KS, Prochaska LJ. 2002. Biochemical and biophysical properties of purified phospholipid vesicles containing bovine heart cytochrome c oxidase. Protein Expr Purif. 26(1):122-130. eng.
Ohashi K, Hayashi T, Sakamoto M, Iuchi H, Suzuki H, Ebisawa T, Tojo K, Sasano H, Utsunomiya K. 2016. Aldosterone-producing adrenocortical carcinoma with prominent hepatic metastasis diagnosed by liver biopsy: a case report. BMC endocrine disorders. 16:3.
Pashirova TN, Zueva IV, Petrov KA, Lukashenko SS, Nizameev IR, Kulik NV, Voloshina AD, Almasy L, Kadirov MK, Masson P et al. 2018. Mixed cationic liposomes for brain delivery of drugs by the intranasal route: The acetylcholinesterase reactivator 2-PAM as encapsulated drug model. Colloids and surfaces B, Biointerfaces. 171:358-367.
Petersen GH, Alzghari SK, Chee W, Sankari SS, La-Beck NM. 2016. Meta-analysis of clinical and preclinical studies comparing the anticancer efficacy of liposomal versus conventional non- liposomal doxorubicin. Journal of Controlled Release.
Pianovski MA, Maluf EM, de Carvalho DS, Ribeiro RC, Rodriguez-Galindo C, Boffetta P, Zancanella P, Figueiredo BC. 2006. Mortality rate of adrenocortical tumors in children under 15 years of age in Curitiba, Brazil. Pediatr Blood Cancer. 47(1):56-60. eng.
Pudipeddi M, Serajuddin AT. 2005. Trends in solubility of polymorphs. Journal of pharmaceutical sciences. 94(5):929-939.
Rodrigues MV, Reyes FG, Magalhães PM, Rath S. 2007. GC-MS determination of organochlorine pesticides in medicinal plants harvested in Brazil. Journal of the Brazilian Chemical Society. 18(1):135-142.
Severino P, Moraes LF, Zanchetta B, Souto EB, Santana MH. 2012. Elastic liposomes containing benzophenone-3 for sun protection factor enhancement. Pharmaceutical development and technology. 17(6):661-665.
Severino P, Pinho SC, Souto EB, Santana MH. 2011. Polymorphism, crystallinity and hydrophilic-lipophilic balance of stearic acid and stearic acid-capric/caprylic triglyceride matrices for production of stable nanoparticles. Colloids and surfaces B, Biointerfaces. 86(1):125-130.
Severino P, Souto EB, Pinho SC, Santana MH. 2013. Hydrophilic coating of mitotane-loaded lipid nanoparticles: Preliminary studies for mucosal adhesion. Pharmaceutical development and technology. 18(3):577-581.
Souto EB. 2009. A special issue on Lipid-based delivery systems (liposomes, lipid nanoparticles, lipid matrices and medicines). J Biomed Nanotechnol. 5(4):315-316.
Souto EB, Souto SB, Campos JR, Severino P, Pashirova TN, Zakharova LY, Silva AM, Durazzo A, Lucarini M, Izzo AA et al. 2019. Nanoparticle Delivery Systems in the Treatment of Diabetes Complications. Molecules. 24(23).
Stigliano A, Cerquetti L, Lardo P, Petrangeli E, Toscano V. 2017. New insights and future perspectives in the therapeutic strategy of adrenocortical carcinoma (Review). Oncology reports. 37(3):1301-1311.
Teixeira MC, Carbone C, Souto EB. 2017. Beyond liposomes: Recent advances on lipid based nanostructures for poorly soluble/poorly permeable drug delivery. Prog Lipid Res. 68:1-11.
Westover EJ, Lin X, Riehl TE, Ma L, Stenson WF, Covey DF, Ostlund RE. 2006. Rapid transient absorption and biliary secretion of enantiomeric cholesterol in hamsters. Journal of lipid research. 47(11):2374-2381.
Xu YZ, Zhu Y. 2013. Conventional chemotherapy and emerging targeted therapy for advanced adrenocortical carcinoma. Anti-cancer agents in medicinal chemistry. 13(2):248-253.
Yin F, Guo S, Gan Y, Zhang X. 2014. Preparation of redispersible liposomal dry powder using an ultrasonic spray freeze-drying technique for transdermal delivery of human epithelial growth factor. International journal of nanomedicine. 9:1665-1676. eng.
Zancanella P, Pianovski MAD, Oliveira BH, Ferman S, Piovezan GC, Lichtvan LL, Voss SZ, Stinghen ST, Callefe LG, Parise GA et al. 2006. Mitotane associated with cisplatin, etoposide, and doxorubicin in advanced childhood adrenocortical carcinoma: mitotane monitoring and tumor regression. J Pediatr Hematol Oncol. 28(8):513-524. eng.
Accepted Manusta mu
Figure 1. WAXD diffractograms recorded for (A) mitotane; (B) mannitol; (C) phospholipid, (D) Group A liposomes of (a) control (L); (b) Lipid/Mitotane 1:1; (c) Lipid/Mitotane 2:1; (E) Group B liposomes of (a) control, (b) Lipid/Mitotane 1:1; (c) Lipid/Mitotane 2:1; (F) Group C liposomes of (a) control, (b) Lipid/Mitotane (1:0.25); (c) Lipid/Mitotane (1:1), (d) Lipid/Mitotane (2:1).
Figure 2. SEM analysis of (A) Bulk mitotane; Group A liposomes of (B) Control, (C) Lipid/Mitotane 1:1 and (D) Lipid/Mitotane 2:1; Group B liposomes of (E) control, (F) Lipid/Mitotane 1:1 and (G) Lipid/Mitotane 2:1; Group C liposomes of (H) control, (I) Lipid/Mitotane (1:0.25), (J) Lipid/Mitotane (1:1) and (L) Lipid/Mitotane (2:1). Magnification 500x.
Figure 3. Quantification of mitotane in internal intestinal medium (A), duodenum segment (B) and in external medium (C), recorded after treatment with liposomal formulation (MIT-L), Trigliceril® CM-free solution (MIT-S) and Trigliceril® CM-solution (MIT-T). (standard deviations of n=6)
Figure 4. Plasma concentration of mitotane recorded after chronic treatment with liposomal formulation (MIT-L), Trigliceril® CM-free solution (MIT-S) and Trigliceril® CM-solution (MIT-T). (standard deviations of n=6) Figure 5. Percentage of cumulative concentration of mitotane in plasma recorded after chronic treatment with liposomal formulation (MIT-L), Trigliceril® CM-free solution (MIT-S) and Trigliceril® CM-solution (MIT-T). (standard deviations of n=6)
Accepte owyplatil, les k engelliler
350
300
18000
250
15000
Intensity (a.u.)
Intensity (a.u.)
200
12000
150
9000
100
6000
50
3000
0
0
0
5
10
15
20
25
30
35
40
0
5
10
15
20
25
30
35
40
2 theta
2 theta
(1A)
(1B)
0 5 10 15 20 Accepted Manuscript
300
5000
250
4000
Intensity (a.u.)
200
Intensitu (a.u.)
(c)
3000
150
100
2000
(b)
50
1000
0
(a)
0
5
10
15
20
25
30
35
40
2 theta
10
15
20
25
30
35
40
2 theta
(1C)
(1D - group A)
7000
3500
6000
3000
5000
(d)
Intensity (a.u.)
2500
Intensity (a.u.)
2000
4000
(c)
1500
(b)
3000
1000
2000
(b)
500
1000
0
(a)
(a)
0
5
25
30
35
40
0
0
5
10
15
20
25
30
35
40
2 theta
2 theta
(E - group B)
(F- group C)
20um 14 Mag= 500 x Controle Mitotano LRAC/FEQ/UNICAMP
2um || Mag= 5.00 KX Controle Lip. Crist. LRAC/FEQ/UNICAMP
(2A)
(2B)
Accepted Man
2pm H lag= 5.00 KX Controle Lip. Crist. LRAC/FEQ/UNICAMP
2um Mag= 5.00 K X Cristalino L. M. 2. 1 LRAC/FEQ/UNICAMP
(2C)
(2D)
>
2um || Mag= 5.00 K X Controle Lip. Inter. LRAC/FEQ/UNICAMP
2um H Mag= 5.00 K X Int. L.M.2.1 LRAC/FEQ/UNICAMP
(2E)
(2F)
2um
Mag= 5.00 K X Intermed. L.M 1.1 LRAC/FEQ/UNICAMP
2um
Mag= 5.00 K X Controle Lip. Amorfo LRAC/FEQ/UNICAMP
2μm Mag= 5.00 K X Controle Lip. Amorfo LRAC/FEQ/UNICAMP
2um
Mag= 5.00 K X Amorfo L.M.1.1. LRAC/FEQ/UNICAMP
(2I)
2um | Mag= 5.00 K X Amorfo lip. Mit.2.1 LRAC/FEQ/UNICAMP
(2L)
(2J)
Accepted Manuscript
MIT-L
MIT-S
MIT-T
35
% Mitotane in internal Medium
30
25
20
15
10
5
0
% Mitotane in Duodenum Segment
40 MIT-L Accepted Manuscript
0
5
10
20
40
60
80
120
Time (Minutes)
(3A)
MIT-L
MIT-S
MIT-T
30
25
20
15
10
5
0
0
5
10
20
60
80
120
Time (Minutes)
(3B)
- MIT-S
MIT-T
60
% Mitotane in External Medium
50
40
30
20
10
0
0
5
10
20
40
60
80
120
Time (Minutes)
(3C)
+ MIT-L -MIT-S … MIT-T
Mitotane concentration in plasma (ng/ml)
10
9
8
7
6
5
4
3
2
1
0
0
6
12
18
Time (Days)
21 Accepted Manuscript
0.15
MIT-S
Cumulative amount of mitotane in plasma (ppm)
MIT-T
0.10
MIT-L
0.05
0.00
1
6
12
18
21
Time (Days)
Accepted Manuscript
Table 1. Composition of liposome formulations and feed flow rate conditions.
Table 2. Mass production yield (%R, Percentage of Recovery) of liposomes produced by spray-drying.
Table 3. Mean hydrodynamic diameter and polydispersity index of liposomes determined by dynamic light scattering (n=3).
Table 4. Quantification of phospholipid content in liposomes (n=3).
Table 5. Estimation of mitotane molar proportion in liposomes (n=3). Table 6. Mean hydrodynamic diameter of liposomes and mitotane content as a function of sucrose gradient (n=3).
Accepted Manuscript,
| Formulation | Components concentration (mM) | Ethanol (mL) | Feed flow rate (mL/min) | ||||
|---|---|---|---|---|---|---|---|
| Phospholipid (L) | Mitotane (M) | Mannitol | |||||
| GROUP A | F1 | Mitotane | - | 85 | - | 50 | 20 |
| F2 | Control | 85 | - | - | 50 | 20 | |
| F3 | L/M 1:1 | 170 | 170 | - | 100 | 20 | |
| F4 | L/M 2:1 | 170 | 85 | - | 100 | 20 | |
| GROUP B | F5 | Control | 85 | - | 36 | 100 | 20 |
| F6 | L/M 1:1 | 85 | 85 | 36 | 100 | 20 | |
| F7 | L/M 2:1 | 170 | 85 | 72 | 100 | 20 | |
| GROUP C | F8 | Control | 180 | - | 23 | 100 | 3 |
| F9 | L/M 1:0.25 | 180 | 45 | 92 | 100 | 3 | |
| F10 | L/M 1:1 | 180 | 180 | 92 | Manuscript 100 | 3 | |
| F11 | L/M 2:1 | Accepted 180 | 90 | 92 | 100 | 3 | |
| Formulation | Mass (g) | Recovery (%) | |||
|---|---|---|---|---|---|
| Mi | Mf | ||||
| F1 | Mitotane | 3.336 | 0 | 0 | |
| GROUP A | F2 | Control | 3.336 | 1.581 | 47.39+ |
| F3 | L/M 1:1 | 4.696 | 2.103 | 44.78+ | |
| F4 | L/M 2:1 | 8.032 | 4.063 | 50.58+ | |
| GROUP B | F5 | Control | 3.664 | 2.141 | 58.43± |
| F6 | L/M 1:1 | 10.408 | 4.784 | 45.97± | |
| F7 | L/M 2:1 | 10.752 | 3.517 | 32.71₺ | |
| GROUP C | F8 | Control | 1.976 | 0.512 | 25.91* |
| F9 | L/M 1:0.25 | 4.835 | 0.812 | 16.80* | |
| F10 | L/M 1:1 | 5.392 | 0.829 | 15.38* | |
| Accepted F11 | L/M 2:1 | 4.672 | Manustep 0.301 | 6.45* | |
Mi: before spray drying; Mj. after spray drying; *** , no statistical differences have been recorded by one-way ANOVA among samples within the same group.
| Formulation | Mean size (um) | Polydispersity index | ||
|---|---|---|---|---|
| GROUP A | F1 | Mitotane | - | - |
| F2 | Control | 1.238 ± 0.015 | 0.15± 0.03 | |
| F3 | L/M 1:1 | 1.190±0.032 | 0.19±0.01 | |
| F4 | L/M 2:1 | 1.006± 0.001 | 0.09 ± 0.00 | |
| GROUP B | F5 | Control | 1.592±0.004 | 0.18±0.11 |
| F6 | L/M 1:1 | 0.970 ± 0.002 | 0.17±0.04 | |
| F7 | L/M 2:1 | 0.795 ± 0.102 | 0.16±0.02 | |
| GROUP C | F8 | Control | 1.702 ± 0.006 | 0.22 ±0.01 |
| F9 | L/M 1:0.25 | 1.915± 0.011 | 0.24 ± 0.00 | |
| F10 | L/M 1:1 | 1.195 ± 0.023 (84%) | 0.29± 0.10 | |
| 3.656± 0.107 (16%) | ||||
| F11 | L/M 2:1 | 1.338 ± 0.061 | 0.15±0.08 | |
Accepted Manuscript
| FORMULATIONS | Phosphate level (mM) | |||
|---|---|---|---|---|
| Initial* | Final ** | |||
| GROUP A | F1 | Mitotane | - | - |
| F2 | Control | 85 ± 0.20 | 65.9± 1.13 | |
| F3 | L/M 1:1 | 170± 1.20 | 91.6± 2.20 | |
| F4 | L/M 2:1 | 170± 2.00 | 85.0±2.34 | |
| F5 | Control | 85 ±0.50 | 77.3 ± 1.45 | |
| GROUP B | F6 | L/M 1:1 | 85±0.36 | 56.6±0.75 |
| F7 | L/M 2:1 | 170± 1.20 | 83.0± 0.75 | |
| GROUP C | F8 | Control | 90±0.60 | 79.9± 0.45 |
| F9 | L/M 1:0.25 | 90±0.28 | 79.4±1.23 | |
| F10 | L/M 1:1 | 90±0.13 | 92.0±4.22 | |
| F11 | L/M 2:1 Accepted | 180±0.05 Manuscrits | 83.9± 0.32 | |
*Initial phosphate level - before spray drying;
** Final phosphate level - after spray drying
| Formulation | Phospholipid (mM) | Initial mass (g) | Final mass (g) | Yield (%) | Lipid (nM) | Lipid (g) | Mitotane (g) | Mitotane (mM) | Molar proportion (L/M) | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| A Group | F1 | Mitotane | - | 3.34 | 0.00±0.01 | 0.00 | 0.00 | 0.00 | 0.00 | 0.00 | - |
| F2 | Control | 85 | 3.34 | 1.58±0.12 | 47.39 | 65.9 | 1.55 | - | - | - | |
| F3 | L/M 1:1 | 170 | 9.39 | 2.10±0.03 | 44.78 | 91.6 | 2.16 | no | no | - | |
| F4 | L/M 2:1 | 170 | 8.03 | 4.06±0.23 | 50.58 | 85 | 2.00 | 2.06 | 6.40 | 13.20 | |
| B Group | F5 | Control | 85 | 3.66 | 2.14±0.11 | 58.43 | 77.3 | 1.82 | - | - | - |
| F6 | L/M 1:1 | 85 | 10.41 | 4.78±0.13 | 45.97 | 56.6 | 1.33 | 3.45 | 10.08 | 5.20 | |
| F7 | L/M 2:1 | 170 | 10.75 | 2.32±0.15 | 32.71 | 83 | 1.95 | 0.37 | 1.20 | 71.90 | |
| C Group | F8 | Control | 180 | 1.98 | 1.54±0.21 | 25.91 | 79.9 | 1.88 | - | - | - |
| F9 | L/M 1:0.25 | 180 | 4.84 | 2.44±0.13 | 16.80 | 79.4 | 1.87 | no | no | - | |
| F10 | L/M 1:1 | 180 | 5.39 | 0.83±0.11 | 15.38 | 46.0 | 1.08 | no | no | - | |
| F11 | L/M 2:1 | Accepted 180 | 4.67 | 0.30±0.03 | 6.45 | 83.9 | Manuscript 1.98 | no | no | - | |
| Sucrose gradient | Formulation Group A | Layers | Mean size (nm) | Mitotane determination | |
|---|---|---|---|---|---|
| (a) 15%; 50% | (2) Control | Superior | 39.7±0.2 | YES | Within the sensitivity range |
| Inferior | 1238.1 ±5.3 | No | - | ||
| (4) L/M 2:1 | Superior | 659.5± 5.4 | YES | Within the sensitivity range | |
| Inferior | 1006.5 ± 55.3 | No | - | ||
Accepted Manuscript