An oral malaria therapy: Curcumin-loaded lipid-based drug delivery systems combined with β-arteether

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Journal of Controlled Release 172 (2013) 904–913

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An oral malaria therapy: Curcumin-loaded lipid-based drug delivery systems combined with β-arteether Patrick B. Memvanga a,b, Régis Coco a, Véronique Préat a,⁎ a b

Université catholique de Louvain, Louvain Drug Research Institute, Pharmaceutics and drug delivery group, Avenue Mounier 73, B1.73.12, 1200 Brussels, Belgium University of Kinshasa, Faculty of Pharmaceutical Sciences, Laboratoire de Pharmacie galénique, BP 212 Kinshasa XI, Democratic Republic of Congo

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Article history: Received 19 June 2013 Accepted 1 September 2013 Available online 7 September 2013 Keywords: Curcumin β-arteether Lipid-based formulations Oral delivery Caco-2 cells Antimalarial efficacy

a b s t r a c t Curcumin (CC), a potential antimalarial drug, has poor water solubility, stability and oral bioavailability. To circumvent these pitfalls, lipid-based drug delivery systems (LBDDSs) with a high CC loading (30 mg/g) were formulated. In a biorelevant gastric medium, CC-LBDDSs formed particle sizes in the range of 30–40 nm. During in vitro lipolysis, 90–95% of the CC remained solubilized, whereas 5–10% of the CC precipitated as an amorphous solid, with a high rate of re-dissolution in a biorelevant intestinal medium. The transport of the CC-LBDDS across Caco-2 monolayers was enhanced compared with the transport of free drug because of the increased CC solubility. In Plasmodium berghei-infected mice, modest antimalarial efficacy was observed following oral treatment with CC-LBDDSs. However, the combination therapy of CC-LBDDS with a subtherapeutic dose of β-arteetherLBDDS provided an increase in protection and survival rate that was associated with a significant delay in recrudescence. These findings suggest that the combination of oral CC and β-arteether lipid-based formulations may constitute a promising approach for the treatment of malaria. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Malaria is the most prevalent parasitic disease and the foremost cause of morbidity and mortality in the world [1]. Currently, the treatment of choice for malaria is based on the association of an artemisinin-type compound with another drug [1,2]. The combination antimalarials with different mechanisms of action can ensure very high cure rates and prevent the onset of drug resistance or delay its emergence [1]. Indeed, these combination therapies may reduce the impact of artemisinin-induced dormancy (or quiescence) of a subpopulation of the ring stage parasites, a mechanism that allows the parasites to survive under the pressure of high doses of artemisinins [3,4]. However, non-viable parasites can retain the ability to cytoadhere, which contributes to the disease [5]. Moreover, a variety of reasons, including cost considerations, pharmacokinetic mismatch, resistance, crossresistance and side effects due to the partner drug, can also contribute to treatment failure [1,6]. Numerous investigations have demonstrated that curcumin, a hydrophobic polyphenol extracted from the rhizomes of Curcuma longa, exhibits an antimalarial activity both in vitro and in vivo [7,8]. Therefore, curcumin (CC) has been proposed as an attractive partner drug for β-arteether (an artemisinin derivative) due to its short-life (1–2 h), which closely matches the half-life of artemisinin derivatives [9–11]. CC acts as an adjunctive therapeutic strategy in the artemisinincombination therapies to protect against parasite recrudescence and ⁎ Corresponding author. Tel.: +32 2 764 7320; fax: +32 2 764 7398. E-mail address: [email protected] (V. Préat). 0168-3659/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jconrel.2013.09.001

relapse [6,10–14]. Indeed, CC synergizes with artemisinins as an antimalarial to kill the parasites [6,15] and can also reverse cytoadherence and subsequent sequestration of parasitized erythrocytes by the inhibition of NF-κB activation, which downregulates the proinflammatory cytokine responses and the expression of cytoadhesion molecules in endothelial cells [16,17]. By producing a transient induction of reactive oxygen species in infected red blood cells, CC is able to damage the parasitic DNA [7] and to prime the immune system (the activation of TLR2 and IL-10 and antiparasitic antibody production) [11]. CC also upregulates the cyto- and neuro-protective enzymes and the surface expression of CD36 on monocytes/macrophages, which increases their phagocytic activity [17]. Treatment with CC also inhibits the microtubule activity [18] and the formation of hemozoin, an intraerythrocytic pigment that is essential to the survival of the parasites [6,11,13]. Due to its safety, relative abundance and cost effectiveness, CC is a phytochemical of great interest in the treatment of several diseases, including malaria [16]. However, the major disadvantages of CC when administered orally are its low bioavailability due to its poor water solubility, chemical instability at neutral and alkaline pH, poor absorption associated with its high rate of metabolism and its rapid elimination [16,19]. Encapsulations in nanoparticles [13,14,20], nanoemulsions [21,22], cyclodextrins [23], phospholipid complexes [24] or solid dispersions [25] have been proposed to improve the oral bioavailability and the therapeutic efficacy of CC (see [26] for review). However, these strategies are characterized by several weaknesses, such as high costs, cytotoxicity, limited drug loading and entrapment efficiency, poor scale up and the use of organic solvents. Self-emulsifying lipid-based systems are a simple and effective approach to solubilize hydrophobic drugs in

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the gastrointestinal tract and to protect these drugs from enzymatic and/or chemical hydrolysis, and these systems have been developed to improve the oral bioavailability of CC [27–29]. However, no antimalarial evaluation of CC in these systems has been reported. The objective of this study was to develop novel oral lipid-based drug delivery systems (LBDDSs) with (i) a high CC loading, (ii) a high degree of resistance to lipolysis, (iii) an increased intestinal absorption and (iv) antimalarial efficacy either alone or in combination with β-arteether loaded in lipid-based drug delivery systems. 2. Materials and methods 2.1. Materials Curcumin (~80%), L-α-Phosphatidylcholine from egg yolk (~60%), pancreatin from porcine pancreas (activity 8 × USP specifications), tributyrin and all the other chemicals were obtained from SigmaAldrich (Diegem, Belgium). Unless otherwise indicated, all cell culture media and reagents were purchased from Invitrogen Gibco® (Merelbeke, Belgium). Cremophor EL (polyoxyl 35 castor oil) was kindly provided by BASF (Burgbernheim, Germany). Labrafil M2125CS (linoleoyl polyoxyl glycerides), Labrafil M1944CS (oleoyl polyoxyl glycerides), Labrasol (caprylocaproyl polyoxyl glycerides) and Transcutol HP (diethylene glycol monoethyl ether) were kind gifts from Gattefossé (Saint-Priest, France).

Fig. 1. Pseudo-ternary phase diagram for Labrafil M2125CS-Labrasol-Cremophor EL mixture diluted in 0.1 N HCl, pH 1.2 (1:100, w/v). The gray area represents the range of existence of a nanoemulsion. The red dot highlights the selected self-nanoemulsifying system: Labrafil M2125CS-Labrasol-Cremophor EL at weight ratio of 1:1:1.

2.2. Simulated media Fasted state simulated gastric fluid (FaSSGF) contained 80 μM sodium taurodeoxycholate, 20 μM lecithin, 0.1 mg/ml pepsin and 34.2 mM sodium chloride (pH 1.6) [30]. Fasted state simulated intestinal fluid (FaSSIF) contained 50 mM Tris-maleate, 150 mM sodium chloride, 5 mM calcium chloride dihydrate, 5 mM sodium taurodeoxycholate and 1.25 mM lecithin, pH 6.8 [31]. The medium in the receiver compartment of transport studies contained Hank's balanced salt solution (HBSS) + 10 mM Hydroxyethyl piperazine ethanesulfonic acid (HEPES) + 1% (w/v) bovine serum albumin (BSA) [32,33]. The lipolysis media contained 50 mM Tris-maleate, 150 mM sodium chloride, 5 mM calcium chloride dihydrate, 5 mM sodium taurodeoxycholate and 1.25 mM lecithin, pH 7.5 [31,34]. 2.3. The development of CC lipid-based self-emulsifying systems The solubility of CC in various excipients was estimated by dissolving increasing quantities of CC in 10 g of each excipient at room temperature (20 ± 2 °C). After 2 h of stirring, the solubilization of CC was observed visually. The absence of CC crystals was confirmed using a microscope [35]. The solubility of CC in modified biorelevant media (FaSSGF and FaSSIF) and in the receiver compartment of the transport experiment was determined by dispersing a large amount of free CC in these solutions at 37 °C [36]. Over a period of 2 h, aliquots from the biorelevant media were centrifuged for 15 min at 5000 ×g and 37 °C. The supernatants were then diluted with methanol: 3.6% glacial acetic acid (73:27, v/v) and assayed for CC content by HPLC analysis. All experiments were performed in triplicate. Based on the solubility study, the pseudo-ternary phase diagram (Fig. 1) was constructed using the water titration method. Different mixtures of the surfactants (Labrasol and Cremophor EL at different weight ratios from 10:0 to 0:10) were prepared with stirring at room temperature. The oil phase (Labrafil M2125CS) was then added at ratios (w/w) of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10. To determine the feasibility of the self-(nano)emulsification, 1 g of each solution (surfactants + oil) was slowly titrated with 0.1 N HCl, pH 1.2 (100 ml, 37 °C), gently stirred and visually examined for transparency. Another series of self-emulsifying systems was assessed with varying concentrations of Labrafil M2125CS: Labrasol: Transcutol HP mixture (2:2:1, w/w)

from 70 to 100% (w/w) and of Cremophor EL from 0 to 30% (w/w). The droplet size was determined at 37 °C by photon correlation spectroscopy using a Zêtasizer Nano ZS model ZEN 3600 (Malvern Instrument Ltd, UK). Finally, to determine the maximum loading content of the CC in the lipid-based systems, its solubility in the selected formulations was determined in triplicate. 2.4. Preparation and characterization of CC-LBDDS 2.4.1. Preparation of CC-LBDDS The CC-LBDDSs were prepared by mixing under agitation (400 rpm, 30 min, 20 °C) the appropriate quantities of excipients (see Table 1). Thirty milligrams of CC was then added to 1 g of each LBDDS preconcentrate (drug loading = 30 mg/g) and mixed for dissolution under agitation (400 rpm, 120 min, 20 °C), protected from light. 2.4.2. Self-emulsification of CC-LBDDS in gastrointestinal and transport media The size of droplets, the solubility and stability of CC-loaded LBDDS in FaSSGF, FaSSIF, lipolysis and transport (HBSS) media were assessed as previously described [35]. Briefly, 1.03 g of CC-LBDDS was gently stirred with 36 ml of lipolysis medium or 250–1000 ml of gastrointestinal and

Table 1 Composition and characteristics of selected CC-LBDDSs.

Composition (mg) Labrafil M2125CS Labrasol Cremophor EL Transcutol HP CC (mg/g excipient) Droplet size (nm), polydispersity index FaSSGF FaSSIF pH 6.8 FaSSIF pH 7.5 (lipolysis medium) HBSS a

Formulation A

Formulation B

100 100 100

100 100 100 50 30

30 a

38.0 49.2 50.2 32.1

± ± ± ±

3.1 (0.27) 1.2 (0.17) 2.8 (0.14) 0.1 (0.03)

Polydispersity index (PDI) are given in the parentheses (n = 3).

42.0 56.4 61.8 29.5

± ± ± ±

2.7 (0.29) 0.5 (0.31) 2.3 (0.18) 0.3 (0.09)

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transport media at 37 °C. After 20 min, the samples were removed for particle size analysis by photon correlation spectroscopy. To evaluate the solubility and the stability of CC, the samples or supernatants from centrifugation (5000 ×g, 15 min, 37 °C) were diluted twice in a mixture of methanol: 3.6% glacial acetic acid (73:27, v/v). The quantity of CC present in the solution of the various media with or without centrifugation was assayed by HPLC analysis. All the measurements were performed in triplicate. 2.4.3. Stability of CC-LBDDS over 180 days The stability of CC in the lipid formulations selected was studied for 180 days at room temperature. During storage, the self-emulsifying systems were kept in sealed glass vials and protected from light. The presence or absence of drug re-precipitation was initially observed visually and then by microscopy [35]. One milliliter of the lipid-based systems was then centrifuged (5000 ×g, 30 min, 20 °C). After dispersion of the supernatant in 0.1 N HCl and an appropriate dilution in a methanol: 3.6% glacial acetic acid (73:27, v/v) mixture, the quantity of CC present in the solution was determined in triplicate by HPLC. The efficiency of self-emulsification and the droplet size in the gastric medium were also assessed. 2.5. CC analysis by HPLC An Agilent 1100 Series HPLC system with a diode array and multiple wavelength detector was used for CC analysis. The column was an EC 250/2 Nucleodur 100-5 C18 ec (Macherey-Nagel, Düren, Germany). The sample (50 μl) was eluted with the mobile phase composed of methanol: 3.6% glacial acetic acid (73:27, v/v) [27]. The different samples were analyzed after an appropriate dilution in the mobile phase. The system was run isocratically at a flow rate of 0.2 ml/min, and sample detection was achieved at 428 nm. The retention time of the CC was approximately 6 min under these conditions. The limits of detection (LOD) and of quantification (LOQ) of CC were 5 ng/ml and 15 ng/ml, respectively. The coefficients of variation (CVs) for intra- and inter-assay were all within 5%. 2.6. Lipolysis of CC-LBDDS Dynamic in vitro lipolysis was performed using a pH-stat titration system (Metrohm Titrando 842; Software Tiamo 1.3, Herisau, Switzerland) as previously described [34,36]. Briefly, 1.03 g of CC-LBDDS was dispersed in 36 ml of a lipolysis medium and stirred for 15 min at 37 °C. The enzymatic digestion was initiated by the addition of 4 ml of freshly prepared pancreatin extract (lipase activity of 1000 tributyrin units per ml of digest). The fatty acids released during in vitro lipolysis were automatically titrated with 0.2 M NaOH to maintain a pH of 7.5. At different times (15, 30, 45 and 60 min), aliquots were sampled, and a lipolysis inhibitor (0.5 M 4-bromophenylboronic, 9 μl/ml of digestion medium) was immediately added. Subsequently, the aliquots were centrifuged for 60 min at 37 °C and 48,000 ×g to separate the sample into an aqueous and a pellet phase. The samples obtained from each separated phase were assayed for the CC content by HPLC analysis. Control experiments with the lipolysis medium alone were also performed. 2.7. Analysis of the pellet obtained after lipolysis The dissolution of the pellet obtained from centrifugation from all the media sampled at the end of lipolysis was performed at 37 °C using the USP II Apparatus with the paddle rotating at 100 rpm. After dispersion of two pellets in 10 ml of water, 500 ml of intestinal medium (pH 6.8, 37 °C) was added. At each sampling time point, the samples were filtered through 0.45-μm filters (Acrodisc®, Pall, France) and then analyzed by HPLC for drug content. These experiments were conducted in triplicate.

To elucidate the solid state of the CC present in the isolated pellet, X-ray powder diffraction was performed on a Siemens D5000 diffractometer using the Kα radiation of Cu (λ = 1.5418 Å). The 2θ range between 2° and 60° was scanned at a rate of 0.02°/s with an applied voltage and current of 40 kV and 40 mA, respectively. The isolated pellets (CC pellets) were spread on a zero-loss sample holder (Si single crystal) and then allowed to air dry. Control experiments were conducted using blank pellets obtained from the lipolysis of unloaded-LBDDS and blank pellets spiked with the corresponding amount of crystalline drugs present in the CC pellets [36,37]. 2.8. In vitro intestinal cell models used to study the transport of CC 2.8.1. Cell cultures Caco-2 cells (clone 1), obtained from the University of MilanoBicocca, Italy, were used [38]. The cells were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% (v/v) heat inactivated fetal bovine serum (Hyclone®, Thermo Scientific, UK), 1% (v/v) L-glutamine and 1% (v/v) non-essential aminoacids at 37 °C in an atmosphere of 10% CO2. Caco-2 cells were used between passage numbers x + 17 and x + 30 [35,38]. The human Burkitt's lymphoma Raji-B cell line (American Type Culture Collection, VA, USA) was maintained in RPMI 1640 supplemented with 10% (v/v) heat inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) non-essential amino acids at 37 °C under a 5% CO2 humidified atmosphere. Raji-B cells were used at passage numbers 106–110 [38,39]. 2.8.2. Cytotoxicity The viability of the Caco-2 cells was assessed after the incubation of 2 × 104 Caco-2 cells/well with 100 μl of unloaded-LBDDS dispersed in the culture medium in a 96-well tissue culture plate (Corning Costar®). After 2 h of incubation, the supernatants of each well were removed and kept at 4 °C until the LDH assay. The cells were then incubated for 3 h with 100 μl of 0.5 mg/ml MTT [35]. The IC50 values of the lipid formulations were calculated using the GraphPad Prism®5. The LDH activity released from the cytosol of damaged cells was measured according to the manufacturer's instructions. Each experiment was performed in triplicate.

2.8.3. Caco-2 and FAE monolayers Caco-2 cells (5 × 105 cells/well) were seeded on 12-well cell culture inserts with a 1 μm pore diameter and 0.9 cm2 area (Corning Costar®, NY, USA) and were grown in supplemented DMEM + 1% PEST for 21 days [35,36,38]. The culture medium was changed every 2 days. For transport studies, only Caco-2 cell monolayers with initial transepithelial electrical resistance (TEER) values higher than 400 Ω cm2 were used. The inverted follicle-associated epithelium (FAE) model (including M-like cells) was obtained by co-culturing Caco-2 and Raji-B cells [36,38,39]. Briefly, after 5 days of Caco-2 seeding at a density of 5 × 105 cells/well onto 12-well cell culture inserts with a 3 μm pore diameter and 0.9 cm2 area (Corning Costar®), the inserts were inverted and a piece of silicon tube was plated on the basolateral side of each insert. The inverted inserts were transferred into pre-filled Petri dishes with supplemented DMEM + 1% PEST and maintained for 11 days. The basolateral medium was refreshed every 2 days. Raji-B cells (2.5 × 105 cells/well) were added to the basolateral compartment of the inserts. The co-cultures were maintained inverted for 5 days. Monocultures of Caco-2 cells, cultivated as described above but without the addition of Raji-B cells, were used as controls. After removing the silicon tube, the inserts were placed in 12-well multiwell plates (Becton Dickinson, France) in their original orientation for the transport experiment. Permeation studies were conducted on cell monolayers with initial TEER values greater than 200 Ω cm2 for the monocultures and greater than 100 Ω cm2 for the co-cultures.

2.8.4. Permeation studies of free CC and CC-LBDDS across intestinal in vitro models The apical (A) to basolateral (B) transport experiments across Caco-2 monolayers were conducted by adding 0.5 ml of the CC solution (0.015 mg/ml in HBSS + 3 mM MES + 4% [v/v] methanol) or 0.5 ml of dispersed formulations in HBSS (1.03 mg/ml of CC-LBDDS i.e. 0.03 mg/ml of CC, unless otherwise stated) on the apical compartment of the inserts and 1.2 ml of HBSS + 10 mM HEPES + 1% BSA in the basolateral compartment. For the basolateral to apical transport experiments (B to A), 1.2 ml of CC solution (0.015 mg/ml) was added at the basolateral side, while the apical side was filled with 0.5 ml of HBSS + 10 mM HEPES + 1% BSA. After 2 h, sampling of the acceptor compartment (basolateral for A to B transport or apical for B to A transport) was performed to determine the permeation of CC in solution or loaded in LBDDS. The amount of CC that had crossed the Caco-2 monolayers was determined by HPLC. The CC apparent permeability coefficient (Papp) was calculated according to the following equation: Papp ¼

dQ 1  ; dt Co A

where dQ/dt (transport rate) is the amount of CC (μg) appearing per time unit (s) in the receiver compartment, Co is the initial concentration in the donor compartment (μg/ml) and A is the surface area of the monolayer (A = 0.9 cm2). The integrity of cell monolayers after 2 h of exposure to the CCLBDDSs was assessed by TEER measurements using an electrode connected to an EVOM® volt ohm meter (World Precision Instruments, USA). During the transport studies in the FAE models, A to B transport was investigated as described above. 2.8.5. Intracellular CC in the cell monolayer After the transport studies, the intracellular amount of CC in the Caco-2 cells was evaluated quantitatively by HPLC and qualitatively by confocal laser scanning microscopy. For the HPLC analysis, the cells were washed three times with HBSS + 3 mM MES, detached from the plates by trypsinization and then centrifuged (1500 ×g, 10 min). The supernatant was discarded, and the cells in the pellet were lysed in HBSS + 3 mM MES + 1% Triton (w/v) for 120 min at 37 °C. The amount of CC in the Caco-2 monolayers was then quantified. For the confocal microscopy study, inserts fixed in 2% (v/v) buffered paraformaldehyde (pH 7.4, 30 min) were washed twice in PBS. To visualize the cell boundaries, actin was immunostained with 250 μl of rhodamine–phalloidin (4 U/ml) (Molecular Probes, OR, USA) in PBS for 30 min protected from light. After washing in PBS, the inserts were cut and mounted on glass slides with 4′,6′-diamidino-2-phenylindole (DAPI) (1:20) to visualize cell nuclei [40]. Images were captured using a Carl Zeiss™ confocal microscope equipped with a multi-Argon laser. The fluorescence of CC was detected at 488 nm [41]. The data were analyzed by the Axio Vision software (version 4.8) to obtain y–z, x–z and x–y views of the cell monolayers.

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the absorbance of the supernatants was measured on HumanLyzer 2000 (Human, Germany) at 540 nm. The percentage of hemolysis was determined by the following formula: %Hemolysis ¼

ast‐anc  100; apc‐anc

where ast = absorbance of sample-test, apc = absorbance of positive control, anc = absorbance of negative control. All the experiments were performed in triplicate. 2.10. In vivo antimalarial efficacy of CC-LBDDS The in vivo evaluation of the antimalarial activity of CC-LBDDS in Plasmodium berghei ANKA infection was performed by the curative test as described previously [35,43]. Briefly, on day 0, mice were inoculated intraperitoneally with 1 × 107 P. berghei parasitized erythrocytes. Three days after infection (4–8% parasitemia), the mice were orally administered 0.1 ml of CC-LBDDS (at 0, 60 and 100 mg/kg/day) or a CC solution in dimethyl sulfoxide (DMSO) (at 0 and 100 mg/kg/day), once daily, for 4 consecutive days. Four others groups of mice received CC-LBDDS (at 0, 60, 80 and 100 mg/kg/day) and β-arteether-LBDDS (at 0 and 12 mg/kg/day) in combination. β-arteether (AE)-loaded LBDDSs were composed of groundnut oil (300 mg), Maisine 35-1 (300 mg), Cremophor EL (300 mg), ethanol (100 mg), AE (200 mg) (Formulation C) or of sesame oil (300 mg), Maisine 35-1 (300 mg), Tween 80 (300 mg), ethanol (100 mg), AE (200 mg) (Formulation D) [35,36]. The final group of mice was infected but not treated. Parasite counts were made on days 3, 7, 12 and 28 post-infection, and survival times were also recorded. All animal experiments were approved by ethical committee of animal use of the University of Kinshasa. Antimalarial efficacy was assessed by the parasitemia level, the activity, the mean survival time and the survival rate of the mice for up to 4–6 weeks following inoculation. Parasitemia was monitored by light microscopy (oil immersion, 1000× magnification) by examining thin smears of blood from the tail veins of the mice, fixed with methanol and stained with Giemsa. The parasitemia level was determined by counting, in random fields of the microscope, the number of parasitized erythrocytes per 1000 erythrocytes. The average percent antimalarial activity (equal to the percent suppression) was determined according to the following formula [44]: Activity ¼ 1−

Mean parasitemia of treated group : Mean parasitemia of control group

2.11. Statistical analyses Significant differences between the droplet sizes, permeability coefficients and percent distribution of CC into the various digestion phases were compared by one-way ANOVA with Tukey's post-hoc test (significance set at of p b 0.05). The cumulative survival rates from each treatment group were compared by the Kaplan–Meier survival analysis.

2.9. In vitro hemolytic study

3. Results and discussion

To estimate the damage caused by the formulations on the erythrocyte membrane, a hemolysis test was performed as previously described [18,42]. Briefly, the blood from human healthy volunteers was centrifuged for 10 min at 2000 ×g and the plasma discarded. The erythrocytes were washed three times and then diluted 12-fold with isotonic phosphate buffer saline (PBS). Two milliliters of this erythrocyte suspension were incubated with 2 ml of the unloaded-LBDDS in dispersion in PBS (0–10 mg/ml). Incubation was carried out at 37 °C for 30 min under agitation. PBS was used as a negative control and Triton X-100 (1%, w/v) as a positive control. After centrifugation (2000 ×g, 5 min),

3.1. Solubilization of CC in various excipients and in biorelevant media To select the excipients for the self-emulsifying systems, the solubility of CC in different excipients was determined (n = 3). CC exhibited poor solubility (b25 mg/g) in most of the lipid vehicles and surfactants (ethyl oleate, sesame oil, groundnut oil, Maisine 35-1, Labrafil M2125CS, Labrafil M1944CS, Tween 80 and Cremophor EL). This solubility reached approximately 85 mg/g in Labrasol. In the tested co-solvents, PEG 400 yielded the highest solubility for CC (~140 mg/g), followed by Transcutol HP (~100 mg/g) and ethanol (~10 mg/g).

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Due to their emulsion forming capability, Cremophor EL and Labrasol were selected as surfactants. Labrafil M2125CS was also chosen because of its miscibility with Cremophor EL and Labrasol. After lipolysis, Labrafil M2125CS can also release oleic and linolenic acid that could contribute to the antimalarial activity [45–47]. Transcutol HP was selected as a solubilizer and absorption promoter; PEG-400 was not chosen due to its high aqueous partition, which caused the precipitation of a large amount of CC upon dilution (data not shown). The solubility of CC in the simulated fluids containing bile salts and phospholipids or albumin dramatically increased compared with water (solubility = 11 ng/ml). Indeed, up to 0.5 μg/ml, 5.4 μg/ml and 4.2 μg/ml of CC were dissolved in FaSSGF, FaSSIF (pH 6.8 and pH 7.5) and HBSS + 10 mM HEPES + 1% BSA, respectively, after 2 h. 3.2. The development of oral CC self-emulsifying systems To obtain self-emulsifying systems, pseudo-ternary phase diagrams were constructed using a water titration method with three different mixtures: Labrafil M2125CS-Labrasol-Transcutol HP, Labrafil M2125CSCremophor EL-Transcutol HP and Labrafil M2125CS-Labrasol-Cremophor EL. Labrafil M2125CS-Labrasol-Cremophor EL provided the highest selfnanoemulsion area (particle size b 100 nm), in agreement with previous results (data not shown, [48]); hence, it was selected as the desirable mixture for the self-nanoemulsifying formulation development. When the aforementioned selected mixture contained relatively high quantities of Cremophor EL, it formed a viscous gel that slowly dissolved over time. In addition, its cytotoxicity was enhanced as the concentrations of Labrasol increased (data not shown). Based on self-emulsification efficiency and cytotoxicity, a first self-nanoemulsifying system (Formulation A) was retained and is highlighted by a dot in the phase diagram (Fig. 1). The droplet size of Formulation A in gastric media (1%, w/v) was approximately 30 nm (Polydispersity index, PDI b 0.25). The mixture of Labrafil M2125CS, Labrasol and Transcutol HP (2:2:1, v/v) was previously shown to be well-tolerated in rats [49] but was not able to self-nanoemulsify and yielded an unstable emulsion in 0.1 N HCl (a droplet size of 3 μm on average). The incorporation of increasing concentrations of Cremophor EL (0 to 30%, w/w) facilitated its self-nanoemulsification and decreased the size of the resulting oil droplets from 3 μm to 35 nm. Hence, a second self-nanoemulsifying system (Formulation B) containing Labrafil M2125CS, Labrasol, Transcutol HP and Cremophor EL (2:2:1:2, w/w) was selected. The composition of the two selected LBDDSs is detailed in Table 1. The solubility of CC in the selected self-emulsifying systems was then evaluated. Up to 42 mg/g of CC were solubilized in both Formulation A and Formulation B. However, to avoid the rapid precipitation of CC after dilution in the aqueous medium, the CC loading was reduced to 30 mg/g (70% of saturation solubility).

In 250 ml of FaSSGF, 80–95% of the CC (~96–114 μg/ml) contained in LBDDS was dissolved after 1 h, whereas more than 96% of CC (~115 μg/ml) was dissolved in the FaSSIF (pH 6.8) (Fig. 2). The dilution of each medium up to 1000 ml did not significantly change the quantity of solubilized CC (p N 0.05, Fig. 2). After 1 h, the total CC content was more than 95% in gastric and intestinal media, indicating an improvement in CC stability (Fig. 2). Over periods of 1 h and 3 h, CC-LBDDS (1.03 to 4.12 mg/ml) showed a similar solubility and stability in the transport medium (HBSS buffer) and in the intestinal medium (data not shown). The stability of free CC in the transport media is also crucial to avoid an erroneous estimation of its permeability. Consistent with results previously reported [50], approximately 50–60% of the CC was degraded in the HBSS buffer (pH 7.4) after 2 h. The impact of this degradation in the donor compartment was suppressed to less than 5% by the adjustment of the pH of HBSS to 6.5 with 3 mM MES. Additionally, the use of HBSS + 10 mM HEPES + 1% BSA (pH 7.4) in the receiver chamber was highly effective at stabilizing more than 85% of the CC, which agrees with previously reported results [51]. A preliminary study of stability was conducted at room temperature (20 ± 2 °C) and protected from light. Over 180 days, the percentage of CC remaining in the formulations was greater than 98.5% (data not shown). None of the LBDDS samples showed any change in color or appearance under the above-described storage conditions. Moreover, no change in the droplet size and in the self-nanoemulsification efficiency was observed (data not shown). 3.4. In vitro dynamic lipolysis 3.4.1. In vitro lipolysis To determine whether CC precipitates due to intestinal digestion of the lipid vehicles present in LBDDS, a dynamic lipolysis procedure was

3.3. Dilution of CC-LBDDS in gastrointestinal and transport media To verify whether the CC-loaded LBDDSs could self-emulsify in the gastrointestinal tract, 1.03 g of the formulation was diluted in 250– 1000 ml of gastro-intestinal or in 36 ml of the lipolysis medium. The size of the droplets, the solubility and the stability of CC were then assessed. In the gastrointestinal medium, Formulation A and Formulation B dispersed spontaneously to form nanoemulsions with sizes of 30–40 nm (PDI b 0.25). Dilution did not induce changes in the size of droplets, confirming the kinetic stability that characterizes self-nanoemulsifying drug delivery systems (SNEDDSs). Additionally, no phase separation was observed after centrifugation for 15 min at 5000 ×g. The incorporation of CC in the self-emulsifying systems did not change significantly the size and the polydispersity index of droplets compared to unloaded-LBDDS (p N 0.05, Table 1). In the lipolysis medium, CC-LBDDSs formed particles with a size of approximately 60 nm and a polydispersity index of 0.15 (Table 1).

Fig. 2. Solubilization of CC-loaded LBDDS in FaSSGF (A) and in FaSSIF (B) after 60 min. The total CC (□) and the solubilized CC (■) are expressed as the percentage of CC recovered before and after centrifugation. Data are presented as the mean ± SD, n = 3.

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performed, as previously described [34,36]. The course of NaOH addition as a function of time during the lipolysis of CC-LBDDS is shown in Fig. 3A. The quantity of NaOH used to titrate the fatty acids released by the lipid formulations ranged from 1.15 to 1.35 mmol for both the formulations. After centrifugation of the digested formulations, a pellet and an aqueous phase were isolated. The amount of the non-precipitated CC in the aqueous phase and the amount of the precipitated CC in the pellet as a function of time are shown in Fig. 3B. For all the formulations, 90– 95% (27–28.5 mg) of the CC was present in the aqueous phase at each time point of the lipolysis, and 5–10% (1.5–3 mg) was present in the pellet phase. The dispersion of CC-LBDDS in the lipolysis medium

Fig. 3 (continued)

(36 ml, no lipase) maintained the CC in a solubilized state; no precipitation was observed after 30 min.

Fig. 3. In vitro dynamic lipolysis of Formulation A (●, ○) and Formulation B (■, □). (A) Quantity of 0.2 M NaOH added to titrate the fatty acids that were released during lipid digestion. (B) Distribution profile of CC in the aqueous phase (open shapes and dotted lines) and in the pellet phase (filled shapes and lines) as a function of lipolysis time. (C) Dissolution rate of CC in pellets at the end of lipolysis (60 min). (D and E) The X-ray powder diffraction pattern of (a) crystalline, (b) CC pellet, (c) blank pellet spiked with CC and (d) blank pellet from the lipolysis of Formulation A (D) and Formulation B (E). The numbers over the peaks indicate d-spacings.

3.4.2. X-ray powder diffraction and CC dissolution in pellets after lipolysis To determine the solid state of the precipitated CC, X-ray powder diffraction was performed on the CC pellets. These results show similar characteristic reflections between the typical X-ray powder diffraction of crystalline CC and of the blank pellets spiked with CC, but not with CC present in the pellets, suggesting that the CC precipitated in an amorphous form and/or in molecular dispersion during in vitro lipolysis (Fig. 3D and E). This phenomenon was also reported with cinnarizine [37], halofantrine [52] and simvastatine [53], but to our knowledge, this result is the first report of the phenomenon with CC. The transformation of crystalline CC to amorphous solids had already been reported after encapsulation in PLGA nanoparticles [20,56] and in solid dispersions [25]. The difference observed in the organization of the fatty acids phase between the CC pellets and the blank pellets spiked with CC suggests a possible intermolecular interaction between the drug and the colloidal structure during lipolysis (Fig. 3D and E). Potential re-dissolution of the precipitated CC in the intestinal medium prior to absorption was assessed by performing a dissolution test of the pellet obtained from the lipolysis of CC-LBDDS. Interestingly, 0.8–1 mg (~ 30–70%) of the CC present in these pellets was re-dissolved in the intestinal medium after 60 min (n = 3, Fig. 3C). The re-dissolution of CC could occur as lipid digestion progresses. The drug micellization in various colloidal structures containing bile salts, phospholipids and lipid digestion products and the amorphization of crystalline CC may have a strong influence on the re-dissolution of CC pellets.

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its stability, a solution of 1% BSA in HBSS + 10 mM HEPES was used as a receiver medium [32,51]. In these conditions, the Papp (A to B) of CC was 3.85 ± 0.45 × 10−6 cm/s at 37 °C (Fig. 4A). A similar permeability of 2.93 × 10−6 cm/s has been observed by Wahlang et al. [50]. Yu and Huang also reported a CC Papp value in the same rank order (7.1 ×10− 6 cm/s and 8.4 × 10− 6 cm/s) [22,54]. Each report indicated a high level of permeability of CC (Papp N 10− 6 cm/s). The existence of potential active efflux was also investigated during the transport. The Papp (B to A) of CC was 3.68 × 10−6 cm/s (Fig. 4A). The ratio Papp (B to A)/Papp (A to B) corresponds to 0.96, indicating that CC is not a substrate of the Pg-P efflux transporter [50,54,55].

Fig. 4. (A) Apparent permeability coefficient of the free CC across Caco-2 monolayers in the apical to basolateral (A to B) and the basolateral to apical (B to A) directions. (B) Apparent permeability and transport rate of CC-loaded LBDDS across Caco-2 monolayers with two different drug concentrations (0.03 and 0.05 mg/ml). (C) The influence of M cells on the transport of CC-LBDDS in Caco-2 monolayers and in the follicle-associated epithelium model (co-cultures). Each value represents the mean ± SD (n = 3–6).

3.5. Caco-2 cell in vitro studies 3.5.1. Cytotoxic assessment of the LBDDS To select the range of concentrations used for the permeation studies, the cytotoxicity of the LBDDS was investigated using the MTT test. The IC50 ranged between 2.0 and 3.0 mg/ml (data not shown). The presence of co-solvent (Transcutol HP) in Formulation B did not result in a significant difference in the metabolic activity of Caco-2 cells compared to Formulation A. After exposure of cells with 3 mg/ml of LBDDSs, less than 20% of the LDH was released (data not shown). 3.5.2. Free CC transport across Caco-2 cell monolayers To evaluate the permeability of free CC (0.015 mg/ml in HBSS + 3 mM MES + 4% methanol), absorptive (A to B) and secretive (B to A) transports across Caco-2 cell monolayers were performed for 120 min. To improve the recovery of CC by the enhancement of its solubility and

3.5.3. The transport of CC-LBDDS across Caco-2 and FAE monolayers To determine the intestinal permeability of CC formulated in LBDDS, transport studies were carried out in Caco-2 cells. Based on cytotoxicity studies, Caco-2 monolayers were incubated for 120 min at 37 °C with 1.03 mg/ml of CC-LBDDS, which corresponded to 0.03 mg/ml of CC. The TEER values before and after incubation with the CC-LBDDSs did not change (p N 0.05). As shown in Fig. 4B, the Papp of CC was 1.10 ± 0.07 × 10− 6 cm/s (Formulation A) and 1.20 ± 0.06 × 10− 6 cm/s (Formulation B). The quantity of CC transported across the Caco-2 monolayers ranged between 1.4 and 1.6% (0.21 and 0.24 μg) of the donor CC-LBDDS. Transcutol HP, the absorption enhancer employed in Formulation B, did not significantly increase the transport of CC across Caco-2 monolayers compared to Formulation A (p N 0.05). The transport of CC in suspension (0.15 mg/ml in HBSS) was below the limit of detection (data not shown). CC is known to be retained in enterocytes [50]. To determine the amount of CC within Caco-2 cells, CC was quantified by HPLC after cell lysis. Approximately 1.6% of the CC from LBDDS (i.e., 0.24 μg) was present in Caco-2 monolayers after a 2 h incubation, suggesting that the amount of CC transported across Caco-2 cells may increase beyond this period of time. Fig. 5 shows the confocal microscopy images corresponding to the cellular uptake of CC in Caco-2 cells. CC-loaded LBDDS was taken up more by enterocytes than the CC in suspension. An intracellular staining was observed, which confirms an increased uptake of CC from LBDDS. The effect of the concentration of CC-LBDDS on CC permeation across the Caco-2 cell monolayer was investigated. At 0.03 and 0.05 mg/ml of CC loaded in LBDDS, the transport rates were 3.40 × 10−2 ng/s and 4.01 × 10−2 ng/s for Formulation A and 3.62 × 10−2 ng/s and 4.54 × 10−2 ng/s for Formulation B. These results indicate that the transport rates were similar at the same concentration of CC (p N 0.05) but were not linearly dependent on drug concentration (Fig. 4B). Using the inverted M cell model, no significant difference in the Papp of CC between the co-culture and monoculture (Papp 1.3 to 1.5 × 10−6 cm/s) (p N 0.05, Fig. 4C) was detected, confirming that the uptake of the encapsulated drug in LBDDS was not enhanced by M cells [36]. Before and after the experiment, the average TEER was not modified for the monocultures and for the co-cultures (p N 0.05). A series of complex processes or factors (e.g., bile salts, phospholipid micelles, lipid and surfactant digestion) occurring in vivo may largely influence the predictive permeability of CC-LBDDS observed in the in vitro Caco-2 cell system. Therefore, we attempted to assess the permeability of CC across intestinal cells after a lipid digestion of CCLBDDS. However, the cytotoxic effects of the lipolysis buffer (bile salts and lipase) prevented the realization of this experiment. Appropriate experimental conditions could not be obtained even after the addition of lipase inhibitors, thermal inactivation of enzymes or a dilution of digested CC-LBDDS to non-cytotoxic concentrations of enzymes and bile salts to maintain cell integrity (data not shown, [57,58]). 3.6. Hemolysis test To check if emulsions transported to systemic blood circulation could cause damage on the membrane of erythrocytes which is the

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Fig. 5. Confocal laser scanning microscopy images of the inserts after 2 h of incubation with Formulation A (Panel A), Formulation B (Panel B) and CC suspension (Panel C). Green fluorescence indicates the localization of CC in cell monolayers (excitation wavelength = 488 nm). Cell membranes are immunostained (red) with rhodamine– phalloidin and cell nuclei (blue) with DAPI.

main target in malaria therapy, hemolytic activity of unloaded-LBDDS was assessed as previously described [18,42]. All the formulations (0–10 mg/ml in PBS) showed negligible hemolytic effect (1.2–3.7%). Nevertheless, this in vitro hemolytic study does not exactly predict in vivo observations due to lipid digestion in gastrointestinal tract. Therefore, the estimated in vitro erythrocyte toxicity may be largely reduced in vivo. 3.7. In vivo antimalarial efficacy of CC-LBDDS To determine whether treatment of CC-LBDDSs could delay parasitemia in an established infection, their antimalarial efficacy was investigated in a P. berghei mouse model of malaria. The results are summarized in Fig. 6 and Table 2. At a dosage of 100 mg/kg for four

Fig. 6. Survival of mice treated with CC-DMSO (at 100 mg/kg), CC-LBDDS (at 0, 60 and 100 mg/kg) (Panel A and B) or with CC-LBDDS (at 0, 60, 80 and 100 mg/kg) in combination with AE-LBDDS (at 0 and 12 mg/kg) (Panel C and D).

consecutive days, parasitemia suppression by CC-loaded LBDDSs ranged between 40 and 50% (Table 2). The mean survival time was approximately 14 days, whereas the survival rate was 43% and 0% at day-14 and day-21, respectively. No significant differences in the antimalarial efficacy of Formulation A and Formulation B were observed (p N 0.05). Oral delivery of CC-LBDDS at 60 mg/kg for 4 days decreased the survival rate to less than 25% at day-14 with a mean survival time of approximately 9 days.

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Table 2 Antimalarial activity of formulations and mean survival time of mice. Formulation

Drug

A

CC

B

CC

C+A

AE + CC

D+B

AE + CC

DMSO Untreated

CC –

a b

Dose (mg/kg/day) AE

CC

– – – – – – 0 12 12 12 12 0 12 12 12 12 – –

0 60 100 0 60 100 0 0 60 80 100 0 0 60 80 100 100 –

% Activity a

Mean survival time (days) b

11.5 20.8 46.0 10.6 24.4 43.0 8.7 99.8 99.8 99.8 99.8 9.6 99.5 99.5 99.7 99.8 32 –

7.3 9.6 14.0 8.3 10.3 13.9 8.1 18.7 20.7 22.9 25.1 7.7 18.9 21.7 22.9 24.9 9.3 6.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.6 4.1 4.9 1.4 4.4 4.2 2.3 5.3 4.8 5.2 3.7 1.1 6.0 4.7 4.9 3.7 4.5 0.8

Average activity of formulations was determined on day 4 of treatment. Mean survival time of mice determined during an observation period of 28 days.

Administered in DMSO at 100 mg/kg for 4 days, CC decreased parasitemia by 32% with a mean survival time of 9 days (Table 2), which is in agreement with previous studies [10,11]. The survival rate of mice was 30% at day-14 and 0% at day-21. These results contrast with a survival rate of 29% at day-21 reported by Reddy et al. [8]. Some differences in the experimental conditions (administration protocols and parasitemia levels) may account for these observations. At 100 mg/kg, the efficacy of CC-DMSO was lower than the efficacy of CC-LBDDS in both parasite suppression and survival time (p b 0.05). The possibility of re-precipitation and degradation of CC dissolved in DMSO occurring in the gastrointestinal tract may reduce the intestinal absorption and therefore the activity of CC. The LBDDS without CC prolonged survival time up to 11 days postinfection for 15% of the mice compared with 6–7 days post-infection in untreated mice. This finding suggests that lipid vehicles may have some inherent antimalarial activity and immunomodulatory properties [35,45,47,59]. Indeed, C18 fatty acids (linolenic, linoleic and oleic acid) inhibit parasite growth both in vitro and in vivo [46,47]. Fatty acids may also stimulate a protective immune response by the activation of neutrophils, Th2 type CD4+ T cells and other effector cells to increase the clearing of parasitemia [47,59]. These results demonstrate that, at the dose of 100 mg/kg, LBDDS (and DMSO) containing CC have a moderate effect on the reduction of parasitemia and are not able to delay the course of an established infection on mice. Hence, to overcome an incomplete cure and recrudescence occurring during this monotherapy, a combinatorial approach using a subtherapeutic dose of AE-LBDDS (12 mg/kg) [35] and CCLBDDS was investigated. When administered, combinations of AE and CC-loaded lipid-based systems drastically decreased blood parasitemia (~0%) (data not shown) but were not fully effective in suppressing recrudescence (Fig. 6 C and D). Nevertheless, they significantly enhanced the survival rate of mice compared with their respective monotherapy groups. Indeed, the combination of Formulation C (12 mg/kg) + Formulation A (100 mg/kg) resulted in a survival rate of 60% in comparison with a survival rate of 15% for Formulation C (12 mg/kg) + Formulation A (0 mg/kg) (Fig. 6C). The co-administration of Formulation D with Formulation B at 12 mg/kg + 0 mg/kg and 12 mg/kg + 100 mg/kg led to a 15% and 45% 28-day survival, respectively (Fig. 6D). Similar results were previously obtained after oral administration of Formulation C and Formulation D (alone) at the subtherapeutic dose of 12 mg/kg [35]. The moderate antimalarial activity and the

immunomodulatory effect of CC (and fatty acids to a lesser extent) may explain the increasing rate of survival (45–65%), regardless of the high recrudescence of parasites on day-12 (~ 2% parasitemia on average). No significant differences in survival time were observed when Formulation C +Formulation B or Formulation D + Formulation A were used at the different doses (p N 0.05 and data not shown). The potential of AE-CC combination therapy in experimental malaria have been demonstrated previously [10–12]. In these reports, CC was orally administered in DMSO or intravenously administered in liposomes and AE intramuscularly administered. The present study shows the utility of LBDDS in the oral delivery of CC: enhanced drug's solubilization and transport across Caco-2 monolayers as well as increased antimalarial efficacy. The suitability of oral AE-LBDDS in the malaria therapy has been also demonstrated previously [35]. Oral AE-LBDDS constitute a promising alternative to the marketed oil-based intramuscular injectable formulations that are characterized by slow and erratic absorption [2]. In order to be able to appreciate the potential effect of oral AE-LBDDS and CC-LBDDS when administered in combination, a subtherapeutic dose of each drug was tested in P. berghei-infected mice. The results clearly demonstrate for the first time that the oral administration of both CC-LBDDS and AE-LBDDS display a significant anti-malarial efficacy against P.berghei-infected mice. The results are supportive but not conclusive of a synergistic effect of CC in an artemisinin-combination therapy. In this combination, an increased dose of AE (to 18–24 mg/kg) and/or CC (to 150–200 mg/kg) may lead to complete cure and long-term protection. Additionally, the start of treatment at a very early stage of the disease may also favor a quick resolution of symptoms and prevention of hyperparisitemia, which would reduce mortality. Additional in vivo studies are needed to confirm these hypotheses and to assess the oral bioavailability of CC loaded in LBDDS. 4. Conclusion In this study, two LBDDSs containing a high CC loading (30 mg/g) were formulated. In a gastrointestinal medium, CC-LBDDSs spontaneously self-emulsify to form nanosized particles. During in vitro lipolysis, 5–10% of CC precipitated from LBDDS as an amorphous solid with a high rate of re-dissolution in FaSSIF. Due to the increased CC solubility, LBDDS enhanced the transport of CC across Caco-2 cell monolayers in comparison with the transport of free drug. In mice, a modest antimalarial efficacy was observed following treatment with CC-LBDDS (100 mg/kg). However, the combination therapy of CC-LBDDS (80– 100 mg/kg) with a subtherapeutic dose of AE-LBDDS (12 mg/kg) showed an increased protection and survival rate associated with a significant delay in recrudescence. The combination of oral CC and AE lipid-based formulations may constitute a promising approach for the treatment of malaria. Acknowledgments The authors thank the Université Catholique de Louvain (Bourse de la coopération au développement) for financing and supporting the project and the scholarship of Patrick Bondo Memvanga. We are also indebted to Dr Pascal Somville (UCB Pharma, Belgium) for access to the pH-stat titration system. The scientific and technical support rendered by Pierre Eloy (Institute of Condensed Matter and Nanosciences, Université Catholique de Louvain) during the realization of X-ray powder diffraction is acknowledged. We are grateful to Dr Stomy Karhemere (Institut Nationale de Recherches Biomédicales, Democratic Republic of Congo) for providing the facilities for the in vivo study. The technical assistance rendered by Guy Midingi is acknowledged. We extend our thanks to Gattefossé and BASF for kind gifts of excipients used in this study.

References [1] Who Press, World Malaria Report, 2012. [2] World Health Organization, Guidelines for the Treatment of Malaria, 2010. [3] A. Codd, F. Teuscher, D. Kyle, Q. Cheng, M. Gatton, Artemisinin-induced parasite dormancy: a plausible mechanism for treatment failure, Malar. J. 10 (2011) 56. [4] B. Witkowski, J. Lelievre, M.J. Lopez Barragan, V. Laurent, X.Z. Su, A. Berry, F. BenoitVical, Increased tolerance to artemisinin in Plasmodium falciparum is mediated by a quiescence mechanism, Antimicrob. Agents Chemother. 54 (2010) 1872–1877. [5] K.R. Hughes, G.A. Biagini, A.G. Craig, Continued cytoadherence of Plasmodium falciparum infected red blood cells after antimalarial treatment, Mol. Biochem. Parasitol. 169 (2010) 71–78. [6] G. Padmanaban, V.A. Nagaraj, P.N. Rangarajan, Artemisinin-based combination with curcumin adds a new dimension to malaria therapy, Curr. Sci. 102 (2012) 704–711. [7] L. Cui, J. Miao, L. Cui, Cytotoxic effect of curcumin on malaria parasite Plasmodium falciparum: inhibition of histone acetylation and generation of reactive oxygen species, Antimicrob. Agents Chemother. 51 (2007) 488–494. [8] R.C. Reddy, P.G. Vatsala, V.G. Keshamouni, G. Padmanaban, P.N. Rangarajan, Curcumin for malaria therapy, Biochem. Biophys. Res. Commun. 326 (2005) 472–474. [9] A. Martinelli, L.A. Rodrigues, P. Cravo, Plasmodium chabaudi: efficacy of artemisinin + curcumin combination treatment on a clone selected for artemisinin resistance in mice, Exp. Parasitol. 119 (2008) 304–307. [10] D.N. Nandakumar, V.A. Nagaraj, P.G. Vathsala, P. Rangarajan, G. Padmanaban, Curcumin-artemisinin combination therapy for malaria, Antimicrob. Agents Chemother. 50 (2006) 1859–1860. [11] P.G. Vathsala, C. Dende, V.A. Nagaraj, D. Bhattacharya, G. Das, P.N. Rangarajan, G. Padmanaban, Curcumin-arteether combination therapy of Plasmodium bergheiinfected mice prevents recrudescence through immunomodulation, PLoS One 7 (2012) e29442. [12] N.P. Aditya, G. Chimote, K. Gunalan, R. Banerjee, S. Patankar, B. Madhusudhan, Curcuminoids-loaded liposomes in combination with arteether protects against Plasmodium berghei infection in mice, Exp. Parasitol. 131 (2012) 292–299. [13] F. Akhtar, M.M. Rizvi, S.K. Kar, Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice, Biotechnol. Adv. 30 (2012) 310–320. [14] A.P. Nayak, W. Tiyaboonchai, S. Patankar, B. Madhusudhan, E.B. Souto, Curcuminoidsloaded lipid nanoparticles: novel approach towards malaria treatment, Colloids Surf. B: Biointerfaces 81 (2010) 263–273. [15] J.G. Bilmen, S.Z. Khan, M.H. Javed, F. Michelangeli, Inhibition of the SERCA Ca2+ pumps by curcumin. Curcumin putatively stabilizes the interaction between the nucleotide-binding and phosphorylation domains in the absence of ATP, Eur. J. Biochem. 268 (2001) 6318–6327. [16] B.B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Curcumin: the Indian solid gold, Adv. Exp. Med. Biol. 595 (2007) 1–75. [17] P.N. Mimche, D. Taramelli, L. Vivas, The plant-based immunomodulator curcumin as a potential candidate for the development of an adjunctive therapy for cerebral malaria, Malar. J. 10 (Suppl. 1) (2011) S10. [18] R. Chakrabarti, P.S. Rawat, B.M. Cooke, R.L. Coppel, S. Patankar, Cellular effects of curcumin on Plasmodium falciparum include disruption of microtubules, PLoS One 8 (2013) e57302. [19] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Bioavailability of curcumin: problems and promises, Mol. Pharm. 4 (2007) 807–818. [20] J. Shaikh, D.D. Ankola, V. Beniwal, D. Singh, M.N. Kumar, Nanoparticle encapsulation improves oral bioavailability of curcumin by at least 9-fold when compared to curcumin administered with piperine as absorption enhancer, Eur. J. Pharm. Sci. 37 (2009) 223–230. [21] K. Ahmed, Y. Li, D.J. McClements, H. Xiao, Nanoemulsion- and emulsion-based delivery systems for curcumin: encapsulation and release properties, Food Chem. 132 (2012) 799–807. [22] H. Yu, Q. Huang, Improving the oral bioavailability of curcumin using novel organogel-based nanoemulsions, J. Agric. Food Chem. 60 (2012) 5373–5379. [23] K. Desai, Curcumin cyclodextrin combination for preventing or treating various diseases. Patent US 2010/0779103. [24] K. Maiti, K. Mukherjee, A. Gantait, B.P. Saha, P.K. Mukherjee, Curcumin phospholipid complex: preparation, therapeutic evaluation and pharmacokinetic study in rats, Int. J. Pharm. 330 (2007) 155–163. [25] S.W. Seo, H.K. Han, M.K. Chun, H.K. Choi, Preparation and pharmacokinetic evaluation of curcumin solid dispersion using Solutol HS15 as a carrier, Int. J. Pharm. 424 (2012) 18–25. [26] M.M. Yallapu, M. Jaggi, S.C. Chauhan, Curcumin nanoformulations: a future nanomedicine for cancer, Drug Discov. Today 17 (2012) 71–80. [27] J. Cui, B. Yu, Y. Zhao, W. Zhu, H. Li, H. Lou, G. Zhai, Enhancement of oral absorption of curcumin by self-microemulsifying drug delivery systems, Int. J. Pharm. 371 (2009) 148–155. [28] S. Setthacheewakul, S. Mahattanadul, N. Phadoongsombut, W. Pichayakorn, R. Wiwattanapatapee, Development and evaluation of self-microemulsifying liquid and pellet formulations of curcumin, and absorption studies in rats, Eur. J. Pharm. Biopharm. 76 (2010) 475–485. [29] Y.D. Yan, J.A. Kim, M.K. Kwak, B.K. Yoo, C.S. Yong, H.G. Choi, Enhanced oral bioavailability of curcumin via a solid lipid-based self-emulsifying drug delivery system using a spray-drying technique, Biol. Pharm. Bull. 34 (2011) 1179–1186. [30] E. Jantratid, N. Janssen, C. Reppas, J.B. Dressman, Dissolution media simulating conditions in the proximal human gastrointestinal tract: an update, Pharm. Res. 25 (2008) 1663–1676.

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[31] C.J. Porter, W.N. Charman, In vitro assessment of oral lipid based formulations, Adv. Drug Deliv. Rev. 50 (Suppl. 1) (2001) S127–S147. [32] G. Krishna, K. Chen, C. Lin, A.A. Nomeir, Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2, Int. J. Pharm. 222 (2001) 77–89. [33] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Optimized conditions for prediction of intestinal drug permeability using Caco-2 cells, Eur. J. Pharm. Sci. 10 (2000) 195–204. [34] J.F. Cuine, C.L. McEvoy, W.N. Charman, C.W. Pouton, G.A. Edwards, H. Benameur, C.J. Porter, Evaluation of the impact of surfactant digestion on the bioavailability of danazol after oral administration of lipidic self-emulsifying formulations to dogs, J. Pharm. Sci. 97 (2008) 995–1012. [35] P.B. Memvanga, V. Préat, Formulation design and in vivo antimalarial evaluation of lipid-based drug delivery systems for oral delivery of beta-arteether, Eur. J. Pharm. Biopharm. 82 (2012) 112–119. [36] P.B. Memvanga, P. Eloy, E.M. Gaigneaux, V. Préat, In vitro lipolysis and intestinal transport of beta-arteether-loaded lipid-based drug delivery systems, Pharm. Res. 30 (2013) 2694–2705. [37] P.J. Sassene, M.M. Knopp, J.Z. Hesselkilde, V. Koradia, A. Larsen, T. Rades, A. Mullertz, Precipitation of a poorly soluble model drug during in vitro lipolysis: characterization and dissolution of the precipitate, J. Pharm. Sci. 99 (2010) 4982–4991. [38] A. des Rieux, E.G. Ragnarsson, E. Gullberg, V. Préat, Y.J. Schneider, P. Artursson, Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium, Eur. J. Pharm. Sci. 25 (2005) 455–465. [39] A. des Rieux, V. Fievez, I. Theate, J. Mast, V. Préat, Y.J. Schneider, An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells, Eur. J. Pharm. Sci. 30 (2007) 380–391. [40] R. Coco, L. Plapied, V. Pourcelle, C. Jerome, D.J. Brayden, Y.J. Schneider, V. Preat, Drug delivery to inflamed colon by nanoparticles: comparison of different strategies, Int. J. Pharm. 440 (2013) 3–12. [41] C.F. Chignell, P. Bilski, K.J. Reszka, A.G. Motten, R.H. Sik, T.A. Dahl, Spectral and photochemical properties of curcumin, Photochem. Photobiol. 59 (1994) 295–302. [42] G. Vandermeulen, L. Rouxhet, A. Arien, M.E. Brewster, V. Préat, Encapsulation of amphotericin B in poly(ethyleneglycol)-block-poly(ɛ-caprolactone-cotrimethylenecarbonate) polymeric micelles, Int. J. Pharm. 309 (2006) 234–240. [43] J.F. Ryley, W. Peters, The antimalarial activity of some quinolone esters, Ann. Trop. Med. Parasitol. 64 (1970) 209–222. [44] D.A. Fidock, P.J. Rosenthal, S.L. Croft, R. Brun, S. Nwaka, Antimalarial drug discovery: efficacy models for compound screening, Nat. Rev. Drug Discov. 3 (2004) 509–520. [45] N.M. Carballeira, New advances in fatty acids as antimalarial, antimycobacterial and antifungal agents, Prog. Lipid Res. 47 (2008) 50–61. [46] M. Krugliak, E. Deharo, G. Shalmiev, M. Sauvain, C. Moretti, H. Ginsburg, Antimalarial effects of C18 fatty acids on Plasmodium falciparum in culture and on Plasmodium vinckei petteri and Plasmodium yoelii nigeriensis in vivo, Exp. Parasitol. 81 (1995) 97–105. [47] L.M. Kumaratilake, B.S. Robinson, A. Ferrante, A. Poulos, Antimalarial properties of n-3 and n-6 polyunsaturated fatty acids: in vitro effects on Plasmodium falciparum and in vivo effects on P. berghei, J. Clin. Invest. 89 (1992) 961–967. [48] W. Li, S. Yi, Z. Wang, S. Chen, S. Xin, J. Xie, C. Zhao, Self-nanoemulsifying drug delivery system of persimmon leaf extract: optimization and bioavailability studies, Int. J. Pharm. 420 (2011) 161–171. [49] J.L. Delongeas, G.V. de Conchard, A. Beamonte, H. Bertheux, C. Spire, C. Maisonneuve, N. Becourt-Lhote, F. Goldfain-Blanc, N. Claude, Assessment of Labrasol/Labrafil/Transcutol (4/4/2, v/v/v) as a non-clinical vehicle for poorly water-soluble compounds after 4-week oral toxicity study in Wistar rats, Regul. Toxicol. Pharmacol. 57 (2010) 284–290. [50] B. Wahlang, Y.B. Pawar, A.K. Bansal, Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model, Eur. J. Pharm. Biopharm. 77 (2011) 275–282. [51] M.H. Leung, T.W. Kee, Effective stabilization of curcumin by association to plasma proteins: human serum albumin and fibrinogen, Langmuir 25 (2009) 5773–5777. [52] N. Thomas, R. Holm, A. Mullertz, T. Rades, In vitro and in vivo performance of novel supersaturated self-nanoemulsifying drug delivery systems (super-SNEDDS), J. Control. Release 160 (2012) 25–32. [53] N. Thomas, R. Holm, M. Garmer, J.J. Karlsson, A. Mullertz, T. Rades, Supersaturated self-nanoemulsifying drug delivery systems (Super-SNEDDS) enhance the bioavailability of the poorly water-soluble drug simvastatin in dogs, AAPS J. 15 (2013) 219–227. [54] H. Yu, Q. Huang, Investigation of the absorption mechanism of solubilized curcumin using Caco-2 cell monolayers, J. Agric. Food Chem. 59 (2011) 9120–9126. [55] X.L. Hou, K. Takahashi, K. Tanaka, K. Tougou, F. Qiu, K. Komatsu, K. Takahashi, J. Azuma, Curcuma drugs and curcumin regulate the expression and function of P-gp in Caco-2 cells in completely opposite ways, Int. J. Pharm. 358 (2008) 224–229. [56] K.K. Chereddy, R. Coco, P.B. Memvanga, B. Ucakar, A. des Rieux, G. Vandermeulen, V. Préat, Combined effect of PLGA and curcumin on wound healing activity, J. Control. Release 171 (2013) 208–215. [57] M.B. Gangloff, R.P. Glahn, D.D. Miller, D.R. Van Campen, Assessment of iron availability using combined in vitro digestion and Caco-2 cell culture, Nutr. Res. 16 (1996) 479–487. [58] D.A. Garrett, M.L. Failla, R.J. Sarama, Development of an in vitro digestion method to assess carotenoid bioavailability from meals, J. Agric. Food Chem. 47 (1999) 4301–4309. [59] C. Carrillo, C.M. Del Mar, H. Roelofs, G. Wanten, S.R. Alonso-Torre, Activation of human neutrophils by oleic acid involves the production of reactive oxygen species and a rise in cytosolic calcium concentration: a comparison with N-6 polyunsaturated fatty acids, Cell. Physiol. Biochem. 28 (2011) 329–338.

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