Formulation of oryzalin (ORZ) liposomes: In vitro studies and in vivo fate

European Journal of Pharmaceutics and Biopharmaceutics 82 (2012) 281–290

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Research paper

Formulation of oryzalin (ORZ) liposomes: In vitro studies and in vivo fate Rui M. Lopes a, M. Luísa Corvo a, Carla V. Eleutério a, Manuela C. Carvalheiro a, Effie Scoulica b, M. Eugénia M. Cruz a,⇑ a b

iMed.UL – Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisboa, Portugal School of Medicine, Laboratory of Clinical Bacteriology, Parasitology, University of Crete, Heraklion, Greece

a r t i c l e

i n f o

Article history: Received 16 March 2012 Accepted in revised form 26 June 2012 Available online 5 July 2012 Keywords: Dinitroanilines Oryzalin Leishmaniasis Liposomes Nanomedicines

a b s t r a c t Oryzalin (ORZ) is a dinitroaniline that has attracted increasing interest for the treatment of leishmaniasis. The possible use of ORZ as an antiparasitic agent is limited by low water solubility associated with an in vivo rapid clearance. The aim of this work was to overcome these unfavorable pharmaceutical limitations potentiating ORZ antileishmanial activity allowing a future clinical use. This was attained by incorporating ORZ in appropriate liposomes that act simultaneously as drug solvent and carrier delivering ORZ to the sites of Leishmania infection. The developed ORZ liposomal formulations efficiently incorporated and stabilised ORZ increasing its concentration in aqueous suspensions at least 150 times without the need of toxic solvents. The incorporation of ORZ in liposomes reduced the in vitro haemolytic activity and cytotoxicity observed for the free drug, while ORZ exhibits a stable association with liposomes during the first 24 h after parenteral administration, significantly reducing ORZ blood clearance and elimination from the body. Simultaneously, an increased ORZ delivery was observed in the main organs of leishmanial infection with a 9–13-fold higher accumulation as compared to the free ORZ. These results support the idea that ORZ performance was strongly improved by the incorporation in liposomes. Moreover, ORZ liposomal formulations can be administrated in vivo in aqueous suspensions without the need of toxic solvents. It is expected an improvement in the therapeutic activity of liposomal ORZ that will be tested in future work. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Leishmaniasis is a disease caused by over 20 different species and subspecies of protozoan parasites of the genus Leishmania that affects about 12 million people worldwide [1]. The transmission of this disease occurs through hematophagus vectors and, depending on the causative species, human leishmaniasis can manifest as cutaneous (CL), mucocutaneous (MCL) or visceral (VL) forms, being VL fatal if left untreated [2,3]. Although prevalent in tropical and

Abbreviations: CL, cutaneous leishmaniasis; MCL, mucocutaneous leishmaniasis; VL, visceral leishmaniasis; TFL, Trifluralin; ORZ, Oryzalin; DPPC, Dipalmitoylphosphatidylcholine; DPPG, Dipalmitoylphosphatidylglycerol; DMPC, Dimyristoylphosphatidylcholinel; DMPG, Dimyristoylphosphoglycerol; FBS, foetal bovine serum; THP-1, human monocytic cell line; I.E., incorporation efficacy; L.C., loading capacity; Ø, mean diameter; PI, polydispersity index; f, zeta potential; TFA, trifluoroacetic acid; RBCs, red blood cells; Tt, phase transition temperatures; Lip-14C-ORZ, ORZ– liposome associated labelled with 14C; 3H-Lip, cholesterol–liposome associated labelled with 3H; Free-14C-ORZ, free ORZ labelled with 14C; % ID, percentage of injected dose. ⇑ Corresponding author. iMed.UL – Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Av. Prof. Gama Pinto, 1649-003 Lisboa, Portugal. Tel.: +351 217500764. E-mail address: [email protected] (M.E.M. Cruz). 0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejpb.2012.06.013

subtropical regions, leishmaniasis is also endemic to southern Europe [4]. In Europe, South America, Asia and Africa, co-infection with leishmania and HIV is becoming an emerging problem and has already been reported in 35 of the 88 VL-endemic countries [5,6]. For more than 70 years, the first-line treatment in most countries has been injectable pentavalent antimonials (PentostamÒ and GlucantimeÒ). The treatment is lengthy, potentially toxic and painful; it has become ineffective in parts of India and Nepal as resistance has been developed. Second-line treatments include drugs like diamidinepentamidine, paromomycin and amphotericin B, lipid formulations of amphotericin B (AmBisomeÒ) and the first oral drug miltefosine. However, their use is also limited either due to toxicity and/or long treatment courses, high costs in addition to the emergence of resistance [7]. For these reasons, there is an urgent need of new antileishmanial drugs. Dinitroanilines like Trifluralin (TFL) and Oryzalin (ORZ) are widely used in herbicide formulations. Their herbicidal effect is due to an antimitotic activity that in turn is determined by the binding of the dinitroanilines to tubulins, the main structural component of microtubules [8]. The interaction of dinitroanilines with tubulins is extremely specific: these substances efficiently bind to tubulins of plant and protozoan and practically do not bind to

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animal and fungal tubulins [9]. Due to this specificity, this group of compounds includes promising antiparasitic agents with proven activity against tubulin of several parasitic protozoa, such as Trypanosoma spp., Plasmodium falciparum, Toxoplasma gondii and Leishmania spp. [9–12]. Previous studies had already shown that ORZ and TFL are active in vitro against Leishmania tropica, Leishmania major, Leishmania donovani and Leishmania infantum [10,13,14]. Nevertheless, the use of ORZ (Fig. 1) as an antiparasitic agent may be limited by its low water solubility (e.g., ORZ aqueous solubility of 2.5 mg/L). This weakness may create difficulties on formulation development: the need to use organic solvents that may lead to undesirable side effects, the heterogeneous results in biological assays, which may be difficult to interpret and the need to administer higher, and possibly toxic dosages for in vivo purposes. One of the strategies currently being used to overcome this situation is the incorporation of drugs in liposomes. Liposomes are phospholipid based synthetic vesicles that allow the entrapment of both hydrophilic and hydrophobic drugs. These delivery systems not only enable the safe transport of high drug concentrations into living organisms, but also provide a possible mean of drug targeting to specific cells or organs. In Leishmania infections, a specific targeting arises from the observed fact that conventional liposomes are rapidly removed from the circulation by the mononuclear phagocyte system cells (MPS) after intravenous (i.v.) administration [15,16]. The MPS includes macrophages from liver, spleen or bone marrow, which act as obligate host cell systems for Leishmania parasites, which are in turn specifically targeted by conventional liposomes. Since Black et al. [17] first description of the strategic utility of the i.v. administration of liposomal pentavalent antimony, liposome application on the treatment of leishmaniasis has been studied for a wide variety of antileishmanial agents. Liposomes incorporating current antileishmanial drugs (pentavalentantimonials [18], paromomycin [19] and miltefosine [20]) or new agents (atovaquone [21], harmine [22]) have shown improved antileishmanial performance against experimental VL as compared to the free drugs. The advantages of using new formulations are limited due to the existence of resistant strains (e.g., pentavalent antimonials, miltefosine) or due to the early stage of research (e.g., atovaquone, harmine and paromomycin). The commercialised liposomal amphotericin B, AmBisomeÒ, not only improved the drug therapeutic efficacy but also reduced its liver and kidneys toxicity [23]. This formulation is considered the first choice treatment for patients who are unresponsive to antimonials [24]. However, the cost of such treatment is currently too high for the massive use in developing countries [25,26]. In view of the present scenario, it will be interesting to explore the potentialities of alternative antileishmanial agents and to develop appropriate formulations [27].

To the best of our knowledge, there are no studies concerning the development of liposomal formulations for ORZ. However, the successful application of liposomes for dinitroanilines incorporation was already described in the literature for TFL. Liposomal formulations incorporating TFL showed superior activity in vivo as compared to the free drug in a murine visceral model of infection (L. donovani) [28] as well as in the treatment of experimental canine leishmaniasis (L. infantum) [29], while no sterilisation of the parasites was achieved. The aim of this work is the development of liposomal formulations of ORZ, the evaluation of their in vitro behaviour and their pharmacokinetic and biodistribution profile in the perspective of their future application as a new antileishmanial therapy. 2. Materials and methods 2.1. Materials ORZ was purchased from Supelco (Bellefonte, USA), and pure phospholipids (Dipalmitoylphosphatidylcholine – DPPC; Dipalmitoylphosphatidylglycerol – DPPG; Dimyristoylphosphatidylcholine – DMPC and Dimyristoylphosphoglycerol – DMPG) were supplied by Avanti Polar Lipids, Inc. (Alabaster, USA). Radiolabeled [14C] ORZ (4-aminosulfonyl-2,6-dinitro-N,N-di-n-propylaniline-Ph-UL14 C, specific activity: 6.5 mCi/mmol, uniformly labelled on the aromatic ring) was an offer from Dow AgroSciences (Indianapolis, USA). Radiolabeled [3H]Chol ([1a,2a(n)-3H]Cholesterol, specific activity: 44  103 mCi/mmol) was obtained from Amersham Radiochemicals (Amersham, UK). Acetonitrile (HPLC grade) was from Merck. RPMI 1640 media (20 mM HEPES), penicillin– streptomycin and foetal bovine serum (FBS) were purchased from Sigma–Aldrich (USA). LIVE/DEAD viability kit was obtained from Molecular Probes (UK). All other reagents were analytical grade. 2.2. Cell lines and animals The human monocytic cell line THP-1 was maintained in culture in RPMI 1640 medium, supplemented with 10% heat inactivated FBS, L-glutamine, Penicillin 100 U/mL and Streptomycin 100 lg/mL, pH 7.4 at 37 °C, 5% CO2. Promastigotes of L. infantum MHOM/TN/80/IPT1/LEM 235 were grown in RPMI 1640 supplemented with 10% FCS, L-glutamine and antibiotics, at 26 °C. CD1 male mice weighting 25–30 g were obtained from Charles River, Barcelona, Spain. Animals were fed with standard laboratory food and water ad libitum. All animal experiments were carried out with the permission of the local animal ethical committee, and in accordance with the Declaration of Helsinki, the EEC Directive (86/609/EEC) and the Portuguese laws D.R. no. 31/92, D.R. 153 IA 67/92, and all following legislations. 2.3. Preparation of ORZ liposomal formulations

Fig. 1. ORZ chemical structure (3,5-dinitro-N0 ,N0 -dipropylsulfanilamide, C12H18N4O6S). ORZ is a yellow-orange crystalline solid with a molecular weight of 346.36, soluble in water at 2.5 mg/L at pH 7/25 °C and pKa of 8.6.

The incorporation of ORZ in liposomes was performed by the dehydration-rehydration method (DRV) [30,31] with some modifications. Briefly, the appropriate amounts of phospholipids (10 mM) and ORZ (86.5–865 lg/mL) were dissolved in chloroform and dried on a Büchi rotary evaporator RE-111 (Büchi, Switzerland) until a homogeneous film was formed. The film was dispersed with water, and the resultant suspension was frozen and lyophilised overnight in a Moduyo freeze-dryer (Edwards, Germany). The lyophilised powder was rehydrated in two steps: first with a trehalose–citrate buffer (10 mM citrate, 135 mM NaCl, 30 mM trehalose) pH 5.5 (2/ 10 of the final volume) followed by mild vortexing and left at room temperature for 30 min. The hydration was completed with the addition of the remaining volume (8/10 of the final volume) of

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citrate buffer (10 mM citrate, 145 mM NaCl, pH 5.5). Liposomes were down-sized by sequential extrusion through polycarbonate filters ranging from 0.8 to 0.1 lm in pore size in a 10 mL extrusion barrel (LipexBiomembranes, Canada). Non-incorporated ORZ was separated from the liposome dispersion by size exclusion chromatography in a PD-10 column (Biorad, USA). When needed for in vitro and in vivo studies, the eluted liposomes were concentrated by ultra-centrifugation (49,000g, 1 h 30 min) in a Beckman L8-60M ultracentrifuge (Beckman Instruments, Inc., USA) and suspended in citrate buffer (except when otherwise specified). ORZ liposomal formulations were evaluated in terms of incorporation efficacy (I.E.), loading capacity (L.C.) and ORZ and lipid yield determined as described by the equations below.

I:E: ð%Þ ¼ ð½ORZf=½LipfÞ=ð½ORZi=½LipiÞ  100 L:C: ðg=molÞ ¼ ½ORZ=Lipf ORZ yield ð%Þ ¼ ð½ORZf=½ORZiÞ  100 Lipid yieldð%Þ ¼ ð½Lipf=½LipiÞ  100 where [Lip]f and [ORZ]f represents lipid and ORZ concentration in the final liposomal formulations and [Lip]i and [ORZ]i represents lipid and ORZ concentration in the initial liposomal suspension.

2.4. Liposome characterisation 2.4.1. Size and zeta potential measurements Liposome mean diameter (Ø) and polydispersity index (PI) were determined by quasi-elastic laser light scattering in a Malvern Zetasizer 1000HSA (Malvern Instruments; UK). The zeta potential (surface charge, f) was determined by laser Doppler spectroscopy in a Zetasizer 2000 (Malvern Instruments, UK).

2.4.2. ORZ and lipid quantification Liposomal ORZ content was determined using a HPLC method. The HPLC system consisted of a System Gold (Beckman Instruments, Inc., USA), a Midas Spark 1.1 autoinjector and a Diode-Array 168 detector (Beckman Instruments, Inc., USA). ORZ analysis was performed by UV detection at a fixed wavelength of 284 nm. Separation of ORZ and lipid and ORZ quantification was carried out in a Nucleosil C18, 5 lm (150  4.6 mm) analytical column (Supelco, USA) eluted with a mobile phase consisting of acetonitrile with 0.1 M trifluoroacetic acid (TFA):water with 0.1 M TFA (60:40 (v/ v)) at a flow rate of 1 mL/min. The samples and standards were prepared in mobile phase and loaded in the column with a Midas type 830 auto-sampler with a 20 lL sample loop. Phospholipid determination was performed as described by Rouser et al. [32] based on quantification of inorganic phosphorous.

2.5. Stability studies Liposomal ORZ stability evaluation was performed in different conditions: as a suspension at room temperature and as a freezedried cake. In the first study, the pellets obtained after ultracentrifugation were suspended in citrate buffer without trehalose (citrate 10 mM/NaCl 140 mM, pH 5.5) and kept at room temperature during 72 h. For the latter, the pellets obtained after ultracentrifugation were suspended in trehalose–citrate buffer and lyophilised overnight. All lyophilised formulations were re-hydrated with water under stirring to its original volume. In these studies, stability evaluation was performed in terms ORZ/lipid ratio variation with storage time, ORZ recovery and Ø. All values were determined after separation of the non-incorporated ORZ by size exclusion chromatography.

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2.6. Differential scanning calorimetry (DSC) studies The phase transition behaviour of the ORZ liposomal formulations was performed on a calorimeter DSC Q200 (TA Instruments, DE, USA). Approximately 10 lL of liposomal formulations (lipid concentration ca. 50 mM) were accurately measured into aluminium pans, which were hermetically sealed and then measured against an empty reference pan. The pans were heated and the thermograms were recorded at temperature range of 18–30 °C at a heating rate of 0.5 °C/min. 2.7. In vitro assays 2.7.1. Haemolytic activity evaluation The haemolytic activity was determined using EDTA-preserved peripheral human blood, according to Esteves et al. [33]. Briefly, blood was centrifuged to remove the plasma, and the red blood cells (RBCs) were washed three times in PBS. After the final wash, RBCs were distributed in 96-well microplates (100 lL/well) and an equal volume of free or liposomal ORZ (both diluted in PBS in concentrations between 500 and 6 lM) was added. After incubation at 37 °C for 1 h, the plates were centrifuged (800g, 10 min) and the supernatants recovered. The absorbance of the supernatant was measured at 540 nm with the reference filter at 620 nm on a microplate reader (ELx800, Biotek, USA). In each plate, a positive and negative control wells were added. The negative control consisted in the normal haemolysis achieved by incubation of RBCs with PBS and the positive control (100% haemolysis) consisted in the incubation with water. The absorbance of liposomal and free ORZ solutions was also determined and used as control. The percentage of haemolytic activity of each formulation at different concentrations was estimated using [(A  A0)/(Amax  A0)]  100, where A0 is the negative control haemolysis and Amax corresponds to 100% haemolysis (positive control). The haemolytic activity was also evaluated by the determination of HC50 value (drug concentration that lyses 50% of RBCs) calculated using sigmoidal regression analysis. 2.7.2. Cytotoxicity evaluation As a quantitative measurement of the cell damage after incubation with different concentrations of liposomal and free ORZ, dual staining with SYBR-14 and propidium iodide was used. THP-1 cells (1  106 cells/mL) were incubated with different concentrations (50–0.4 lM) of liposomal and free ORZ. After an incubation period of 72 h, approximately 4  106 cells were stained with propidium iodide and SYBR-14 using the LIVE/DEAD viability kit (Molecular Probes, The Netherlands) according to the manufacturer’s recommendations. The stained THP-1 cells were then analysed by flow cytometry on an Epics Elite model flow cytometer (Coulter, Miami, FL). Excitation of both dyes was done at 488 nm. Differential monitoring of the dyes was achieved by reading the green fluorescence of SYBR-14 at 545 nm and the red fluorescence of PI at 645 nm. At least 10,000 cells were analysed per sample and each staining experiment was repeated three times. Data analysis was performed on fluorescence intensities that excluded cell autofluorescence and cell debris. The cytotoxic effect was evaluated by the determination of CC50 value (drug concentration that kills 50% of THP-1 cells) calculated using sigmoidal regression analysis. 2.7.3. Intracellular amastigote drug susceptibility assay THP-1 cells were differentiated with 1 lM retinoic acid (Sigma) for 72 h at 37 °C/5% CO2. Cells were then washed twice in PBS and once in plain RPMI medium and incubated overnight with L. infantum (LEM 235) promastigotes at 4:1 parasite/cell ratio at 37 °C/5% CO2. Following incubation, cells were harvested, collected in RPMI medium and carefully layered on 4 mL of Histopaque 1077 (Sigma). Free promastigotes were removed by centrifugation at

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1000g for 20 min. The opaque cell layer containing the mononuclear cells was collected, washed thrice in PBS and once in plain RPMI, and resuspended at a concentration of 4  105 cells/mL. The infected cell culture was seeded (200 lL/well) in 24-well plates (Cellstar, Greiner) and incubated for 48 h at 37 °C and 5% CO2 in the absence or in the presence of an equal volume (200 lL) of free ORZ and their liposomal formulations at several dilutions (50–6 lM). After incubation, the well contents were fixed with methanol, stained with Geimsa and observed under a microscope to determine the number of infected cells in 100 cells counted. The IC50 (drug concentration that reduce the infection in 50%) was calculated using sigmoidal regression analysis. 2.8. Pharmacokinetic and biodistribution studies 2.8.1. Comparative study: liposomal versus free ORZ Male CD1 mice weighing between 25 and 30 g were separated randomly into groups of 5. To one group, 200 lL of a ORZ liposomal formulation (DMPC:DMPG 7:3 (F1), 0.15 lmol of ORZ injected) was administrated via mice lateral tail vein. The ORZ liposomal formulation was prepared as described above (see Section 2.3) using traces of 14C-ORZ (4.1  106 counts per minute/mL) and of 3H-cholesterol (1.8  106 counts per minute/mL). As a comparison, a solution of free ORZ with traces of 14C-ORZ (0.15 lmol of ORZ injected) prepared in TweenÒ80:citrate buffer (40:60, v/v) was also evaluated in all studies. Groups of five animals were sacrificed at 30 min and 1, 2, 4, 6 and 24 h post-dosing. Blood samples were collected from the orbital sinus into EDTA-containing tubes. Liver, spleen, heart, lungs and kidneys were removed from animals immediately and rinsed with PBS to remove excess of blood. At selected time-points, the gall bladder was removed, transferred to a scintillation vial and weighed. Following rinsing, organs were quickly dried, weighed and finely minced. Then, 0.05 g of each organ sample and 0.05 mL of blood were added to scintillation vials. All samples were digested overnight at 60 °C with 0.1 mL of perchloric acid (72%) and 0.2 mL of Perhydrol (hydrogen peroxide, 30%). Samples were neutralised with 0.1 mL of acetic acid, and 10 mL of scintillation cocktail was added. All samples were counted for radioactivity in a Beckman LS 5000 scintillation counter (Beckman Instruments, Inc., USA). 2.9. Statistical analysis Data presented are expressed as mean (±) and standard deviation (S.D.). Statistical analysis was performed using ANOVA Single Factor. The acceptable probability for a significant difference between mean values was p < 0.05 (Fcrit < F). 3. Results 3.1. Optimisation of ORZ liposomal formulations Due to the importance of the lipid composition on the incorporation parameters of a hydrophobic drug like ORZ, the incorpora-

tion of this drug was evaluated as a function of phospholipids with two different phase transition temperatures (Tt) (DMPC: DMPG, Tt = 23 °C and DPPC:DPPG, Tt = 42 °C) and two lipid molar ratio (7:3 and 9:1). The results presented in Table 1 show that ORZ incorporation was highly influenced by the lipid composition of the formulation. In fact F1 and F2 (lower Tt. values) provided formulations with higher L.C. (around 30 g/mol) when compared to those of F3 (higher Tt), which presented 16 g/mol for L.C. The reduction in DMPG molar ratio in the formulation resulted in a decrease in the zeta potential from 41 mV (F1) to 19 mV (F2), although this surface charge variation did not affect ORZ incorporation or liposome size. In order to determine whether the incorporation parameters could be further improved, namely if the L.C. could be increased, the saturation profile of F1 was studied. This was done by evaluating the influence of [ORZ/Lip]i on incorporation parameters (Fig. 2). The results show that the L.C. increases with increasing [ORZ/ Lip]i reaching a plateau at [ORZ/Lip]i of 44 g/mol. Irrespectively of the amount of ORZ incorporated in liposomes, the size of liposomes and zeta potential were not affected. All formulations presented a highly negative surface charge (ca. 35 mV) and a mean size of about 0.13–0.15 lm with a PI < 0.15. 3.2. Stability studies of ORZ liposomal formulations The ability of the liposomes to retain the drug and to keep their size during storage was assessed in two different storage conditions: in suspension at room temperature and after freeze-drying. The stability was assessed by the evaluation of variations on the L.C., incorporated ORZ, mean size and PI. The ORZ liposomal formulations F1 and F2 were kept in suspension at room temperature for 72 h. For the two formulations, and under the scheduled conditions, over 90 ± 5% ORZ remains in liposomal form. Moreover, less than 5% change in particle size and in polydispersity index was observed (data not shown). This led us to conclude that liposomes can be safely used under these storage conditions, during at least 72 h. The stability after freeze-drying was evaluated for the F1, F2 and F3 ORZ liposomal formulations in the presence of 30 mM trehalose used as a cryoprotectant. This evaluation was performed 48 h after lyophilisation and immediately after reconstitution of the freeze-dried liposomes with water. The results obtained indicate that ORZ recovery was highly dependent on the lipid composition, particularly the Tt of lipid constituents. The F1 and F2 formulations (lower Tt) provided a higher recovery of ORZ (from 82% to 87%, respectively) as compared to formulation F3 (29%). In contrast, the results for F1 and F2 prove that the stability was not affected by the variations on surface charge of the liposomes. In addition, no significant variation in liposome size was observed for all formulations, with an average final size of 0.16 lm corresponding to an increase of 0.01 to 0.03 lm of the initial size; however, the PI increased from 0.15 to 0.25. The properties of the reconstituted liposomes remained unchanged during at least 72 h (after new evaluation) at room temperature.

Table 1 Incorporation parameters for different ORZ liposomal formulations. Formulation lipid composition

L.C.a (g/mol)

I.E. (%)

Lipid yield (%)

ORZ yield (%)

Øb (lm)

Zeta potential (mV)

F1 F2 F3

29 ± 3 30 ± 1 16 ± 4

88 ± 4 88 ± 5 43 ± 4

89 ± 5 92 ± 4 88 ± 5

77 ± 4 81 ± 4 38 ± 5

0.14 ± 0.02 0.14 ± 0.02 0.15 ± 0.01

41 ± 3 19 ± 2 36 ± 4

DMPC:DMPG (7:3) DMPC:DMPG (9:1) DPPC:DPPG (7:3)

The data were expressed as mean ± S.D. (n = 3). a [ORZ/Lip]i = 34.6 g/mol. b For all formulations PI < 0.15.

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F2) were selected for further testing, such as in vitro and in vivo assays. 3.4. In vitro assays Both liposomal and free ORZ were evaluated in vitro for their toxicity to human cells, for their haemolytic activity to human red blood cells (RBCs) and for their antileishmanial activity. The antileishmanial activity was evaluated against the L. infantum intracellular form. From the data of all those assays, we calculated the various concentrations that were able to reduce 50% of the cells activity and the results are displayed in Table 2.

Fig. 2. Influence of initial ORZ/Lipid ratio ([ORZ/Lip]i) on the incorporation parameters (L.C. and I.E.). Lipid concentration in all formulations is 10 mM. I.E. (%) (h) and L.C. (g/mol) (d). All ORZ liposomal formulations used in this study were freshly prepared. Data were expressed as mean ± S.D. (n = 3).

3.3. Thermotrophic behaviour of ORZ liposomes The thermotrophic behaviour of liposomes (F1) with and without ORZ was studied with the aim to evaluate the changes in the conformational properties of the phospholipids induced by the incorporation of ORZ. The respective thermograms are present in Fig. 3. The results show that empty F1 presents a typical Tt at 23.5 °C due to the transition from a gel state to a liquid crystal state of the constituent lipids. The incorporation of ORZ in these liposomes caused a modification of the thermal behaviour resulting in the abolition of the observed Tt of the empty formulation. Accordingly, the enthalpy decreased from 34.9 to 3.49 J/g. These data are compatible with the interaction of ORZ with the acyl groups of phospholipids. Due to their high incorporation parameters and high stability, either in suspension or after freeze-drying, and the fact that they do not undergo transition temperature during manipulation procedures, both DMPC:DMPG liposomal formulations (F1 and

3.4.1. Haemolytic activity and cytotoxicity The assessment of liposomal and free ORZ haemolytic activity was done using RBCs (as a marker of a general membrane toxicity effect), while their cytotoxicity was evaluated using the monocytic THP-1 cell line (macrophage like cell line). ORZ liposomal formulations F1 and F2 as well as the free drug were tested simultaneously. Results presented in Fig. 4 revealed that both ORZ liposomal formulations did not evidence significant haemolytic activity even at the higher concentration tested (500 lM), while the free drug had a considerable haemolytic activity (about 20% of haemolysis at 100 lM). As presented in Fig. 5, free ORZ also had a cytotoxic effect on THP-1 cells (CC50 of 39 lM). However, when the drug was incorporated within a liposome structure, no cytotoxic effect was observed at concentrations up to 50 lM. 3.4.2. Activity against the intracellular form of Leishmania infantum The antileishmanial activity of ORZ liposomal formulations against the intracellular amastigote form of Leishmania was assessed in vitro by infecting THP-1 cells with L. infantum (LEM 235) promastigotes. After treatment with increasing concentrations of liposomal or free ORZ, the percentage of the infected cells was measured (Fig. 6). The results show that after incorporation in liposomes ORZ kept the activity against the intracellular form of L. infantum. While the results show no significant differences between both liposomal formulations (IC50 = 16.4 lM), the incorporation of ORZ in liposomes slightly increased the drug activity of the free form (IC50 = 24.2 lM). After incorporation of ORZ in liposomes, the resulting formulations have advantages over the free drug, namely reduced cytotoxicity and haemolytic activity, and increased intracellular activity (Table 2). 3.5. In vivo assays

Fig. 3. DSC thermograms of F1 lipid composition. F1 with ORZ (solid line) and empty F1 (dashed line). Samples (lipid concentration ca. 50 mM) were recorded at temperature range of 18–30 °C at a heating rate of 0.5 °C/min. All ORZ liposomal formulations used in this study were freshly prepared.

Although both F1 and F2 formulations presented a high ORZ incorporation and similar in vitro results, F1 formulation was selected for in vivo assays (biodistribution studies) due to its high negative surface charge described to favour liposome passive targeting to macrophages in liver and spleen. To better understand the in vivo fate of F1 formulation, a double-labelling approach was used that allowed following the individual fate of ORZ either in free form or associated to liposomes. This double-labelling also allowed to follow ORZ even after its release from liposomes, and concomitantly to follow the fate of liposomes alone. With this purpose, F1 was prepared using simultaneously traces of ORZ uniformly labelled with 14C and traces of cholesterol–lipid associated labelled with 3H. The dual radiolabelled ORZ liposomal formulation F1 will be designated as Lip-14C-ORZ or 3H-Lip when the formulation was followed by the measurements of 14C or 3H, respectively. As a comparison, free ORZ (hereinafter designated Free-14C-ORZ) was also evaluated in all studies.

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Table 2 In vitro biological evaluation of liposomal and free ORZ. Formulation

% Haemolysis at 100 lM/500 lM

Haemolysis HC50 (lM)

Cytotoxicity CC50 (lM)

Intracellular L. infantum IC50 (lM)

ORZ F1 F2

19.6 ± 2.5/54 ± 3.4 0.0/4.8 ± 1.1 0.0/6.1 ± 1.2

425 ± 10 >500 >500

39 ± 4 >50 >50

24.2 16.4 16.4

DMSO concentration < 1% (v/v) in all assays.

Fig. 4. Haemolytic activity induced by liposomal and free ORZ. RBCs were exposed to different concentrations of freeze-dried reconstituted liposomal formulation F1 (j), F2 (N) and of free ORZ (O). Data were expressed as mean ± S.D. (n = 3).

Fig. 6. Antiparasitic activity of liposomal and free ORZ against THP-1 cell line infected with L. infantum. The percentage of infected THP-1 cells was measured in the presence of different concentrations of freeze-dried reconstituted liposomal formulation F1 (j), F2 (N) and of free ORZ (O). Data were expressed as mean ± S.D. (n = 3).

Fig. 5. Cytotoxic effect of liposomal and free ORZ against THP-1 cell line. Cells were exposed to different concentrations of freeze-dried reconstituted liposomal formulation F1 (j), F2 (N) and of free ORZ (O). Data were expressed as mean ± S.D. (n = 3).

Fig. 7. Blood profiles of liposomal versus free ORZ. Healthy mice were injected i.v. with F1 (Lip-14C-ORZ (s) and 3H-Lip (d)) and Free-14C-ORZ (D)). Data were expressed as the percentage of the injected dose detected (% ID/Blood). F1 and free ORZ freshly prepared were administered at an ORZ dose of 0.15 lmol/mouse. Values presented represent mean ± S.D., n = 5, p < 0.05.

3.5.1. Blood profile: liposomal versus free ORZ The blood profiles of dual radiolabelled F1 and Free-14C-ORZ over a 24 h period after i.v. administration are shown in Fig. 7. Both liposomal and free ORZ showed a bicompartimental behaviour regarding blood clearance. Liposomal ORZ (followed both by Lip-14C-ORZ or by 3H-Lip) decreases rapidly in the first 30 min post-administration up to a value of 58% ID followed by a different slope decrease remaining only 20% ID at 24 h. However, the blood levels of Free-14C-ORZ decreased even more rapidly with

only 17% ID at 30 min and only residual amounts were observed at the end of 24 h. In spite of this rapid decrease in blood level, at 4 h post-administration, a small and transient increase in Free-14CORZ was observed. A parallel increase was also observed for Lip-14C-ORZ; however, this occurred at 6 h post administration. The clearance profiles analysed by the two labels, Lip-14C-ORZ and 3H-Lip, that measure the blood levels for the F1 formulation were very similar suggesting a reduced ORZ leakage from the

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liposomes and thus liposome stability. This assumption was confirmed by the constant Lip-14C-ORZ to 3H-Lip ratio (% ID Lip-14CORZ/% ID 3H-Lip) in the blood, with values between 0.95 and 1.12, up to 4 h post-administration indicating that ORZ remained incorporated within the liposome. A small decrease in this ratio was observed from 6 h to 24 h post administration implying that the liposomes remaining in circulation were starting to lose part of the incorporated ORZ. 3.5.2. Biodistribution studies: liposomal versus free ORZ The distribution of both liposomal and free ORZ was evaluated in five main organs, with particular interest in the liver and spleen, as they are the organs foremost affected in visceral leishmaniasis. Results on the accumulation of Lip-14C-ORZ and 3H-Lip as well as Free-14C-ORZ in the liver and spleen, during the 24 h period after i.v. administration, are shown in Fig. 8A and B, respectively. Following F1 administration, a fast and simultaneous accumulation of Lip-14C-ORZ and 3H-Lip in the liver was observed during the first 2 h, where both labels reached their higher values of 14 and 18% ID/g, respectively. Accumulation in the spleen followed a similar pattern with maximal Lip-14C-ORZ and 3H-Lip levels of 21 and

Fig. 8. Liver (A) and spleen (B) biodistribution profiles of liposomal versus free ORZ. Healthy mice were injected i.v. with F1 (Lip-14C-ORZ ( ) and 3H-Lip (j)) and Free-14C-ORZ (h). Data were expressed as the percentage of the injected dose detected per gram of organ (% ID/g of organ). F1 and free ORZ freshly prepared were administered at an ORZ dose of 0.15 lmol/mouse. Values presented represent mean ± S.D., n = 5, p < 0.05.

287

26% ID/g also occurring 2 h post-administration. After this period, Lip-14C-ORZ and 3H-Lip levels in both organs gradually reduced, and 24 h post-administration, the levels of both labels were still about 6 and 8% ID/g in the liver and about 5 and 6% ID/g in the spleen, respectively. Throughout the 24 h period, Lip-14C-ORZ levels in both organs were significantly higher (p < 0.05) as compared to Free-14C-ORZ. In fact, 2 h post administration, when Lip-14C-ORZ reached its higher levels, it presented 9- and 13-fold higher levels in the liver and spleen, as compared to Free-14C-ORZ. The results clearly show that the incorporation of ORZ in liposome significantly enhances liver and spleen accumulation as compared to free ORZ. The levels of Free-14C-ORZ in the liver and spleen were almost constant (4% ID/g) during the first hour post-administration, after which they decreased gradually and at 24 h post-administration only residual amounts were detected in both organs. Despite this gradual reduction, a small and transient increase was observed 4 h post-administration. This effect was also observed for Lip-14C-ORZ 6 h post administration but at a lower extent as compared to Free-14C-ORZ. Other organs like lung, heart and kidney were also analysed for the accumulation of Lip-14C-ORZ and Free-14C-ORZ. The results are presented in Table 3. In all organs, Free-14C-ORZ presented a similar distribution profile as previously observed for liver and spleen, including the transient increase observed 4 h post-administration after which Free-14C-ORZ decreased very rapidly to residual amounts after 6 and 24 h post administration. The highest Free-14C-ORZ levels (3–5% ID) observed at 30 min after administration were not significantly different than the ones observed for the liver and spleen. As for Lip-14C-ORZ, in contrast to that observed in the liver and spleen, no accumulation was observed in these other organs up to 2 h post-administration. During this period, Lip-14C-ORZ levels remain constant and significantly lower in comparison (p < 0.05) with those observed in our main target organs. A small increase in Lip-14C-ORZ levels was also observed 6 h post administration, particularly in the lungs and was maintained up to 24 h post administration. As described for Free-14C-ORZ, this fact may be related to the levels of Lip-14C-ORZ observed at that moment in the blood (20% ID) associated with the highly vascular tissue present in the lungs, which may result in a higher presence of residual blood even after rinsing in buffer. After macroscopic observation of the gall bladder, it was observed that in the groups to which ORZ liposomal formulation was administrated, the organ presented high amount of fluids, which lead us to determine the levels of Lip-14C-ORZ and 3H-Lip in this organ. In all Free-14C-ORZ groups, the gall bladder was empty so it was not possible to measure the amount of accumulated radiolabels. The results obtained for the % ID/g gall bladder of both F1 components (Lip-14C-ORZ and 3H-Lip) are presented in Fig. 9. The results show that the accumulation of both labels in the gall bladder presented two distinctive phases. In a first phase, between 30 min and 2 h post-administration, it was observed an increase in both Lip-14C-ORZ and 3H-Lip followed by a second phase where the levels of both F1 components gradually decreased. These observations are in agreement with those obtained in the liver, spleen and in the blood, including the second-peak phenomenon of Lip-14CORZ at 6 h post administration, which can result from the ORZ enterohepatic circulation.

4. Discussion Leishmaniasis is classified by the WHO as a neglected tropical disease; however, no vaccine is currently available and thus

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Table 3 Biodistribution profile of Lip-14C-ORZ and Free-14C-ORZ in the lungs, heart and kidneys of healthy mice following i.v. administration. Time after administration 30 min Free-14C-ORZ (% ID/g of tissue) Lungs 5.3 ± 1.3 Heart 3.7 ± 1.5 Kidneys 4.8 ± 2.6

1h

2h

4h

6h

24 h

Lip-14CORZ (% ID/ g of tissue)

Free-14CORZ (% ID/ g of tissue)

Lip-14CORZ (% ID/ g of tissue)

Free-14CORZ (% ID/ g of tissue)

Lip-14CORZ (% ID/ g of tissue)

Free-14CORZ (% ID/ g of tissue)

Lip-14CORZ (% ID/ g of tissue)

Free-14CORZ (% ID/ g of tissue)

Lip-14CORZ (% ID/ g of tissue)

Free-14CORZ (% ID/ g of tissue)

Lip-14CORZ (% ID/ g of tissue)

4.1 ± 0.7

4.6 ± 1.6

3.5 ± 0.8

3.4 ± 2.0

5.1 ± 1.1

4.9 ± 0.8

4.9 ± 0.8

0.4 ± 0.1

6.8 ± 0.6

0.3 ± 0.1

6.8 ± 1.2

2.0 ± 0.5

3.7 ± 0.7

1.7 ± 0.3

2.8 ± 1.8

1.8 ± 0.6

4.5 ± 0.8

1.5 ± 0.2

0.3 ± 0.1

2.0 ± 0.2

0.3 ± 0.1

2.0 ± 0.4

3.9 ± 0.6

3.9 ± 0.4

3.5 ± 0.5

1.9 ± 1.3

4.2 ± 0.6

5.0 ± 0.4

2.7 ± 0.6

0.5 ± 0.1

3.3 ± 0.6

0.2 ± 0.1

2.9 ± 0.7

Values represents mean of the % ID/g of organ ± S.D., n = 5.

chemotherapy is the only effective way to treat all forms of this disease. Moreover, current therapy is either toxic or loosing efficacy due to widespread resistance of Leishmania strains. This scenario leads to the urgent need of new antileishmanial drugs [26,27]. Substances, such as dinitroanilines have attracted increasing interest due to the specific mechanism of action (tubulin-binding exerting antimitotic activity on Leishmania spp.) and the low toxicity in mammals [14]. These facts make dinitroanilines potential efficient drugs against Leishmania providing that their unfavourable physicochemical properties (low water solubility and easy sublimation due to low vapour pressure) are overcome either by chemical modification and/or incorporation in drug delivery systems, such as liposomes. This work concerned the development of liposomal formulations of one dinitroaniline (ORZ) that may improve the drug performance both in vitro and in vivo. During the formulation process, it was expected ORZ to be incorporated within the lipid bilayer of liposomes, rather than in the internal aqueous medium, due to its hydrophobicity (aqueous solubility of 2.5 mg/L and octanol/water partition coefficient, log P 3.73 at pH 7) [34]. The DSC studies confirmed this assumption as the incorporation of ORZ abolished the typical Tt of empty liposomes and strongly reduced enthalpy. These findings are compatible with the strong interactions between ORZ and the acyl chains of the phospholipids and have been observed with other molecules, such as cholesterol [35] and other hydrophobic drugs [36,37]. Accordingly, the effect of a variation on the lipid composition of liposomes, namely the acyl chain lengths, on ORZ incorporation was studied. The high incorporation parameters obtained for ORZ in formulations with lipids of lower transition temperature (F1 and F2) were expected due to weaker interactions between the shorter acyl chains of these phospholipids and thus an increased availability for ORZ incorporation. In contrast, due to the well-ordered arrangement of the acyl chains of F3 formulation (longer chain), lower availability for ORZ incorporation was expected, resulting in a difficult insertion and stabilisation of the drug in these liposomes. These observations are in close agreement with those reported in the literature for another dinitroaniline, TFL [29], as well as for other hydrophobic drugs such as rifabutin or dexamethasone [30,38]. This behaviour was reinforced by the fact that the variation on formulations surface charge (F1 versus F2) had no influence on ORZ incorporation, indicating that no apparent electrostatic interactions were present between the drug and the lipid head groups. The lipid composition identified as a key factor for ORZ incorporation was also important for the ORZ stabilisation in the bilayer, either as a suspension at room temperature or as a freeze-dried cake. The association of ORZ with the acyl chains of F1 and F2 evidenced by DSC studies seems to account for the prevention of leakage during, at least, 72 h. The same reason seems to be underlined during the freeze-drying process in the presence of a cryoprotectant (trehalose), avoiding leakage of ORZ. A similar sta-

bilisation process was described for TFL [29], although for ORZ liposomes, a lower trehalose concentration was needed. The specific characteristics of each drug and the particular lipidic compositions of liposomes in each study may account for this difference. The liposomal stability and its ability to retain ORZ were also demonstrated in vivo by dual radiolabel studies. In fact, a constant ratio between the liposomes and the incorporated ORZ was observed in the blood several hours after i.v. administration of liposomal formulation. After optimisation of incorporation and stabilisation of ORZ in liposomes, the in vitro and in vivo performance of the so developed formulation was evaluated. The incorporation in liposomes was responsible for the reduction in haemolytic activity and cell toxicity of free ORZ. The cytotoxic effect observed for the free drug in THP-1 cells (CC50 39 lM) was probably a result of drug crystal precipitation at high drug concentrations due to ORZ low solubility in the cells growth medium. This drawback, already described for ORZ in fibroblasts in vitro [11] was abolished in our study, by its incorporation in liposomes were a stable and homogeneous suspension was obtained. A reduction in ORZ cytotoxicity on THP-1 cells was already observed after ORZ incorporation in other drug delivery systems, such as solid lipid nanoparticles [39]. Regarding the observed ORZ haemolytic activity to RBCs (HC50 425 lM), no data is available in the literature; however, several new ORZ derivatives induced signs of haemolysis and/or renal dysfunction when i.v. administrated to rats [40]. Through incorporation in liposomes, all these deleterious effects of free ORZ were absent (HC50 > 500 lM; CC50 > 50 lM), demonstrating the protective role of liposomes and the possibility to administer this drug without noxious effects. Similar protective role of liposomes was already described for several classes of drugs, including antineoplasic (vincristine and cantharidin) [41,42], bactericidal (rifampicin) [43] and antifungal (nystatin) [44] drugs. The incorporation of several current antileishmaniasis drugs (amphotericin B or miltefosine) in liposomes was also proved to be crucial in the reduction of their cytotoxic or haemolytic effects [45,46]. The retention of therapeutic activity was another crucial factor to be evaluated, for the estimation of the putative validity of this formulation as therapeutic agent. As promastigotes are not the clinically relevant form of the parasite, a more realistic evaluation of the intracellular form was done in a macrophage like cell line (THP-1) infected with L.infantum amastigotes. From these experiments, we could conclude that ORZ retain its intracellular activity even when incorporated in liposomes. Despite the recognised therapeutic benefit of the use of dinitroanilines against leishmaniasis, there are only a few reports on dinitroanilines pharmacokinetics and biodistribution after parenteral administration, and none of them, associated with liposomes or other drug delivery systems [47,48]. The in vivo behaviour of our ORZ liposomal formulations was also evaluated in the present

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The so described properties of ORZ liposomal formulation, both in vitro and in vivo, are indicative of an enhancement in ORZ performance and advisable that should be further tested for evaluation of their therapeutic interest as antileishmanial agents. 5. Conclusion

Fig. 9. Gall bladder biodistribution profile of F1. Healthy mice were injected i.v. with F1 (Lip-14C-ORZ ( ) and 3H-Lip (j)). Data were expressed as% ID/g of organ. F1 freshly prepared was administered at an ORZ dose of 0.15 lmol/mouse. Values presented represent mean ± S.D., n = 5.

Liposomal formulations containing ORZ were optimised by selection of the appropriated preparation method, lipid composition and experimental conditions. These formulations are stable in different storage conditions and have demonstrated superior biopharmaceutical properties over free ORZ. Incorporation of ORZ in liposomes proved to be important in reducing the haemolysis of red blood cells and the cytotoxic activity in THP-1 cells observed for free ORZ and that they are active against intracellular leishmanial infection. In vivo studies demonstrated the efficacy of the liposomal formulation to passively target ORZ to the main organ of leishmanial infections (liver and spleen). These results seem to indicate that ORZ liposomes could be candidates as therapeutic agents against leishmaniasis. Further studies in leishmaniasis animal models are needed to prove this hypothesis. Acknowledgements

work. It was demonstrated that ORZ liposomes were highly advantageous, as compared to the free drug, in terms of blood profile and biodistribution, significantly increasing ORZ accumulation in the main affected organs in leishmaniasis. In fact, it was demonstrated that, 2 h post administration, liposomal ORZ presented a substantial accumulation in the liver and spleen (9- and 13-fold higher levels) as compared to the free drug. This increased accumulation was due to the well described mechanism of conventional liposome capture by MPS cells that are mainly present in the liver (Kuppfer cells) and spleen [49]. The superiority of the liposomal formulation in targeting ORZ was observed throughout the 24 h evaluation period. This clearly demonstrates that ORZ incorporation in liposomes not only increased the drug accumulation in the target organs but also allowed ORZ to remain in these organs for a longer period of time, which may prove beneficial for leishmaniasis treatment. Moreover the incorporation of ORZ in liposomes reduced the drug’s fast elimination from the body. Indeed, while for free ORZ 85% ID was removed from the blood circulation 30 min post administration with only 13% ID detected in all organs, for Lip-14C-ORZ from the 45% ID removed from circulation, 34% ID were detected in all organs. More important is that 30% of those were accumulated in liver and spleen. These differences may arise from the free ORZ rapid excretion from the body or accumulation in other tissues or organs. A rapid excretion of another dinitroaniline, TFL, was already observed after oral administration with a high accumulation in faeces and urine [50]. Another study also demonstrated TFL high accumulation in fat after intraperitoneal and intramuscular administration [51]. A relevant aspect of this study was the observation of a second ORZ blood peak which could be an indication that this drug was reabsorbed into circulation. This second peak phenomenon could be explained by ORZ reflux into circulation from another organ/tissue (possibly from fat tissues) or by enterohepatic recirculation. While ORZ accumulation in fat was not evaluated, the observed accumulation of Lip-14C-ORZ (and also 3H-Lip) in the gall bladder could reinforce the enterohepatic recirculation. This is an important metabolic pathway for many hydrophobic compounds and has been reported previously for several dinitroanilines, including TFL [50]. The second peak phenomenon allows ORZ (or possible derivative) to circulate for a longer time in the blood, thus increasing its accumulation in the liver and spleen which could contribute to an increase in ORZ therapeutic efficacy.

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