International Medical Microbiology Physicochemical and Biopharmaceutical Characterization of N-Iodomethyl-N,N-Dimethyl-N-(6,6-1 Diphenylhex-5-En-1-Yl) Ammonium Iodide and A Promising Antileishmania Delivery System

Cutaneous leishmaniasis (CL) is an infectious disease endemic in tropical and subtropical countries. The current drugs have severe drawbacks that restrict their use and en hance the need for better drugs. Recently, the N-iodometh -yl-N,N-dimethyl-N-(6,6-diphenylhex-5-en-1-yl) ammonium iodide (C 6 I) was identified as a promising compound for the topical treatment of CL. The need for oral drugs with poten tial use to treat cutaneous, mucosal and visceral leishmani -asis, in the present work were determined the pharmaceuti cal and some biopharmaceutical properties of C 6 I as possi ble oral treatment and based on this results a nanoformula tion was elaborated, characterized and tested in in vitro and in vivo model of the antileishmanial activity and toxicological assays. The C 6 I showed crystalline form and good intestinal permeability, its dissolution profile did not change with pH changes. The C 6 I was not mutagenic and genotoxic in vitro , it presented some minors acute toxicological effects. The solid lipid nanoparticles (SLN) used Precirol ® as lipid, it had a size in the nano range scale with a low polydispersity index, and encapsulation efficiency > 60%. The nanoparti cles of C 6 I (PC 6 I) increased the in vitro antileishmanial ac tivity 40-fold than free C 6 I. In turn, the oral administration of C 6 I and PC 6 I (30 mg/kg/d, 28 days) produced complete cure in 42.9% and 71.4%, respectively, with no relapses and no toxicity. The effectiveness of meglumine antimoniate was 100% but the relapse rate was 28.6%. C 6 I and PC 6 I are safe compounds, as demonstrated in in vitro and in vivo assays for toxicological profile. In conclusion, a novel oral quaternary ammonium iodide salt-based formulation with antileishmani al properties was developed. The safety and effectiveness information of PC 6 I formulation showed here supports the further evaluation of efficacy and safety in patients to validate the use of PC 6 I as an alternative for the oral treatment of CL. when administered topically at a dose of 2 mg/kg/day for 15 days [5]. Here we show that free C 6 I, administered orally at a dose of 15 mg/kg/day for 28 days, showed an effectiveness close to 30%. Nev -ertheless, its effectiveness increased to 71% when the C 6 I was incorporated in Precirol® NPs and administered at a higher dose of 30 mg/kg/day. The results were con sistent in the in vivo model, with 71% cure in the group of animals treated with PC 6 I upon oral administration of a dose of 30 mg/kg/day for 28 days. Differences in the effectivity and toxicity between free C 6 I or encapsulated PC 6 I was evident not only because the LC 50 increased two-fold but also because the EC 50 was lowered 40-fold.


Introduction
Current drug therapies for cutaneous leishmaniasis (CL) are unsatisfactory due to numerous drawbacks including severe side effects, reproductive toxicity, prolonged treatment with high doses and decreased effectiveness. Since CL is prevalent in economically dis-

Ethical aspects
The study for intestinal absorption was approved by the Ethical Committee for Animal Welfare of the University of Valencia (Act No. CE 86/609) while the in vivo assays for, in vivo effectiveness and toxicity, maximum tolerable dose and oral toxicity at repeated dose were approved by the Ethical Committee for Animal Experimentation of the Universidad de Antioquia (Act No. 91-2014).
Biopharmaceutical and pharmacological parameters of C 6 I Intrinsic dissolution: One hundred mg of C 6 I were compressed at 700 Pa and a 6.33 mm punch during 1 min. The surface area of the compacts was 3.14 cm 2 [9]. Compacts were placed in a molten beeswax-mold with one face in contact with dissolution medium. Dissolution was conducted in an USP type 2 dissolution apparatus using 900 mL of either 0.1 N HCl, 0.2 M acetate buffer, pH 1.5, purified water pH 7.0 and 0.2 M phosphate buffer, pH 8.0 at 37 ± 1 °C, as dissolution media, respectively, with a paddle rotating at 100 rpm. One mL samples were withdrawn at regular intervals and a plot of absorbance vs. time was constructed. Experiments were done in triplicate. Absorbance was determined using the UV-Vis spectrophotometer (VarioScan Thermo) at 233. The dissolved cumulative amount per surface unit of the compact was plotted against time for each vessel. A standard curve was built using standard solutions used in dissolution media. Intrinsic dissolution rate (IDR) was calculated using Eq. 1: Where j is the IDR, V is the volume of the dissolution medium, c is the concentration, A is the area of the C 6 I disk and t is the time Thermic properties and powder X-ray diffraction: The melting points, purity and crystalline appearance of C 6 I were determined by differential scanning calorimetry (DSC) profile using a DSC 204 F1 (Phoe-nix®-NETZSCH-Gerätebau GmbH). Measurements were in a range of 25 °C to 300 °C with increases in 10 °C per min. The temperature scale was calibrated using an internal reference within pots of aluminum. Tests were performed in a nitrogen atmosphere.
The crystallinity properties of C 6 I were determined by Powder x-ray diffraction (PXRD) using an X-ray diffractometer Rigaku Miniflex® (Rigaku, Tokyo, Japan) with a source of Cu, Ka1 radiation (1.542 A°) and angles ranging between 3° and 50°.

In situ intestinal absorption by doluisio method:
Thirteen adult Wistar male rats were fasted for 18 h (except for drinking) and then anesthetized and immobilized. Afterwards, the abdominal cavity was cut along advantaged communities, new drugs development lack for economic incentives to large pharmaceutical companies and in consequence, leishmaniasis is designated as neglected tropical disease [1,2].
In a previous work was reported that the quaternary halomethylated ammonium salt N-iodomethyl-N,N-dimethyl-N-(6,6-diphenylhex-5-en-1-yl) ammonium iodide (C 6 I), a choline analog, could be considered as a lead compound in the development of a new treatment for CL. The C 6 I has activity against intracellular amastigotes of Leishmania (Viannia) panamensis [3,4]. Moreover, the C 6 I administered topically at a dose of 2 mg/kg/day during 15 days was able to cure hamsters with experimental CL caused by L. (V) braziliensis [5]. The ability of C 6 I to inhibit the choline/ethanolamine kinase of L. (Leishmania) infantum and the production of phosphatidyl choline and to interfere with the in vitro uptake of choline by promastigotes of L. tarentolae was also demonstrated [6]. The organoleptic analyses showed that C 6 I is an odorless, white-colored powder with fine appearance, soft to the touch, crystalline habits with regular plaque shapes and smooth texture, grouped in clusters and particle size varying between 20 and 100 microns. Other physiochemical studies showed that the solubility of C 6 I is high in acetonitrile (1.5 mg/mL), low in methanol, isopropanol and chloroform (< 1.0 mg/mL) and water (0.61 mg/mL). This solubility is not affected by pH changes [5].
The importance of C 6 I is higher when considering the urgency expressed by the World Health Organization of having drugs for oral administration since miltefosine, the only medicine available for oral treatment of leishmaniasis, is not widely available in the market.
In the present work were determined the pharmaceutical properties of C 6 I as well as the usefulness of oral C 6 I to cure experimental CL in hamsters. The in vitro cytotoxicity, mutagenicity and genotoxicity of C 6 I were also determined. Because the low solubility observed previously for C 6 I could difficult the dissolution process in the GI tract and, affect the bioavailability [7], the oral delivery can be improved using nanocarrier systems such as solid lipid nanoparticles (SLNs) that are at the forefront of the potential application mainly due to the low cost and easy industrial production [8]. Thus, in the present work were prepared and characterized SLNs of C 6 I using Precirol® (PC 6 I). The in vitro and in vivo antileishmanial toxicity and activity of PC 6 I were assessed against L. (V) braziliensis, compared to the antileishmanial activity of free C 6 I and with the standard treatment to this disease, meglumine antimoniate (MA). Finally, the in vivo oral toxicity profile was also determined.

Compound
The C 6 I was supplied by L.A Rios and R. Ocampo. Its synthesis was previously described by Duque, et al. 2016 [3].
trations (46 -23 and 12 µg/mL) and incubated during 24 h. Colcemid (0.1 pg/ml) was added and cells were incubated again during 1 h. The cells were centrifuged, resuspended in 0.075 M KCl and incubated at 37 °C for 7 min, fixed 10 min with Carnoy solution, washed and dripping. Slides were dried and stained with 5% Giemsa, pH 6.8. One hundred metaphase cells (46 chromosomes) were analyzed. Structural chromosome aberrations were classified as chromatid breaks (B), chromosome breaks (BB), dicentric chromosomes (DC), ring chromosomes (R) and multiradial chromosomes (MR). Each treatment was performed in two separate experiments using RPMI as negative control and mitomycin-C (10 M) as positive control.
Maximum tolerable dose of C 6 I: Twenty-one female, nulliparous and non-pregnant Wistar rats, 188 ± 12 g weight, were divided into four groups. Three groups (n = 6 each) were treated with a single dose of 5, 50 or 300 mg/kg (1 mL) of C 6 I by the oral route. The forth group (n = 3) received distilled water (control group). Rats were observed during the first 6 h for any potential signs of toxicity and then daily during 14 days. Signs were graded according their nature and severity. Rats were weighed before and every week after doses. At day 14 rats were sacrificed and necropsied. The heart, brain, liver, spleen, kidneys, testicles and epididymis (males) and ovaries (females) were weighed and biopsies were stored in 10% formaldehyde for histopathology analysis [13]. The severity of lesions was graded from 0 to 3, where 0 for no lesion, mild, moderate or severe, respectively. The surviving rats were monitored for mortality, behavior, fur, pain and any sign of illness during the study.

Development and characterization of solid lipid nanoparticles
The Precirol® lipid was selected because it is frequently used in oral formulation, the production method of SLN was selected, first to determine of solubility of C 6 I in Precirol®, one milligram of compound was added to 25 mg of lipid that was heated above 10 °C its melting points. Solubility was verified after that the excess of C 6 I persisted for more than 8 hours [14].
The Precirol® NPs loaded with C 6 I (PC 6 I) were elaborated by evaporation of the emulsion-solvent using 0.6% sodium deoxycholate, 0.5% Tween 20 and 1% soy lecithin as co-surfactants [15]. Fifty mg of Precirol® was dissolved with lecithin and C 6 I (3 mg) in 1 ml of dichloromethane. This organic phase was then added to 5 mL of aqueous phase containing Tween 20 and sodium deoxycholate. Dispersion was performed by sonication for 3 min (Branson Sonifier 250, USA) followed by 5 min of stirring at 12,500 rpm for 4 h at room temperature using a High Speed Mixer (Model L4RT, Silverson Machines, Chesham, UK) until the dichloromethane had completely evaporated. Free C 6 I was separated from the dispersed Precirol® NPs by size exclusion chromatography in a PD-10 column (Bio-Rad Laboratories) using as eluent phosphate buffer saline (PBS), pH 7.4. the medioventral line, and the small intestine was separated. Then, the small intestine was washed with 0.9% saline solution and two different concentration of compound was used, 10 mL of supersaturated or 1/10 diluted C 6 I solution were perfused. The concentration of C 6 I in the intestinal lumen was measured every 5 min for 30 min by sampling 200 µL of the remaining liquid. Samples were immediately centrifuged (1,500 g, 10 min) and the supernatant frozen (-80 °C) until quantification by HPLC using the method described [5]. Samples of 1.0 mg/mL of C 6 I were prepared and run in an Agilent Technologies 1200 chromatograph equipped with a C18 column (Restek C18, 5 µm, 250 × 4.6 mm), with UV-detector. The mobile phase consisted of acetonitrile and C 2 H 3 N-aO 2 0.1 M (60:40, v/v), adjusted to pH 5.0 with acetic acid. Elution was achieved in 12 min at a flow rate of 0.8 mL/min. The injection volume was 10 µL. Compound was monitored at 233 nm. At the end of the sampling, the remaining volume was recovered and the extent of water reabsorption was determined at every sampling time, considering that this process follows zero-order kinetics [10]. The apparent constant absorption rate for C 6 I was obtained from the first-order equation Eq. 2: Where C is the concentration of C 6 I remaining in the intestinal lumen, k a is the apparent rate constant for absorption, and C 0 is the initial concentration of C 6 I.
The k a was transformed in to permeability values by means of Eq. 3: Where R is the effective radius of the intestinal segment corresponding to 0.1784 cm, considering that intestinal segment as a cylinder of 10 mL volume and 100 cm length.
In vitro genotoxicity: Chromosome alterations induced by C 6 I were evaluated in lymphocytes from three healthy donors [12]. Cells cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum and 1% penicillin-streptomycin (10,000 U-10 mg/mL) (complete medium) were stimulated with 200 µL phytohemagglutinin and incubated at 37 °C, 5% CO 2 . After 36 hours, the cells were treated with each of sublethal C 6 I concen-Release of C 6 I in solid lipid nanoparticle: The NPs were incubated in 10 mM PBS release medium, pH 7.4, with horizontal shaking at 37 °C. At 0.5 -1 -2 -4 -8 and 24 h intervals samples were centrifuged at 30,000 × g for 30 min at 4 °C. The released C 6 I was quantified in the supernatants by spectrophotometry in a UV-1700 microplate reader at 233 nm. Quantifications were done in triplicate [15].

Differential scanning calorimetry (DSC):
The DSC measurements were performed on a DSC calorimeter Q200 (TA Instruments, DE, USA). Dispersions of C 6 I, Precirol®, sodium deoxycholate, Tween 20, soy lecithin, empty NPs and PC 6 I nanoparticles were weighed and measured against an empty reference vessel. The samples were heated, and the respective thermograms were recorded in the temperature range of -20 °C to 240 °C at a heating rate of 10 °C/min.

Evaluation of efficacy and safety of C 6 I and PC 6 I
In vitro evaluation of cytotoxicity: The cytotoxicity of free C 6 I, empty NPs and PC 6 I was evaluated in vitro on primary cultures of human monocyte-derived macrophages (MDMhu) and golden hamster peritoneal macrophages (MPha) and human cell lines U937 (ATCC® CRL-1593.2 ™ ), Caco-2 (ATCC® HTB-37 ™ ) and Detroit 551 (ATCC® CCL-110™). Cytotoxicity was determined by cell viability using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) enzymatic micromethod following a methodology described by others [17]. Untreated cells were used as a viability control and doxorubicin was used as a positive control for cytotoxicity. At least two independent experiments were performed, each in triplicate. The % of mortality was used to calculate the half maximal lethal concentration (LC 50 ) using the Probit analysis [18].

In vitro evaluation of anti-Leishmanial activity:
Intracellular amastigotes of L. (V) braziliensis (MHOM/ CO/88/UA301-EGFP) were obtained after infection of U937 cells as described elsewhere [19]. After 24 h of infection the medium was replaced by fresh medium containing each C 6 I, empty NPs or PC 6 I, at concentrations of 100 -12.5 -3.125 -0.78 µg/μL. After 72 h, cells infected and treated were analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, USA). The antileishmanial activity was determined according to the reduction in the number of viable parasites inside the infected cells obtained with each treatment and each concentration, according to the mean fluorescence intensity (MFI). Data obtained in untreated infected cells corresponds to 100% of parasites. All measurements were performed in triplicate, in two independent experiments. The half maximal effective concentration (EC 50 ) was calculated according to the Probit analysis [18]. The NPs were characterized based on particle size and surface charge (PZs) determined by photon correlation spectroscopy (PCS) using a Zetasizer Nano S (Malvern Instruments, UK). Measurements of particle size were performed at 25.0 ± 0.1 °C after dilution of the NPs in purified water (1:100). The results were expressed as the average of particle size and the polydispersion index (PI). In turn, the surface PZ value of the NPs was determined based on the mobility of the particles in an electric field using a Zetasizer Nano Z. For this, the diluted samples in purified water were exposed to 150 mV and the appearance.

Evaluation of in vivo
The incorporation of C 6 I into the NPs was quantified after dissolving the NPs loaded with C 6 I in acetonitrile, which promoted precipitation of the lipid phase. After centrifuging, free C 6 I was measured at 233 nm in a microplate spectrophotometer (FLUOstar Omega). The supernatant from unloaded NPs was used as a blank. The quantification method was validated according to the international guide for validation of analytical procedures [16]. The efficiencies of encapsulation (EE) of C 6 I and drug loading (DL) in the NPs were calculated according to Eq. 4 and Eq. 5: The morphology of the NPs were determined by scanning electron microscopy (SEM, XL-30 Royal Philips Electronics, Amsterdam). All measurements were performed in triplicate.

Stability of solid lipid nanoparticles loaded with C 6 I:
Five mL aliquots of PC 6 I in suspension were stored at 2-8 °C, and the mean particle diameter, PI, zeta potential and DL were determined after 30 and 60 days of storage. The average of particle size was analyzed as previously described. The stability evaluation in terms of EE and DL was determined after separation of unbound C 6 I by size exclusion chromatography, as described above. The effect of autoclaving was also evaluated, for this assessment, the PC 6 I formulation was divided into two aliquots of equal volume. One aliquot was autoclaved at 121 °C for 20 min, while the other aliquot was maintained at 2-8 °C as a reference sample and then, the physicochemical properties (particle diameter, PI, surface charge, DL and EE) were compared. Additionally, the influence of temperature on the physical stability of PC 6 I was evaluated using DLS (Zetasizer Nano S). Samples were diluted with purified water (1:100) in a quartz cell, and particle size analysis was performed while heating the sample from 25 °C to 90 °C at a rate of 0.5 °C/min and then cooling it from 90 °C to 25 °C at the same rate. Particle size was measured every 0.5 °C. This assay was done in triplicated. females, each). Two groups received 0.2 mL of C 6 I or PC 6 I at 30 mg/kg/day, respectively by oral route during 28 days. A third group remained untreated (negative control). Mice were monitored daily and weighted before treatment and weekly during treatment. Blood samples were obtained prior to the first dose (TD0) and at the end of the study (TD28) [22]. At the end of the study animals were sacrificed and necropsy and biopsies were performed. Serum level of aspartic transaminase (AST), ALT, BUN, CRT, albumin (ALB) and alkaline phosphatase (AP) were quantified. The blood cell parameters were also evaluated.

Statistical analysis
Data are expressed as mean ± SD, except for effectiveness of treatments that are expressed as percentage. The Kolmogorov-Smirnov test was used for normality distribution of data and differences between variables were analyzed by Student's t-test using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). Differences were considered significant when p < 0.05.

Results
Biopharmaceutical and pharmacological parameters of C 6 I Intrinsic dissolution: Intrinsic dissolution of pure compounds did not change with different pHs, the value of intrinsic rates (IDR) were similar in tree conditions, 0.24 ± 0.03 at pH 1.5; 0.26 ± 0.04 at pH 7.0 and 0.20 ± 0.05 at pH 8.0 (mg/min/cm 2 ).

Thermic properties and powder X-ray diffraction:
The DSC and powder X-ray diffractometric data are summarized in Figure 1. The C 6 I had a melting point of 160.1 °C, an exothermic peak close to 150 °C, possibly due to the sublimation of iodine ( Figure 1A) and an internal structure predominantly was crystalline confirmed by the XRPD analysis ( Figure 1B).

In situ Intestinal absorption by Doluisio method:
The individual absorption rate constant (k a ) and Papp permeability values of C 6 I obtained using a saturated solution and a 1/10 dilution solution were similar value CO/88/UA301-EGFP)/100 mL. When the ulcer was developed in the skin, hamsters were distributed in five experimental groups. Two group of hamsters (n = 7 each) received by oral route 1 mL of C 6 I or PC 6 I at a dose of 30 mg/kg/day for 28 days. A third group (n = 7) was treated with MA at a dose of 120 mg/kg/day for 10 days, by intramuscular route, that corresponds to the curative dose identified in previous work (cure control) [20]. A fourth group (n = 3) was treated with the empty NPs (vehicle) and the fifth group (n = 3) remained untreated after infected (non healing control).
The clinical effectiveness of each treatment was determined by comparing the lesion sizes before treatment (TD0) with those observed the last day of treatment (TD28 for C 6 I, PC 6 I and NPs), or TD10 for MA) and days 30, 60 and 90 of follow-up after treatment (PTD30, PTD60 and PTD90, respectively). At the end of the study that is PDT90, the outcome was recorded as "cure" (complete disappearance of the lesion), "improvement" (more than 20% of decrease of the area), "failure" (increasing of the area), or "relapse" (reactivation of lesion after an initial cure). The parasitological effectiveness was also determined by quantification of parasite load in the skin samples from the ulcer at the end of the study (PTD90) by RT-PCR, following the protocol described by others [21].

Toxicity in hamsters after treatment:
The toxicity of C 6 I, PC 6 I, NPs and MA was studied based on changes in the weight of hamsters measured every two weeks previous sedation. Additionally, at TD0 and day 8 after the end of treatment (PTD8) hamsters were bled by cardiac punction and serum was separated by centrifuging (5 min at 5,000 × g). Levels of alanine amino transferase (ALT), blood urea nitrogen (BUN) and creatinine (CRT) metabolites were quantified using commercial kits (Biosystems S.A, Barcelona, Spain). Hamsters were monitored daily for food and water consumption, activity and behavior. The appearance of fur, eyes and mucous membranes was also supervised. a day after the compound C 6 I was administered but it was normalized during the same day. A weight gain was observed in rats of all groups. There was no evidence of pathological signs during necropsy and no histological changes were confirmed. Therefore, it is possible to administer at one time a maximum concentration of pure compound of 300 mg/kg without significant effects.

Development and characterization of solid lipid nanoparticles
The C 6 I was not soluble in Precirol® after fusion. Therefore, the formulation of PC 6 I was prepared by emulsion and solvent evaporation processes. The particle size was homogeneous and this was range of nanometer, the encapsulation performance was 68% (2.04 mg of C 6 I ), and the PI values was lower than 0.3 ( Table 2).
The SEM analyses showed that the NPs had spherical and homogeneous shape and some aggregates were present ( Figure 2).

Stability of solid lipid nanoparticles loaded with C 6 I:
Storage of the NPs under refrigeration condition generated a slight decrease of the particle size of the NPs during the first month. On the other hand, the zeta potential increased in the first month but decreased in the second month. Unloaded NPs were not affected at the same extent by extensive refrigeration.
The heating processes by autoclaving did affect the particle sizes and the zeta potential decreased (Table 2). Additionally, DLS analysis showed that by increasing the temperature at 90 °C there was a gradual reduction in the particle size. Although the original size was not recovered after the cooling phase ( Figure 3A), the heating and cooling process did not affect significantly of size of particles the values were always within the nano range. without statistically significant differences, the permeability process in intestinal wall was independent of concentration (p > 0.05) ( Table 1).

In vitro mutagenicity:
There was no significant increase in the number of revertant colonies of the TA98 and TA100 S. typhimirium strains in the three concentrations of C 6 I compared to the negative controls. The test was done without exogenous metabolic activation system (p > 0.05). The positive controls AzNa and 4-NQO increased two times in the mutagenicity index compared to C 6 I and negative control.
In vitro genotoxicity: None structural chromosome aberrations were identified in the different concentrations of C 6 I evaluated. No statistically significant differences were found between the subtoxic concentrations of C 6 I and the negative control.
Maximum tolerable dose of C 6 I: During the 14 days of observation after oral administration of three different concentrations of C 6 I (5, 50 or 300 mg/kg), there were no deaths or evidence of clinical signs associated with severe toxicity or changes in the normal behavior of rats. A soft but not diarrheic stool was observed  to 0.6 mg/mL, with a SD of 0.08 and a coefficient of variation of 3.1%. The limit of quantification (LOQ) and the limit of detection (LOD) were 0.03 and 0.009 mg/mL, respectively, with a confidence interval of 99.25%.

Differential scanning calorimetry (DSC):
The melting point (mp) of Precirol® and C 6 I were 70-80 °C and 163 °C, respectively. However, in the PC 6 I, the melting point peak corresponding to C 6 I was absent, possibly due to a structural change of the compound within the NPs from its crystalline form to an amorphous or molecularly dispersed state or the concentration of the compound is lower than of the lipid, which did not allow looking its peak. The Figure 4 shows the thermograms of each of the components of formulation, unloaded NPs and PC 6 I.

Evaluation of toxicity and effectiveness of C 6 I and PC 6 I
In vitro cytotoxicity and antileishmanial activity: To compare the results of free C 6 I and encapsulated, first, the cytotoxic effects on fibroblast (Detroit 551) and MD-Mhu the doses was LC 50 < 100 μg/mL for C 6 I while PC 6 I was not cytotoxic for all the cell types evaluated, with a LC 50 > 200 μg/mL (Table 3). In relation with the effectivity, C 6 I had an EC 50 17.6 ± 1.0 μg/mL while PC 6 I showed Release of C 6 I in solid lipid nanoparticle: There was a rapid release phase in the first 30 min followed by an exponential phase of release in the following 22 hours (Figure 3B). The efficiency of release of C 6 I was ≥ 94% after 20 hours of observation. The quantification method for C 6 I showed a sensitivity and accuracy according to the concentration range. The linearity varied from 0.03875   rate of 71.4% for PC 6 I treatment. Although all hamsters treated with intramuscular injections of MA showed cure during the study, only 5/7 (71.4%) hamsters remained cured at the end of the study while the last two hamsters had relapses of their ulcers. As expected, any hamster from the vehicle treated or untreated groups resolved their lesions during the study ( Figure 5A). The appearance of lesion in hamsters prior and after treatment with C 6 I and PC 6 I and at the end of the study are shown in Figure 5B, Figure 6.
The parasite load expressed as at the end of the study in the ulcers by group of treatment was 644.4 ± 953.8 parasites/mg of tissue (mean ± SD, n = 7) and 303.2 ± 288.6 parasites/mg of tissue in hamsters treated with C 6 I and PC 6 I, respectively. These difference was statistically significant (p < 0.01).

Toxicity in hamsters after treatment:
The hamsters gained weight during the study and no statistically significant differences were observed among groups (Figure 7). Serum levels for all animals of ALT, BUN, and CRT were within the range of normal values for all animals both before (TD0) and after (PTD8) the all treatments ( Figure 8).

Oral toxicity at repeated doses (28 days):
No deaths or disabling events were neither registered nor clinical signs associated with toxicity of the compounds. The behavior of the mice and their physical appearance were normal even after the administration of the substances. Mice were attentive to the medium, exploring in the cages, grooming each other or adopting typical positions to rest or sleep. Mice treated with free C 6 I at 30 mg/kg/day showed alterations at the gastrointestinal level with soft stools from the middle of the study to the end without affect the body weight. At contrary, at the end of the study all treated with free C 6 I or PC 6 I presented weight gain. At necropsy, only hepatic liver-type changes were reported in two animals of the PC 6 I group. At the histological level, slight vacuolar degeneration was observed in the kidney, liver and small an EC 50 of 0.4 ± 0.2. The ratio of cytotoxicity and activity (LC 50 /EC 50 ) showed an IS of 0.53 and higher than 500 μg/ mL for C 6 I and PC 6 I, respectively. These results suggest that the selectivity of C 6 I for the parasite increased significantly when was incorporated into the Precirol® NPs ( Table 3).

Evaluation of in vivo effectiveness:
After experimental infection, hamsters developed a single lesion ranged from 113.5 to 199.3 mm 2 . The treatment of hamsters with free C 6 I, administered orally at a dose of 30 mg/kg/ day for 28 days, resulted in complete healing of ulcers in 3 animals (42.9%) at the end of the study ( Figure 5). The remained hamsters that did not cure showed improvement with a reduction in the size of their lesions in 90.4, 38.4, 37.05 and 8.6%. On the other hand, the treatment of hamsters with PC 6 I, administered orally at the same dose during 28 days increased the cure rate to 85.7% corresponding to 6 of 7 hamsters with complete healing of ulcers. The remained hamster of this group showed a decrease in the lesion of 95.9% (hamster PC 6 I-5, Figure 5). One of cured hamster had relapse at the end of the study (hamster PC 6 I-7, Figure 5) for a definitive cure in the three different pH, additionally the DSC and DRX analysis confirmed that C 6 I presented a crystalline structure, as described by others authors [7,8]. This crystalline lattice is mainly due to the presence of aromatic rings, which tend to form strong and organize crystalline structure, compounds with this type of structure as usual they present low solubility and dissolution [8,9].
The Doluisio method showed good intestinal permeability of C 6 I, with Papp one order of magnitude greater than that showed by metoprolol (5.6 × 10-6 cm/s) and in the same range of ibuprofen, which are drugs with good intestinal permeability [10]. On the other hand, rate constants (k a ) were the first order, which showed the intestinal absorption of C 6 I was a passive diffusion process, since there were no statistically significant differences between the constants of the two concentra-intestine while slight hyperplasia was observed in liver and spleen of some hamsters treated with both C 6 I and PC 6 I. The hematological parameters and serum levels of ALT, AST, CRT and BUN were within the reference values indicating no alterations in the kidney and liver function for both treatments.

Discussion
One of the objectives pursued for the treatment of leishmaniasis is the development of more effective and safer active pharmaceutical ingredients that can be administered topical or orally to ensure greater acceptance by patients. Previously studies of the quaternary halomethylated ammonium iodide C 6 I meets the requirements mentioned previously. Our study continued with the pharmacological characterization; the C 6 I exhibited low dissolution profile with a IDR value similar and creatinine (c) in serum of treatment groups before (TD0) and 8 days after treatment ended (PTD8) in hamsters with cutaneous leishmaniasis. Data are shown as median ± SD. No significant differences were seen between groups (p > 0.05). Precirol ® nanoparticles incorporating C 6 I (PC 6 I); meglumine antimoniate (MA); Empty Precirol ® nanoparticles (VE); Infected and untreated hamsters (I/U). No differences were observed neither among individuals in each group nor between groups. Dotted lines in red delimit the reference values.
for less ordered or amorphous crystals, the fusion of the substance requires much less energy than that for crystalline substances. Additionally, the crystallization processes during the production are complex and strongly dependent on the lipid composition and production conditions as they directly affect the location of the active compound within the particle and its release profile [35]. After 24 hours the 96% of C 6 I was released, being exponential and faster during the first hour possibly due to the presence of molecules of the compound on the surfaces of the particle. After 2 hours the released was delayed possibly due to the diffusion of C 6 I dissolved in the nucleus of the nanoparticles in the dissolution medium.
In this work, the release profile showed that C 6 I improved its solubility in solution when it was incorporated into Precirol® NPs compared to the results of the intrinsic dissolution of pure free C 6 I. According with the IDR value, 0.24 mg of compounds per centimeter was dissolved each minute, while the release and dissolution of C 6 I in the nanoparticles occurred rapidly in the first hours and more than half of the compound was in solution. According to Noyes-Whitney and Ostwald-Freundlich principles, the particle size in the nanometer range can lead to an increased dissolution velocity and saturation solubility for a nanoformulation. Previous studies with a number of poorly soluble drugs have demonstrated that particle size reduction can lead to an increased rate of dissolution and higher oral bioavailability [8,29,36].
On the side of toxicity and effectiveness, our results showed a significant decrease in the toxicity but an increase of antileishmanial activity for intracellular amastigotes of L. (V) braziliensis of C 6 I when incorporated in Precirol® NPs. In this case, the EC 50 of PC 6 I was 40-fold lower than free C 6 I. Additionally, previous studies in vivo demonstrated that C 6 I was able to cure hamsters with CL by L. (V) panamensis when administered topically at a dose of 2 mg/kg/day for 15 days [5]. Here we show that free C 6 I, administered orally at a dose of 15 mg/kg/day for 28 days, showed an effectiveness close to 30%. Nevertheless, its effectiveness increased to 71% when the C 6 I was incorporated in Precirol® NPs and administered at a higher dose of 30 mg/kg/day. The results were consistent in the in vivo model, with 71% cure in the group of animals treated with PC 6 I upon oral administration of a dose of 30 mg/kg/day for 28 days. Differences in the effectivity and toxicity between free C 6 I or encapsulated PC 6 I was evident not only because the LC 50 increased two-fold but also because the EC 50 was lowered 40-fold. Several hypotheses may explain how the SLN improve de effectivity of compound. We hypothesize that the solubility and dissolution of pure compound was low so that the concentration available in polar and physiological solutions is few. Possibly, nanoparticles loaded with C 6 I increased the dissolution of compound and improved the solubility in polar solutions, for example, culture medium, and thus, more compound was avail-tions tested [23]. The passive diffusion of C 6 I is favored by its small molecular weight, positive charge and moderate polarity. Some authors report that compounds with positive charge and molecular weight below 400 Da could access at intestinal epithelium through paracellular pathway [24,25]. Together these results and those reported previously [5] allowed to classify the C 6 I as type 2 compound according to the biopharmaceutical classification system (BCS), with low solubility and good intestinal bioavailability. In turn, the bioavailability of type 2 compounds can affect the amount of drug in solution as well as the amount of C 6 I that can be absorbed at gastrointestinal level; therefore, type 2 compounds should adapt new strategies to improve their gastrointestinal biopharmaceutical profile, with the purpose to increase the amount of compound bioavailable at systemic level [26].
In acute and repeated doses were evidenced slight edema, vascular congestion and vacuolar degeneration. Despite these signs, organ size and serum metabolites related to hepatic function were normal. On the other hand, C 6 I did not show mutagenic activity in the S. typhimurium model (TA98 and TA100 strains) and nor chromosomal alterations in human lymphocytes. These results are similar to those showed by other quaternary ammonium compounds [27].
In this work, we use Precirol® not only because it allows the manufacture of NPs easily and economically and by storage conditions, but mainly because it allows the controlled release of drugs [8,28]. The Precirol® NPs improved the pharmaceutical profile (higher effectiveness and lower toxicity) and the biopharmaceutical profile (bioavailability) for C 6 I as demonstrated by others [29,30].
The C 6 I was solubilized in a small amount of dichloromethane before the incorporation in the lipid phase. Using 3 mg of C 6 I, the percentage of encapsulation was higher than 60%. Although the zeta potential was outside the desired range (+30 to -30) [31], the obtained values still indicate good colloidal stability, considering the steric stabilization given by the Tween 20 used as surfactant [32].
The stability tested showed, the storage condition, autoclave conditions and DLS test did not change significantly the mainly characteristics of formulation as size and encapsulation capacity. Authors reported the SLNs as stable systems in different conditions [33]. In contrast, the zeta potential was significantly lower which may be due to a reorganization of the polymorphic transitions of the lipid matrices with subsequent rearrangement of the compound within the formulation [8,28,29].
The DSC analyses showed a rearrangement of the particles compared with the bulk product. The change in the DSC profile could be due to possible defects in the lipid network, leading to a decrease in their crystallinity compared with their bulk counterparts [34]. Thus, ductive organs, as well as the blood chemistry profile without evident toxic manifestations. Although, two animals had fatty liver and at the histological level, events such as congestion and mild to moderate vacuolar degeneration were observed, it is known that NP systems induce oxidative stress in the liver [39]. Additionally, the presence of a cationic surfactant may generate some cytotoxicity [40].
In conclusion, despite the low solubility and the dissolution profile shown by C 6 I, the compound possesses other physicochemical, pharmacokinetic and pharmaceutical properties that make it suitable for the oral formulation. The C 6 I showed good intestinal permeability and potential no toxicity after oral administration in the murine model. The genotoxicity or mutagenicity of C 6 I were absent. The Precirol® NPs was a suitable strategy that improved not only the bioavailability and toxicity but also the effectiveness of the C 6 I both in vitro and in vivo. Indeed, the cure rate of hamsters with experimental CL increased from 30% to 71% after daily administration for up to 28 days. Results showed here confirm that the PC 6 I formulation has high potential to become a new drug for the treatment of CL. able to enter in the infected cell. In the case in vivo test it is possible that compound was soluble in GI solutions and thus, the dissolved C 6 I is able to cross the intestinal wall and to reach the systemic circulation and its target [8,14,29,37]. Furthermore, SLNs used for oral administration offer several benefits over conventional formulations including increased solubility, enhanced stability, improved epithelium permeability and bioavailability, prolonged half-life, tissue targeting, and minimal side effects as well as the potentiating effect of the antileishmanial activity [8,28].
The in vivo activity of free C 6 I or incorporated in the Precirol® NPs (PC 6 I) was tested in the hamster because this is the most adequate animal model for CL by Leishmania species of Viannia subgenus. The effectiveness was compared with the intramuscular MA instead miltefosine because MA is able to resolve CL in the hamster model infected with L. (V) braziliensis at the dose used in this study [20] while miltefosine has not shown any cure [38]. Despite the use of different administration routes, both treatment with the salts and the MA are systemic and this validates the use of MA as a control. On the other hand, although the days of treatment were different (28 vs. 10 for the salts vs. MA), the efficacy of the treatments was evaluated based on the outcome of the cure observed after the treatment was finished. In this way, day 0 of follow-up began one day after the end of the treatment, regardless of the duration of the treatment. Moreover, the treatment for all groups was safe during 28 days of administration and during 90 days of follow-up; the hamsters gained weight and levels of ALT, BUN, and CRT were within the range of normal values for all animals both before (TD0) and after (PTD8) treatment.
Additionally, in all assays where the safety profile was tested, the compound was safe. This profile was studied in rats and mice as recommended in the international guides; in this way it is also guaranteed that the non-toxicity of the substance does not depend on the species tested. We also showed that this activity is selective, in vitro being potentially non-toxic for colon Caco-2 cells and for primary cultures of human macrophages huMDM, which suggest that C 6 I does not affect the colon cells during the process of intestinal absorption nor the host cells for Leishmania parasites during treatment. Moreover, the NPs improved the cytotoxicity of the free C 6 I increasing the LC 50 in 21 times for the U937 and MPha. The LC 50 for Caco-2 and huMDM were similar. The in vivo oral acute toxicity results defined the maximum concentration to evaluate the effect after oral administration at repeated doses (28 days). There were no deaths or changes in body weight, as well as anatomical changes at necropsy that could be associated with a possible toxicity for C 6 I. The PC 6 I formulation presented a pattern of safety as shown by results obtained in organs such as heart, spleen, intestine, brain and repro-