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In vivo evaluation of the efficacy, toxicity and biodistribution of PLGA-DMSA nanoparticles loaded with itraconazole for treatment of paracoccidioidomycosis
Journal of Drug Delivery Science and Technology 45 (2018) 135–141
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Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
In vivo evaluation of the efficacy, toxicity and biodistribution of PLGA-DMSA nanoparticles loaded with itraconazole for treatment of paracoccidioidomycosis
T
Elaine P. Cunha-Azevedoa,c, Karen R. Py-Daniela, Marigilson P. Siqueira-Mourab, Anamélia L. Boccaa, Maria S.S. Felipea, Antonio C. Tedescob, Osmindo R. Pires Juniora, Carolina M. Luccia, Ricardo B. Azevedoa,∗ a b c
Biological Sciences Institute, Universidade de Brasília, Brasília DF 70910-900, Brazil Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto SP 14040-901, Brazil Health and Sciences Biological Institute, Centro Universitário de Belo Horizonte, Belo Horizonte MG 31910-900, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung mycosis Polymeric nanoparticles Dimercaptosuccinic acid Itraconazole
Itraconazole(ITZ) – an antifungal agent of the azole class – is clinically used to treat a variety of fungal infections. Among them, paracoccidioidomycosis (PCM), a fungal infection caused by Paracoccidioides brasiliensis, is the most prevalent systemic fungal infection in Brazil as well as in many countries in South America. Conventional treatments of PCM with itraconazole include oral solution and capsule formulations, both associated to many side effects such as nausea, vomiting, abdominal pain, diarrhea, headaches and mild alopecia. Drug delivery systems enables the development of new pharmaceutical formulations for the controlled delivery of drugs, which can avoid many of the problems related to low efficacy and side effects of traditional drugs. In this study, using HPLC, we determined the biodistribution of ITZ administered both, as a nanoparticle presentation (PLGA nanoparticle coated with DMSA – ITZ-NANO), as well as oral solution (Free ITZ) in healthy mice. We also compared the therapeutic efficiency as well as toxicity of both forms of ITZ presentations in the treatment of PCM infected mice using clinical, biochemical and histological approaches. Biodistribution demonstrated higher accumulation of ITZ in lung, liver and spleen of ITZ from ITZ-NANO. ITZ-NANO treatment of chronic PCM was capable of eliminate fungal cells from lungs and avoided the side effects of alopecia and increment of liver enzymes concentration. The proposed treatment, with a lower administration dose of ITZ-NANO and decreased number of administrations, demonstrated promising potential with higher success rate, and less stress for the patient with usage of this nanomaterial.
1. Introduction Itraconazole (ITZ) – an antifungal agent of the azole class – is clinically used to treat a variety of fungal infections, such as those caused by Aspergillus spp., Candida spp., Cryptococcus neoformans, Blastomyces spp., Histoplasma spp., Coccidiodes spp., and Paracoccidioides spp. [1], [2] Itraconazole acts by impairing the ergosterol synthesis, essential component of the fungal cell membrane [3]. Paracoccidioidomycosis (PCM), the fungal infection caused by Paracoccidioides brasiliensis, is the most prevalent systemic fungal infection in Brazil as well as in many countries in South America, and is reported as the eighth leading cause of mortality from infectious disease among chronic infectious and parasitic diseases [4].
In traditional fungal infection treatments, administration of high doses of itraconazole occurs over long periods, resulting most of time in severe side effects such as nausea, vomiting, abdominal pain, diarrhea, headaches and mild alopecia. It can also cause hepatotoxicity and thereby increase the levels of bilirubin and transaminases [5]. The occurrence of side effects and the toxicity of this drug increase the necessity of different approaches for specific drug delivery and alternative therapeutic protocols [6]. Nanobiotechnology enables the development of new pharmaceutical formulations for controlled delivery of drugs, which can avoid many of the problems related to low efficacy and side effects of traditional drugs. Some of the advantages of using nanoparticles for drug delivery include: enhanced stability, controlled release and enhanced
∗ Corresponding author. Universidade de Brasília, Biological Sciences Institute, Departamento de Genética e Morfologia, Laboratório de Morfologia e Morfogênese, Campus Darcy Ribeiro, Asa Norte, Brasília DF 70910-900, Brazil. E-mail address: [email protected] (R.B. Azevedo).
https://doi.org/10.1016/j.jddst.2018.02.014 Received 2 October 2017; Received in revised form 8 February 2018; Accepted 20 February 2018 Available online 08 March 2018 1773-2247/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Animals
bioavailability [7]. Previous studies from our group, with magnetic nanoparticles coated with dimercaptosuccinic acid (DMSA), indicated that this acid is capable of targeting nanoparticles to the lungs [8,9]. A proposed approach is to use such properties of DMSA to target nanoparticles to the lung to treat pulmonary infections such as PCM. Amaral et al. (2009) described efficient treatment of PCM in lungs with amphotericin B loaded nanoparticles coated with DMSA, emphasizing lung-targeting role of DMSA [10]. The aim of this work was to 1) determine the biodistribution of ITZ administered either as a poly(lactic-co-glycolic) acid (PLGA) nanoparticle containing ITZ and coated with DMSA (PLGA nanoparticle coated with DMSA – ITZ-NANO), or ITZ in oral solution presentation (Free ITZ) in healthy mice; 2) to verify the therapeutic efficacy of both forms of ITZ in the treatment of PCM infected mice. Nanoparticle biodistribution was determined by ITZ quantification in different organs by HPLC. To determine therapeutic efficacy, animals were infected with PCM and treated with ITZ-NANO or Free ITZ and clinical, biochemical and histological aspects were analyzed.
Animals used in this study were female BALB/c mice ageing 10–14 weeks and weighing 20–22 g, purchased from the University of Campinas, SP, Brazil. The mice were housed in polypropylene cages under controlled conditions of luminosity and received food and water ad libitum. All animal procedures performed in this study were approved by the Animal Care and Use Committee of the University of Brasília (UnB), Brasília – Federal District, Brazil (UnBDoc: 12155/2007) and European Community guidelines as accepted principles for the use of experimental animals were adhered to. The study was divided in two major approaches: a biodistribution in healthy animals and the treatment of PCM infected animals. 2.4. ITZ biodistribution Animals received a single dose of either 1) ITZ-NANO containing 3 μg of ITZ per gram of body weight (administered intraperitoneally) or 2) Free ITZ oral solution with 50 μg of ITZ per gram of body weight (administered by gavage). At 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h after administration of ITZ-NANO or Free ITZ (n = 5/group for each treatment/time), animals were anaesthetized (ketamine 60 mg/kg and xylazine 7.5 mg/kg), blood was collected by heart puncture and was transferred to tubes containing EDTA. After 30 min at room temperature, tubes were centrifuged for 5 min at 500×g and plasma was stored at −80 °C for future analyses. After the animal's death, liver, spleen, lung and kidney were excised, washed with cold 0.9% NaCl (w/v) solution, blotted on filter paper, weighted and stored at −80 °C for future analyses.
2. Material and methods 2.1. Drugs and chemicals Itraconazole (ITZ), poly(lactic-co-glycolic acid, 50:50) (PLGA 50:50), polyvinyl alcohol (PVA) and dimercaptosuccinic acid (DMSA) were purchased from Sigma Aldrich (Sigma–Aldrich Co., St. Louis, MO, USA) and Itraconazole internal standard (5OR-R51012) was purchased from Fitzgerald (Fitzgerald Industries International Inc., Concord, MA, USA). Acetonitrile, methanol and tetrahydrofuran used for the HPLC analyses were HPLC grade and purchased from Mallinckrodt (Mallinckrodt Inc., Hazelwood, MO, USA). Trifluoroacetic acid was purchased from VETEC (Duque de Caxias, RJ, Brazil). Milli-Q water was obtained from a Barnstead* EASYpure* II Thermo Scientific (San Jose, CA, USA) and was used to prepare aqueous solutions.
2.4.1. HPLC instrumentation The chromatographic equipment (Shimadzu-Prominence, Kyoto, Japan) comprised of on-line degasser (Model DGU 20A5), solvent delivery module (Model LC-20AT), autosampler (Model SIL-20AHT), column oven (Model CTO-20A), fluorescence detector (Model RF10AXL) and system controller CBM-20A. Reverse-phase C18 column ACE AQ (25 × 0.4 cm, 5 μm particle size) (ACE, Aberdeen, Scotland) with a pre-column (1 × 0.4 cm, 5 μm particle size). (ACE, Aberdeen, Scotland) were used.
2.2. ITZ-NANO and free ITZ preparation DMSA-PLGA nanoparticles containing itraconazole (ITZ-NANO) were synthesized and characterized as described by Cunha-Azevedo et al. (2011) [11] and were prepared using a modified emulsificationevaporation technique. Briefly, an organic solution of PLGA and ITZ was prepared. This organic phase was poured slowly into an aqueous solution of polyvinyl alcohol (PVA) 1% (w/v) which was homogenized using an Ultraturrax emulsifier. After this, a double emulsion was formed with 1% PVA (w/v) solution and the organic solvent was removed from the solution by continuous stirring at room temperature inducing polymer precipitation as nanospheres. The nanoparticles were isolated by centrifugation and were washed three times with distilled water. The formulation was suspended in PBS solution. DMSA was adsorbed to PLGA nanoparticle with the final concentration of 0,05 mol/L and stored at 4 °C. All procedures were carried out in a sterile hood. The nanoparticles formed had an average size of 174 nm, efficiency of incorporation of itraconazole into 72,8% and zeta potential of −40 mV [11]. ITZ concentration loaded on PLGA was limited to optimum synthesis conditions of nanoparticles adequate for an intravenous vehicle. Nonetheless, doses used were in accordance with previous publications [12]. An oral solution sugar-free of itraconazole for gavage administration (Free ITZ-“Xarope sem açúcar”) was manipulated by Farmacotecnica® (Brasilia, DF, Brazil), in order to simulate the conventional treatment. Optimum Free ITZ dosage was chosen based on literature [13].
2.4.2. HPLC analysis The mobile phase was obtained from the mixture of 0.12% (v/v) TFA in Milli-Q water (pump A) and 0.12% (v/v) TFA in acetonitrile (pump B) at 1:1 proportions. Fluorimetric measurements were carried out in a 12 μL flow cell at 260 and 365 nm excitation and emission wavelength, respectively. The injection volume was 10 μL and the flowrate during the assays was 1 mL/min at working pressure of 80 kgf/cm2. Analysis was performed using column temperature of 30 °C. Software LCsolution (version 1.24 SP1 Shimadzu, Tokyo, Japan) was used for data processing and identification of chromatographic parameters. For ITZ quantification in plasma, 200 μL of plasma were transferred to microcentrifuge tubes containing 20 μL of 20% ZnSO4 (w/v), and vortexed for 1 min. Then 700 μL of methanol was added for ITZ extraction, followed by agitation on a vortex for 3 min and centrifugation for 5 min at 7000×g. Liquid phase was transferred to a 2 mL volumetric flask and the process was repeated with 1 mL of methanol. For ITZ quantification in tissue, whole spleen, lung and kidneys and 400 mg of liver (due to large mass of liver) were homogenized individually in 1.5 mL microcentrifuge tubes with hand pestles and extracted with the same process used for plasma. Prior to extraction of plasma and tissue, 40 μL of Itraconazole Internal Standard (0.3 μg/mL) were added to tubes. The use of internal standards reduces errors associated to loss occurred during extraction. Biodistribution of ITZ from ITZ-NANO and Free ITZ was performed using HPLC detection method described and validated by Py-Daniel et al. (2014) [14]. In the cited method, the recovery of ITZ with the methodology described above was established as 136
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approximately ∼95% for biological samples used in the present work. 2.5. Treatment of PCM infected mice Paracoccidioides brasiliensis, isolate Pb18, was sub-cultured in liquid YPD medium (10 g of yeast extract, 20 g of peptone, 20 g of dextrose, 1 L of distilled water) at 36 °C in a rotary shaker (220 rpm). After 5 days of culture yeast cells were collected by centrifugation, the supernatant was discarded and the cells were washed twice in sterile PBS. The cellular viability was determined by vital staining with 0.05% (w/v) green-Janus. Cell counts were determined with a haemocytometer and the inoculum suspension adjusted to 3 × 107 viable fungi/mL. Animals were randomly divided into four groups of 10 animals as follows: group I (non-infected control group treated intraperitoneally with PBS); group II (infected control group treated intraperitoneally with PBS); group III (infected group treated daily with Free ITZ, 1 mg/animal/day, per gavage); group IV (infected group treated every three days with ITZNANO, 60 μg/animal intraperitoneally). Animals from groups II, III and IV were initially anaesthetized (ketamine 60 mg/kg and xylazine 7.5 mg/kg) and challenged with 3 × 106 viable cells of P. brasiliensis virulent strain Pb18 by intravenous route. Thirty days post-infection, all animals initiated treatment. After 30 days of treatment, 5 animals from each group were euthanized, and the other 5 continued with treatment up to 60 days when they were also euthanized. All animals were clinically monitored at a daily basis and on the 30th or 60th day of treatment, they were weighted and photographed. Animal were then euthanized and lung fragments were collected and fixed in 4% buffered paraformaldehyde (w/v)) to be processed in accordance with the technique of paraffin embedding. Sections (5 μm) were stained with hematoxylin-eosin (HE) to evaluate the morphology and inflammatory response of the organ, and with Grocott-Gomori stain for identification of the fungus. Sections were analyzed on a light microscope (Axiophot, Zeiss, Germany), and images were captured by Moticam 2300 (Meyer Instruments, USA) camera and Axio Vision program 40v 4.6.1.0. © Copyright.
Fig. 1. A) %Dose of ITZ per g of tissue time profile in tissues after intraperitoneal administration of ITZ-NANO in female BALB/c mice. Results are expressed as means ± standard desviation. 1.B) %Dose of ITZ per g of tissue time profile in tissues after oral administration of Free ITZ in female BALB/c mice. Results are expressed as means ± standard desviation.
(57.09 ± 27.91% of dose per gram of tissue), though no statistical difference was observed among the times points 4, 8, 12 and 24 h after ITZ-NANO administration. After 24 h, a statistical reduction of the ITZ concentration for ITZ-NANO treatment was observed when compared with earlier time points (35.80 ± 10.54% of dose per gram of tissue). The splenic ITZ uptake after Free ITZ treatment was extremely low during the entire experiment period with maximum uptake at 2 h (0.19 ± 0.01% of dose per gram of tissue) though no statistical difference was observed among the times points from 0.25 to 8 h. In spleen, the maximum ITZ uptake found in ITZ-NANO treatment was 300 times higher than that obtained after Free ITZ injection. In the kidney, the ITZ levels after ITZ-NANO injection were very low and statically equivalent at all interval times, ranging from (0–0.7% of injected dose/g of organ). After Free ITZ administration, the ITZ concentration reached its highest level at 1 h (0.56 ± 0.15% of injected dose/g of organ) though no statistical difference was observed among the times points from 0.5 to 4 h. At 8 h, a reduction of the ITZ concentration was observed after Free ITZ treatment (0.08 ± 0.13% of injected dose/g of organ). In the liver of animals treated with ITZNANO, the ITZ level attained a maximum peak at 4 h (21.28 ± 6.92% of injected dose/g of organ) though no statistical difference was observed from 2 to 72 h. With Free ITZ treatment, at 30 min a maximum peak was observed (2.03 ± 0.65% of injected dose/g of organ) though no statistical difference was observed from 0.25 to 1 h. For the ITZNANO treated group, the ITZ uptake in liver was constant from 2 to 72 h with no statistical difference among the tested times. A statistically significant decline of hepatic ITZ concentration in Free ITZ-treated mice was observed after 2 h. In the blood, the maximum ITZ concentration occurred at 1 h (0.27 ± 0.06% of injected dose/g of organ) though no statistical difference was observed from 0.5 h to 4 h after Free ITZ administration (data not shown). In contrast, it was not possible to detect any amount of ITZ after ITZ-NANO administration, considering that the limits of detection and quantification of ITZ in plasma were of 0.039 and 0.117 μg/mL, respectively.
2.6. Statistical analysis Data from biodistribution and treatment of PCM infected mice were analyzed to verify statistical significance. Statistical analysis of ITZ recovered from organs was analyzed by ANOVA using Tukey test, for comparisons between organs at the same time point, and Scheffe's test for comparisons between different time points for the same organ. Values were considered significant if P < 0.05. Data of treatment of PCM infected mice were analyzed using the Prism 3.0 program. For results involving 2 groups, the unpaired Student t-test was used. In comparisons between 3 or more groups, data were treated by analysis of variance with the Tukey test. The results were considered significant when P < 0.05 (5%). 3. Results 3.1. ITZ biodistribution studies The tissue distribution of ITZ after ITZ-NANO or Free ITZ administration in the different organs is shown in Fig. 1. In the lungs, the maximum ITZ uptake was achieved after 2 h for ITZ-NANO (64.38 ± 18.90% of injected dose/g of organ) though no statistical difference was observed among the time points of 1, 2 and 4 h. For free ITZ, maximum uptake was observed at 4 h (0.40 ± 0.08% of injected dose/g of organ) though no statistical difference was observed among the time points from 0.5 to 4 h. The maximum ITZ uptake found in the lungs with ITZ-NANO treatment was approximately 160 times higher than that observed after Free ITZ injection. In the spleen, the maximum ITZ uptake was achieved after 8 h 137
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Fig. 2. Photomicrographs of lungs from mice after 30 days of treatment. In A a normal lung without infection, B and C show lungs of infected and untreated mice. D, E and F show the profile of lungs obtained from infected mice and treated with Free ITZ.The arrows indicate thick parenchyma and inflammatory infiltrate (B), mass of fungi (C, D, E and F), free parenchyma in inflammatory infiltrate (F), parenchyma with small inflammatory infiltrate (G), region free of inflammatory infiltrate (H) and region with inflammatory infiltrate (upper arrow) and alveoli free of infiltrate (bottom arrow) (I).
(Fig. 4C and D) presented normal pelage, similar to the uninfected mice control group. Animals infected and not treated had no alterations on pelage.
3.2. Treatment of PCM infected mice 3.2.1. Antifungal activity of ITZ -NANO in vivo In order to evaluate antifungal activity of ITZ-NANO and Free ITZ treatments, histopathological examinations of lungs were performed (Fig. 2). Fig. 2A presents the lung photomicrography of a non-infected control animal, with very thin alveoli and no inflammatory cells infiltrates. Lung parenchyma of infected animals with no further treatment showed thick inflammatory infiltration (Fig. 2B) and granulomas with fungal cells (Fig. 2C and D). The histopathological analysis of the lungs of animals infected and treated for 30 days with Free ITZ demonstrated large inflammatory infiltrates, parenchymal granulomas and presence of fungal cells (Fig. 2E and F). On the other hand, animals infected and treated for the 30 days with ITZ-NANO revealed a moderate inflammatory infiltrates, however, granulomas and fungal cells were not observed (Fig. 2G, H and I). In lungs of animals treated with Free ITZ, inflammatory infiltrate and parenchymal thickening were still observed after 60 days of treatment. However, no granulomas with fungal cells were present (Fig. 3A and B). Lungs of animals treated with ITZ-NANO for 60 days presented a great reduction in areas with inflammatory infiltration when compared to lungs of animals treated for 30 days. In fact most lung parenchyma was similar to that of healthy animals and alveoli were free of infiltrates (Fig. 3C and D).
4. Discussion In this study, we developed a PLGA nanoparticle coated with DMSA loaded with ITZ in order to reduce the dosage administered, number of administrations and consequently the side effects usually caused by the administration of ITZ during the conventional treatment of fungal PCM infection. To verify nanoparticle targeting to the lungs, we performed the biodistribution of intraperitoneally injected ITZ-NANO coated with DMSA and a conventional (oral) treatment with free ITZ in healthy mice. In previous studies, animal treated with free ITZ presented the ratio of ITZ for lung:plasma of ∼3:1, 10:1 for liver:plasma and over 25:1 for fat:plasma, confirming ITZ potential for tissue penetration [15,16]. In the present study, ITZ-NANO biodistribution rendered higher amount of ITZ in lungs and liver to plasma than previous reported (∼64:1 for lungs:plasma and 21:1 for liver:plasma). It is worth to note that only residual plasma concentrations (values below limit of quantification) were found for ITZ when ITZ-NANO was used. Low plasma concentration of ITZ after ITZ-NANO administration indicates that ITZ is not being promptly liberated. ITZ is thus maintained in nanoparticle until degradation of polymer resulting in controlled release. Low kidney concentrations also corroborate with gradual and controlled release of ITZ and could also be explained by metabolization of ITZ rendering undetected metabolites. The observed ITZ concentration in lungs from ITZ-NANO can be explained possibly by the role of DMSA targeting to lungs [8,9] and/or by nanoparticle entrapment in the first capillary bed encountered after parenteral administration [17].
3.2.2. Clinical parameters analysis Animals treated with Free ITZ, which presented a decrease of approximately 14.00 ± 0.53% on the 30th day of treatment. On the contrary, animals treated with ITZ-NANO showed a body weight loss of only 1.60 ± 0.03% upon the 30th day of treatment (Table 1). The clinical symptoms of mice were monitored daily after beginning of treatment. As can be seen of Fig. 4, animals treated with Free ITZ, upon the 30th day of treatment, presented ventral alopecia and irregular spiked dorsal pelage (Fig. 4A and B). Animals treated with ITZ-NANO 138
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Fig. 3. Photomicrographs of lungs at 60 days of treatment A and B: lungs of infected animals treated with Free ITZ. A: parenchyma with thick inflammatory infiltrate; B: another area of the same slice, showing a region free of inflammatory infiltrate. In C and D, lungs of infected animals treated with ITZ-NANO, showing a small infiltrate (arrow) (C) and an apparently normal area in the same slice of C (D).
released of ITZ from ITZ-NANO, and/or a lower concentration of free drug reaching the liver. In correlation to biodistribution results, a maximum concentration in liver was found in 4 h with the observation of 21.28% of ITZ from injected dose/gram of organ, rendering the approximate mass of 11 μg in organ, indicating that encountered dosage is not toxic to liver. An important parameter for identification of ITZ-NANO toxicity was performed by continuous examination of clinical aspects of mice. No visual differences were observed between control animals and animals treated with ITZ-NANO. These data also suggest that administration of ITZ by ITZ-NANO attenuated the side effects in animals, most likely to decreased concentration injected and controlled release of the drug and/or decreased stress levels of animals, due to administrations of ITZNANO every three days. These findings corroborate with the clinical applicability of nanoparticles in nanobiotechnology and reinforce the advantages of enhancing the specificity of the treatments, providing a higher quality of life for patients. Free ITZ, with high daily doses not only was less efficient in eradicating fungal cells from the lungs, but lead to side effects such as alopecia and increased concentration of liver enzymes. These results were corroborated by biodistribution in which a small amount of ITZ from oral solution reached the lungs, and only for a short period after administration. The results achieved with ITZ-NANO were satisfactory since the purpose of incorporation of drugs in nanoparticles is, other than to promote at least the same efficiency of conventional drug treatment, to reduce side effects and promote a greater incentive for patients in treatment. Specifically in patients undergoing Paracoccidioidomycosis treatment, a very high dropout rate is observed since they are offered under the oral administration form, and the patient must take it several times a day for a long period of time (at least six months) [21].
High liver and spleen concentrations observed when ITZ-NANO was administered are explained mainly by opsonization events that result in nanoparticle identification and removal from the blood by phagocytes as well as tissue macrophages that are in direct contact with the blood (hepatic Kupffer cells and the marginal zone and red-pulp macrophages in the spleen) [18]. Also, ITZ is mainly metabolized in the liver through CYP3A4 into a large number of metabolites [19]. To determine therapeutic efficiency of nanoparticles, the treatment of PCM infected mice was performed with ITZ-NANO or Free ITZ, and clinical and histological aspects were analyzed. As demonstrated, ITZ-NANO in a therapeutic dose of 60 μg per animal administered every three days, because of controlled release of the drug, for a period of thirty days, was effective in controlling pulmonary infection observed in PCM (Fig. 2G, H and 2I). On the other hand, in animals treated with Free ITZ for the same period of time, inflammatory infiltrates were observed as well as granulomas with fungal cells (Fig. 2D, E and 2F). Together these results indicates that treatment with ITZ-NANO rendered a better therapeutic effect than Free ITZ as well as resulted in less stress for the animals, once administration was performed every three days, and the amount of drug injected was much smaller. This result also correlates with the biodistribution results in which 72 h after administration of ITZ-NANO, ITZ was quantifiable in lungs. The described results found with Free ITZ are in accordance with Naranjo et al. (2010) who used this drug to treat chronic PCM, and identified that there was a significant reduction in inflammatory infiltration, but still observed fungal cells in the lungs after four weeks of treatment [20]. No liver toxicity was indicated by biochemical parameters of ITZNANO analysis (data not shown). This was probably due to low concentrations of free circulating ITZ, another indication of controlled
Table 1 Media body mass of treatments groups (non-infected untreated, infected untreated, treated with Free ITZ or ITZ-NANO (n = 5) on days, 0, 30, and 60 after initiation of treatment, and % of modification of body mass from day 0–30 and 30 to 60. Results show the mean ± SD body weight of animals of experimental groups. Treatment groups
Day 0 Non-infected Infected Free ITZ ITZ-NANO
Body Mass Variation (days 0 → 30)
Body Mass (g)
22.89 21.33 20.95 20.84
Day 30 ± ± ± ±
1.22 1.06 1.42 2.82
24.95 22.29 23.80 22.98
± ± ± ±
Body Mass (g)
Body Mass Variation (days 30 → 60)
Day 60 0.49 1.01 2.10 1.38
8% 4% 13% 10%
26.50 23.15 20.44 22.61
139
± ± ± ±
0.70 0.79 0.89 0.32
6% 3% −14 ± 0.53% −1.60 ± 0.03%
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Fig. 4. Physical aspects of the animals after treatment with ITZ-NANO and Free ITZ. In A and B, Free ITZ treated animals showing alopecia. In C and D, animals treated with ITZ-NANO showing a typical pelage of healthy animals.
Therefore the decrement in the number of administrations proposed by this study could enable a greater cooperation from patients, a preponderant factor in the success of treatment protocol.
573880/2008-5.
5. Conclusion
The lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
Transparency declarations
The biodistribution of ITZ administered both as a nanoparticle presentation, as well as an oral solution was evaluated and the presentations and routs of administration had a major impact in the quantification in organs. Biodistribution of ITZ from nanoparticle demonstrated higher accumulation in lung, liver and spleen when compared to free drug. We also compared the therapeutic efficiency as well as toxicity of both forms of ITZ presentations in the treatment of PCM infected mice using clinical, biochemical and histological approaches. It was found that ITZ-NANO was as effective in the treatment of chronic PCM as the free drug, having the advantage of avoiding its known side effects. The ITZ-NANO drug concentration was lower than that administered in free ITZ, which would contribute to the reduction of the total cost of treatment, as well as decrease the time and stress caused to the patient, given that the application would be held every three days. With this in mind, the nanoparticle here presented has a biodistribution and efficacy more favorable to treat PCM than the free drug used in conventional treatment.
Acknowledgements We thank the University of Brasília and University of São Paulo for structural support for the developed study. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.jddst.2018.02.014. References [1] D. Andes, Optimizing antifungal choice and administration, Curr. Med. Res. Opin. 29 (S4) (2013) 13–18. [2] F.A. Silva, F.G. Salum, M.A. Figueiredo, K. Cherubini, Important aspects of oral paracoccidioidomycosis – a literature review, Mycoses (2013) 189–199. [3] J. Fuhren, W.S. Voskuil, C.H. Boel, P.J. Haas, F. Hagen, J.F. Meis, J.G. Kusters, High prevalence of of azole resistance in Aspergillus fumigatus isolates from high-risk patients, J. Antimicrob. Chemother. 70 (2015) 2894–2898. [4] H.C. de Oliveira, P.A. Assato, C.M. Marcos, L. Scorzoni, A.C. de Paula eSilva, J.D. da Silva, J.L. Singulani, K.M. Alarcon, A.M. Fusco-Almeida, M.J. Mendes-Giannini, Paracoccidioides-host interaction: an overview on recent advances in the paracoccidioidomycosis, Front. Microbiol. 6 (2015) 1319. [5] A.F. El-Shershaby, A.I. Dakrory, M.H. El-Dakdoky, J. Ibrahim, F. Kassem, Biomonitoring of the genotoxic and hepatotoxic effects and oxidative stress potentials of itraconazole in pregnant rats, Birth Defects Res. B Dev. Reprod. Toxicol 104 (2015) 55–64. [6] G.Q. Andrés, G. Giusiano, P.A. Ezkurra, A.J. Carrillo-Muñoz, Antifúngicos:
Conflicts of interest The authors declare no conflict of interest. Funding We thank the Brazilian agency National Institute of Science and Technology-Nanotechnology (INCT-Nanobiotecnologia) of Ministry of Science, Technology and Innovation (MCT/CNPq) Grant agreement 140
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Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
In vivo evaluation of the efficacy, toxicity and biodistribution of PLGA-DMSA nanoparticles loaded with itraconazole for treatment of paracoccidioidomycosis
T
Elaine P. Cunha-Azevedoa,c, Karen R. Py-Daniela, Marigilson P. Siqueira-Mourab, Anamélia L. Boccaa, Maria S.S. Felipea, Antonio C. Tedescob, Osmindo R. Pires Juniora, Carolina M. Luccia, Ricardo B. Azevedoa,∗ a b c
Biological Sciences Institute, Universidade de Brasília, Brasília DF 70910-900, Brazil Department of Chemistry, Faculty of Philosophy, Sciences and Letters of Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto SP 14040-901, Brazil Health and Sciences Biological Institute, Centro Universitário de Belo Horizonte, Belo Horizonte MG 31910-900, Brazil
A R T I C LE I N FO
A B S T R A C T
Keywords: Lung mycosis Polymeric nanoparticles Dimercaptosuccinic acid Itraconazole
Itraconazole(ITZ) – an antifungal agent of the azole class – is clinically used to treat a variety of fungal infections. Among them, paracoccidioidomycosis (PCM), a fungal infection caused by Paracoccidioides brasiliensis, is the most prevalent systemic fungal infection in Brazil as well as in many countries in South America. Conventional treatments of PCM with itraconazole include oral solution and capsule formulations, both associated to many side effects such as nausea, vomiting, abdominal pain, diarrhea, headaches and mild alopecia. Drug delivery systems enables the development of new pharmaceutical formulations for the controlled delivery of drugs, which can avoid many of the problems related to low efficacy and side effects of traditional drugs. In this study, using HPLC, we determined the biodistribution of ITZ administered both, as a nanoparticle presentation (PLGA nanoparticle coated with DMSA – ITZ-NANO), as well as oral solution (Free ITZ) in healthy mice. We also compared the therapeutic efficiency as well as toxicity of both forms of ITZ presentations in the treatment of PCM infected mice using clinical, biochemical and histological approaches. Biodistribution demonstrated higher accumulation of ITZ in lung, liver and spleen of ITZ from ITZ-NANO. ITZ-NANO treatment of chronic PCM was capable of eliminate fungal cells from lungs and avoided the side effects of alopecia and increment of liver enzymes concentration. The proposed treatment, with a lower administration dose of ITZ-NANO and decreased number of administrations, demonstrated promising potential with higher success rate, and less stress for the patient with usage of this nanomaterial.
1. Introduction Itraconazole (ITZ) – an antifungal agent of the azole class – is clinically used to treat a variety of fungal infections, such as those caused by Aspergillus spp., Candida spp., Cryptococcus neoformans, Blastomyces spp., Histoplasma spp., Coccidiodes spp., and Paracoccidioides spp. [1], [2] Itraconazole acts by impairing the ergosterol synthesis, essential component of the fungal cell membrane [3]. Paracoccidioidomycosis (PCM), the fungal infection caused by Paracoccidioides brasiliensis, is the most prevalent systemic fungal infection in Brazil as well as in many countries in South America, and is reported as the eighth leading cause of mortality from infectious disease among chronic infectious and parasitic diseases [4].
In traditional fungal infection treatments, administration of high doses of itraconazole occurs over long periods, resulting most of time in severe side effects such as nausea, vomiting, abdominal pain, diarrhea, headaches and mild alopecia. It can also cause hepatotoxicity and thereby increase the levels of bilirubin and transaminases [5]. The occurrence of side effects and the toxicity of this drug increase the necessity of different approaches for specific drug delivery and alternative therapeutic protocols [6]. Nanobiotechnology enables the development of new pharmaceutical formulations for controlled delivery of drugs, which can avoid many of the problems related to low efficacy and side effects of traditional drugs. Some of the advantages of using nanoparticles for drug delivery include: enhanced stability, controlled release and enhanced
∗ Corresponding author. Universidade de Brasília, Biological Sciences Institute, Departamento de Genética e Morfologia, Laboratório de Morfologia e Morfogênese, Campus Darcy Ribeiro, Asa Norte, Brasília DF 70910-900, Brazil. E-mail address: [email protected] (R.B. Azevedo).
https://doi.org/10.1016/j.jddst.2018.02.014 Received 2 October 2017; Received in revised form 8 February 2018; Accepted 20 February 2018 Available online 08 March 2018 1773-2247/ © 2018 Elsevier B.V. All rights reserved.
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2.3. Animals
bioavailability [7]. Previous studies from our group, with magnetic nanoparticles coated with dimercaptosuccinic acid (DMSA), indicated that this acid is capable of targeting nanoparticles to the lungs [8,9]. A proposed approach is to use such properties of DMSA to target nanoparticles to the lung to treat pulmonary infections such as PCM. Amaral et al. (2009) described efficient treatment of PCM in lungs with amphotericin B loaded nanoparticles coated with DMSA, emphasizing lung-targeting role of DMSA [10]. The aim of this work was to 1) determine the biodistribution of ITZ administered either as a poly(lactic-co-glycolic) acid (PLGA) nanoparticle containing ITZ and coated with DMSA (PLGA nanoparticle coated with DMSA – ITZ-NANO), or ITZ in oral solution presentation (Free ITZ) in healthy mice; 2) to verify the therapeutic efficacy of both forms of ITZ in the treatment of PCM infected mice. Nanoparticle biodistribution was determined by ITZ quantification in different organs by HPLC. To determine therapeutic efficacy, animals were infected with PCM and treated with ITZ-NANO or Free ITZ and clinical, biochemical and histological aspects were analyzed.
Animals used in this study were female BALB/c mice ageing 10–14 weeks and weighing 20–22 g, purchased from the University of Campinas, SP, Brazil. The mice were housed in polypropylene cages under controlled conditions of luminosity and received food and water ad libitum. All animal procedures performed in this study were approved by the Animal Care and Use Committee of the University of Brasília (UnB), Brasília – Federal District, Brazil (UnBDoc: 12155/2007) and European Community guidelines as accepted principles for the use of experimental animals were adhered to. The study was divided in two major approaches: a biodistribution in healthy animals and the treatment of PCM infected animals. 2.4. ITZ biodistribution Animals received a single dose of either 1) ITZ-NANO containing 3 μg of ITZ per gram of body weight (administered intraperitoneally) or 2) Free ITZ oral solution with 50 μg of ITZ per gram of body weight (administered by gavage). At 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, and 72 h after administration of ITZ-NANO or Free ITZ (n = 5/group for each treatment/time), animals were anaesthetized (ketamine 60 mg/kg and xylazine 7.5 mg/kg), blood was collected by heart puncture and was transferred to tubes containing EDTA. After 30 min at room temperature, tubes were centrifuged for 5 min at 500×g and plasma was stored at −80 °C for future analyses. After the animal's death, liver, spleen, lung and kidney were excised, washed with cold 0.9% NaCl (w/v) solution, blotted on filter paper, weighted and stored at −80 °C for future analyses.
2. Material and methods 2.1. Drugs and chemicals Itraconazole (ITZ), poly(lactic-co-glycolic acid, 50:50) (PLGA 50:50), polyvinyl alcohol (PVA) and dimercaptosuccinic acid (DMSA) were purchased from Sigma Aldrich (Sigma–Aldrich Co., St. Louis, MO, USA) and Itraconazole internal standard (5OR-R51012) was purchased from Fitzgerald (Fitzgerald Industries International Inc., Concord, MA, USA). Acetonitrile, methanol and tetrahydrofuran used for the HPLC analyses were HPLC grade and purchased from Mallinckrodt (Mallinckrodt Inc., Hazelwood, MO, USA). Trifluoroacetic acid was purchased from VETEC (Duque de Caxias, RJ, Brazil). Milli-Q water was obtained from a Barnstead* EASYpure* II Thermo Scientific (San Jose, CA, USA) and was used to prepare aqueous solutions.
2.4.1. HPLC instrumentation The chromatographic equipment (Shimadzu-Prominence, Kyoto, Japan) comprised of on-line degasser (Model DGU 20A5), solvent delivery module (Model LC-20AT), autosampler (Model SIL-20AHT), column oven (Model CTO-20A), fluorescence detector (Model RF10AXL) and system controller CBM-20A. Reverse-phase C18 column ACE AQ (25 × 0.4 cm, 5 μm particle size) (ACE, Aberdeen, Scotland) with a pre-column (1 × 0.4 cm, 5 μm particle size). (ACE, Aberdeen, Scotland) were used.
2.2. ITZ-NANO and free ITZ preparation DMSA-PLGA nanoparticles containing itraconazole (ITZ-NANO) were synthesized and characterized as described by Cunha-Azevedo et al. (2011) [11] and were prepared using a modified emulsificationevaporation technique. Briefly, an organic solution of PLGA and ITZ was prepared. This organic phase was poured slowly into an aqueous solution of polyvinyl alcohol (PVA) 1% (w/v) which was homogenized using an Ultraturrax emulsifier. After this, a double emulsion was formed with 1% PVA (w/v) solution and the organic solvent was removed from the solution by continuous stirring at room temperature inducing polymer precipitation as nanospheres. The nanoparticles were isolated by centrifugation and were washed three times with distilled water. The formulation was suspended in PBS solution. DMSA was adsorbed to PLGA nanoparticle with the final concentration of 0,05 mol/L and stored at 4 °C. All procedures were carried out in a sterile hood. The nanoparticles formed had an average size of 174 nm, efficiency of incorporation of itraconazole into 72,8% and zeta potential of −40 mV [11]. ITZ concentration loaded on PLGA was limited to optimum synthesis conditions of nanoparticles adequate for an intravenous vehicle. Nonetheless, doses used were in accordance with previous publications [12]. An oral solution sugar-free of itraconazole for gavage administration (Free ITZ-“Xarope sem açúcar”) was manipulated by Farmacotecnica® (Brasilia, DF, Brazil), in order to simulate the conventional treatment. Optimum Free ITZ dosage was chosen based on literature [13].
2.4.2. HPLC analysis The mobile phase was obtained from the mixture of 0.12% (v/v) TFA in Milli-Q water (pump A) and 0.12% (v/v) TFA in acetonitrile (pump B) at 1:1 proportions. Fluorimetric measurements were carried out in a 12 μL flow cell at 260 and 365 nm excitation and emission wavelength, respectively. The injection volume was 10 μL and the flowrate during the assays was 1 mL/min at working pressure of 80 kgf/cm2. Analysis was performed using column temperature of 30 °C. Software LCsolution (version 1.24 SP1 Shimadzu, Tokyo, Japan) was used for data processing and identification of chromatographic parameters. For ITZ quantification in plasma, 200 μL of plasma were transferred to microcentrifuge tubes containing 20 μL of 20% ZnSO4 (w/v), and vortexed for 1 min. Then 700 μL of methanol was added for ITZ extraction, followed by agitation on a vortex for 3 min and centrifugation for 5 min at 7000×g. Liquid phase was transferred to a 2 mL volumetric flask and the process was repeated with 1 mL of methanol. For ITZ quantification in tissue, whole spleen, lung and kidneys and 400 mg of liver (due to large mass of liver) were homogenized individually in 1.5 mL microcentrifuge tubes with hand pestles and extracted with the same process used for plasma. Prior to extraction of plasma and tissue, 40 μL of Itraconazole Internal Standard (0.3 μg/mL) were added to tubes. The use of internal standards reduces errors associated to loss occurred during extraction. Biodistribution of ITZ from ITZ-NANO and Free ITZ was performed using HPLC detection method described and validated by Py-Daniel et al. (2014) [14]. In the cited method, the recovery of ITZ with the methodology described above was established as 136
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approximately ∼95% for biological samples used in the present work. 2.5. Treatment of PCM infected mice Paracoccidioides brasiliensis, isolate Pb18, was sub-cultured in liquid YPD medium (10 g of yeast extract, 20 g of peptone, 20 g of dextrose, 1 L of distilled water) at 36 °C in a rotary shaker (220 rpm). After 5 days of culture yeast cells were collected by centrifugation, the supernatant was discarded and the cells were washed twice in sterile PBS. The cellular viability was determined by vital staining with 0.05% (w/v) green-Janus. Cell counts were determined with a haemocytometer and the inoculum suspension adjusted to 3 × 107 viable fungi/mL. Animals were randomly divided into four groups of 10 animals as follows: group I (non-infected control group treated intraperitoneally with PBS); group II (infected control group treated intraperitoneally with PBS); group III (infected group treated daily with Free ITZ, 1 mg/animal/day, per gavage); group IV (infected group treated every three days with ITZNANO, 60 μg/animal intraperitoneally). Animals from groups II, III and IV were initially anaesthetized (ketamine 60 mg/kg and xylazine 7.5 mg/kg) and challenged with 3 × 106 viable cells of P. brasiliensis virulent strain Pb18 by intravenous route. Thirty days post-infection, all animals initiated treatment. After 30 days of treatment, 5 animals from each group were euthanized, and the other 5 continued with treatment up to 60 days when they were also euthanized. All animals were clinically monitored at a daily basis and on the 30th or 60th day of treatment, they were weighted and photographed. Animal were then euthanized and lung fragments were collected and fixed in 4% buffered paraformaldehyde (w/v)) to be processed in accordance with the technique of paraffin embedding. Sections (5 μm) were stained with hematoxylin-eosin (HE) to evaluate the morphology and inflammatory response of the organ, and with Grocott-Gomori stain for identification of the fungus. Sections were analyzed on a light microscope (Axiophot, Zeiss, Germany), and images were captured by Moticam 2300 (Meyer Instruments, USA) camera and Axio Vision program 40v 4.6.1.0. © Copyright.
Fig. 1. A) %Dose of ITZ per g of tissue time profile in tissues after intraperitoneal administration of ITZ-NANO in female BALB/c mice. Results are expressed as means ± standard desviation. 1.B) %Dose of ITZ per g of tissue time profile in tissues after oral administration of Free ITZ in female BALB/c mice. Results are expressed as means ± standard desviation.
(57.09 ± 27.91% of dose per gram of tissue), though no statistical difference was observed among the times points 4, 8, 12 and 24 h after ITZ-NANO administration. After 24 h, a statistical reduction of the ITZ concentration for ITZ-NANO treatment was observed when compared with earlier time points (35.80 ± 10.54% of dose per gram of tissue). The splenic ITZ uptake after Free ITZ treatment was extremely low during the entire experiment period with maximum uptake at 2 h (0.19 ± 0.01% of dose per gram of tissue) though no statistical difference was observed among the times points from 0.25 to 8 h. In spleen, the maximum ITZ uptake found in ITZ-NANO treatment was 300 times higher than that obtained after Free ITZ injection. In the kidney, the ITZ levels after ITZ-NANO injection were very low and statically equivalent at all interval times, ranging from (0–0.7% of injected dose/g of organ). After Free ITZ administration, the ITZ concentration reached its highest level at 1 h (0.56 ± 0.15% of injected dose/g of organ) though no statistical difference was observed among the times points from 0.5 to 4 h. At 8 h, a reduction of the ITZ concentration was observed after Free ITZ treatment (0.08 ± 0.13% of injected dose/g of organ). In the liver of animals treated with ITZNANO, the ITZ level attained a maximum peak at 4 h (21.28 ± 6.92% of injected dose/g of organ) though no statistical difference was observed from 2 to 72 h. With Free ITZ treatment, at 30 min a maximum peak was observed (2.03 ± 0.65% of injected dose/g of organ) though no statistical difference was observed from 0.25 to 1 h. For the ITZNANO treated group, the ITZ uptake in liver was constant from 2 to 72 h with no statistical difference among the tested times. A statistically significant decline of hepatic ITZ concentration in Free ITZ-treated mice was observed after 2 h. In the blood, the maximum ITZ concentration occurred at 1 h (0.27 ± 0.06% of injected dose/g of organ) though no statistical difference was observed from 0.5 h to 4 h after Free ITZ administration (data not shown). In contrast, it was not possible to detect any amount of ITZ after ITZ-NANO administration, considering that the limits of detection and quantification of ITZ in plasma were of 0.039 and 0.117 μg/mL, respectively.
2.6. Statistical analysis Data from biodistribution and treatment of PCM infected mice were analyzed to verify statistical significance. Statistical analysis of ITZ recovered from organs was analyzed by ANOVA using Tukey test, for comparisons between organs at the same time point, and Scheffe's test for comparisons between different time points for the same organ. Values were considered significant if P < 0.05. Data of treatment of PCM infected mice were analyzed using the Prism 3.0 program. For results involving 2 groups, the unpaired Student t-test was used. In comparisons between 3 or more groups, data were treated by analysis of variance with the Tukey test. The results were considered significant when P < 0.05 (5%). 3. Results 3.1. ITZ biodistribution studies The tissue distribution of ITZ after ITZ-NANO or Free ITZ administration in the different organs is shown in Fig. 1. In the lungs, the maximum ITZ uptake was achieved after 2 h for ITZ-NANO (64.38 ± 18.90% of injected dose/g of organ) though no statistical difference was observed among the time points of 1, 2 and 4 h. For free ITZ, maximum uptake was observed at 4 h (0.40 ± 0.08% of injected dose/g of organ) though no statistical difference was observed among the time points from 0.5 to 4 h. The maximum ITZ uptake found in the lungs with ITZ-NANO treatment was approximately 160 times higher than that observed after Free ITZ injection. In the spleen, the maximum ITZ uptake was achieved after 8 h 137
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Fig. 2. Photomicrographs of lungs from mice after 30 days of treatment. In A a normal lung without infection, B and C show lungs of infected and untreated mice. D, E and F show the profile of lungs obtained from infected mice and treated with Free ITZ.The arrows indicate thick parenchyma and inflammatory infiltrate (B), mass of fungi (C, D, E and F), free parenchyma in inflammatory infiltrate (F), parenchyma with small inflammatory infiltrate (G), region free of inflammatory infiltrate (H) and region with inflammatory infiltrate (upper arrow) and alveoli free of infiltrate (bottom arrow) (I).
(Fig. 4C and D) presented normal pelage, similar to the uninfected mice control group. Animals infected and not treated had no alterations on pelage.
3.2. Treatment of PCM infected mice 3.2.1. Antifungal activity of ITZ -NANO in vivo In order to evaluate antifungal activity of ITZ-NANO and Free ITZ treatments, histopathological examinations of lungs were performed (Fig. 2). Fig. 2A presents the lung photomicrography of a non-infected control animal, with very thin alveoli and no inflammatory cells infiltrates. Lung parenchyma of infected animals with no further treatment showed thick inflammatory infiltration (Fig. 2B) and granulomas with fungal cells (Fig. 2C and D). The histopathological analysis of the lungs of animals infected and treated for 30 days with Free ITZ demonstrated large inflammatory infiltrates, parenchymal granulomas and presence of fungal cells (Fig. 2E and F). On the other hand, animals infected and treated for the 30 days with ITZ-NANO revealed a moderate inflammatory infiltrates, however, granulomas and fungal cells were not observed (Fig. 2G, H and I). In lungs of animals treated with Free ITZ, inflammatory infiltrate and parenchymal thickening were still observed after 60 days of treatment. However, no granulomas with fungal cells were present (Fig. 3A and B). Lungs of animals treated with ITZ-NANO for 60 days presented a great reduction in areas with inflammatory infiltration when compared to lungs of animals treated for 30 days. In fact most lung parenchyma was similar to that of healthy animals and alveoli were free of infiltrates (Fig. 3C and D).
4. Discussion In this study, we developed a PLGA nanoparticle coated with DMSA loaded with ITZ in order to reduce the dosage administered, number of administrations and consequently the side effects usually caused by the administration of ITZ during the conventional treatment of fungal PCM infection. To verify nanoparticle targeting to the lungs, we performed the biodistribution of intraperitoneally injected ITZ-NANO coated with DMSA and a conventional (oral) treatment with free ITZ in healthy mice. In previous studies, animal treated with free ITZ presented the ratio of ITZ for lung:plasma of ∼3:1, 10:1 for liver:plasma and over 25:1 for fat:plasma, confirming ITZ potential for tissue penetration [15,16]. In the present study, ITZ-NANO biodistribution rendered higher amount of ITZ in lungs and liver to plasma than previous reported (∼64:1 for lungs:plasma and 21:1 for liver:plasma). It is worth to note that only residual plasma concentrations (values below limit of quantification) were found for ITZ when ITZ-NANO was used. Low plasma concentration of ITZ after ITZ-NANO administration indicates that ITZ is not being promptly liberated. ITZ is thus maintained in nanoparticle until degradation of polymer resulting in controlled release. Low kidney concentrations also corroborate with gradual and controlled release of ITZ and could also be explained by metabolization of ITZ rendering undetected metabolites. The observed ITZ concentration in lungs from ITZ-NANO can be explained possibly by the role of DMSA targeting to lungs [8,9] and/or by nanoparticle entrapment in the first capillary bed encountered after parenteral administration [17].
3.2.2. Clinical parameters analysis Animals treated with Free ITZ, which presented a decrease of approximately 14.00 ± 0.53% on the 30th day of treatment. On the contrary, animals treated with ITZ-NANO showed a body weight loss of only 1.60 ± 0.03% upon the 30th day of treatment (Table 1). The clinical symptoms of mice were monitored daily after beginning of treatment. As can be seen of Fig. 4, animals treated with Free ITZ, upon the 30th day of treatment, presented ventral alopecia and irregular spiked dorsal pelage (Fig. 4A and B). Animals treated with ITZ-NANO 138
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Fig. 3. Photomicrographs of lungs at 60 days of treatment A and B: lungs of infected animals treated with Free ITZ. A: parenchyma with thick inflammatory infiltrate; B: another area of the same slice, showing a region free of inflammatory infiltrate. In C and D, lungs of infected animals treated with ITZ-NANO, showing a small infiltrate (arrow) (C) and an apparently normal area in the same slice of C (D).
released of ITZ from ITZ-NANO, and/or a lower concentration of free drug reaching the liver. In correlation to biodistribution results, a maximum concentration in liver was found in 4 h with the observation of 21.28% of ITZ from injected dose/gram of organ, rendering the approximate mass of 11 μg in organ, indicating that encountered dosage is not toxic to liver. An important parameter for identification of ITZ-NANO toxicity was performed by continuous examination of clinical aspects of mice. No visual differences were observed between control animals and animals treated with ITZ-NANO. These data also suggest that administration of ITZ by ITZ-NANO attenuated the side effects in animals, most likely to decreased concentration injected and controlled release of the drug and/or decreased stress levels of animals, due to administrations of ITZNANO every three days. These findings corroborate with the clinical applicability of nanoparticles in nanobiotechnology and reinforce the advantages of enhancing the specificity of the treatments, providing a higher quality of life for patients. Free ITZ, with high daily doses not only was less efficient in eradicating fungal cells from the lungs, but lead to side effects such as alopecia and increased concentration of liver enzymes. These results were corroborated by biodistribution in which a small amount of ITZ from oral solution reached the lungs, and only for a short period after administration. The results achieved with ITZ-NANO were satisfactory since the purpose of incorporation of drugs in nanoparticles is, other than to promote at least the same efficiency of conventional drug treatment, to reduce side effects and promote a greater incentive for patients in treatment. Specifically in patients undergoing Paracoccidioidomycosis treatment, a very high dropout rate is observed since they are offered under the oral administration form, and the patient must take it several times a day for a long period of time (at least six months) [21].
High liver and spleen concentrations observed when ITZ-NANO was administered are explained mainly by opsonization events that result in nanoparticle identification and removal from the blood by phagocytes as well as tissue macrophages that are in direct contact with the blood (hepatic Kupffer cells and the marginal zone and red-pulp macrophages in the spleen) [18]. Also, ITZ is mainly metabolized in the liver through CYP3A4 into a large number of metabolites [19]. To determine therapeutic efficiency of nanoparticles, the treatment of PCM infected mice was performed with ITZ-NANO or Free ITZ, and clinical and histological aspects were analyzed. As demonstrated, ITZ-NANO in a therapeutic dose of 60 μg per animal administered every three days, because of controlled release of the drug, for a period of thirty days, was effective in controlling pulmonary infection observed in PCM (Fig. 2G, H and 2I). On the other hand, in animals treated with Free ITZ for the same period of time, inflammatory infiltrates were observed as well as granulomas with fungal cells (Fig. 2D, E and 2F). Together these results indicates that treatment with ITZ-NANO rendered a better therapeutic effect than Free ITZ as well as resulted in less stress for the animals, once administration was performed every three days, and the amount of drug injected was much smaller. This result also correlates with the biodistribution results in which 72 h after administration of ITZ-NANO, ITZ was quantifiable in lungs. The described results found with Free ITZ are in accordance with Naranjo et al. (2010) who used this drug to treat chronic PCM, and identified that there was a significant reduction in inflammatory infiltration, but still observed fungal cells in the lungs after four weeks of treatment [20]. No liver toxicity was indicated by biochemical parameters of ITZNANO analysis (data not shown). This was probably due to low concentrations of free circulating ITZ, another indication of controlled
Table 1 Media body mass of treatments groups (non-infected untreated, infected untreated, treated with Free ITZ or ITZ-NANO (n = 5) on days, 0, 30, and 60 after initiation of treatment, and % of modification of body mass from day 0–30 and 30 to 60. Results show the mean ± SD body weight of animals of experimental groups. Treatment groups
Day 0 Non-infected Infected Free ITZ ITZ-NANO
Body Mass Variation (days 0 → 30)
Body Mass (g)
22.89 21.33 20.95 20.84
Day 30 ± ± ± ±
1.22 1.06 1.42 2.82
24.95 22.29 23.80 22.98
± ± ± ±
Body Mass (g)
Body Mass Variation (days 30 → 60)
Day 60 0.49 1.01 2.10 1.38
8% 4% 13% 10%
26.50 23.15 20.44 22.61
139
± ± ± ±
0.70 0.79 0.89 0.32
6% 3% −14 ± 0.53% −1.60 ± 0.03%
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Fig. 4. Physical aspects of the animals after treatment with ITZ-NANO and Free ITZ. In A and B, Free ITZ treated animals showing alopecia. In C and D, animals treated with ITZ-NANO showing a typical pelage of healthy animals.
Therefore the decrement in the number of administrations proposed by this study could enable a greater cooperation from patients, a preponderant factor in the success of treatment protocol.
573880/2008-5.
5. Conclusion
The lead author affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.
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The biodistribution of ITZ administered both as a nanoparticle presentation, as well as an oral solution was evaluated and the presentations and routs of administration had a major impact in the quantification in organs. Biodistribution of ITZ from nanoparticle demonstrated higher accumulation in lung, liver and spleen when compared to free drug. We also compared the therapeutic efficiency as well as toxicity of both forms of ITZ presentations in the treatment of PCM infected mice using clinical, biochemical and histological approaches. It was found that ITZ-NANO was as effective in the treatment of chronic PCM as the free drug, having the advantage of avoiding its known side effects. The ITZ-NANO drug concentration was lower than that administered in free ITZ, which would contribute to the reduction of the total cost of treatment, as well as decrease the time and stress caused to the patient, given that the application would be held every three days. With this in mind, the nanoparticle here presented has a biodistribution and efficacy more favorable to treat PCM than the free drug used in conventional treatment.
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Conflicts of interest The authors declare no conflict of interest. Funding We thank the Brazilian agency National Institute of Science and Technology-Nanotechnology (INCT-Nanobiotecnologia) of Ministry of Science, Technology and Innovation (MCT/CNPq) Grant agreement 140
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