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Contents lists available at ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb
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Research Paper
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Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation
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Melissa Guada a,b, Hugo Lana a, Ana Gloria Gil c,d, Maria del Carmen Dios-Viéitez a, Maria J. Blanco-Prieto a,b,⇑ a
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/Irunlarrea 1, E-31008 Pamplona, Spain Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, E-31008 Pamplona, Spain Department of Pharmacology and Toxicology, University of Navarra, E-31008 Pamplona, Spain d Drug Development Unit, University of Navarra (DDUNAV), E-31008 Pamplona, Spain b c
a r t i c l e
i n f o
Article history: Received 18 November 2015 Revised 18 January 2016 Accepted in revised form 19 January 2016 Available online xxxx Keywords: Cyclosporine A Lipid nanoparticles Immunosuppression T-lymphocyte Pharmacodynamics Pharmacokinetics Nephrotoxicity Oral route Caco-2 cells Biocompatibility
a b s t r a c t The pharmacodynamic effect and the safety of cyclosporine A lipid nanoparticles (CsA LN) for oral administration were investigated using Sandimmune NeoralÒ as reference. First, the biocompatibility of the unloaded LN on Caco-2 cells was demonstrated. The pharmacodynamic response and blood levels of CsA were studied in Balb/c mice after 5 and 10 days of daily oral administration equivalent to 5 and 15 mg/kg of CsA in different formulations. The in vivo nephrotoxicity after 15 days of treatment at the high dose was also evaluated. The results showed a significant decrease in lymphocyte count (indicator of immunosuppression) for the CsA LN groups which was not observed with Sandimmune NeoralÒ. CsA blood levels remained constant over the time after treatment with LN, whereas a proportional increase in drug blood concentration was observed with Sandimmune NeoralÒ. Therefore, CsA LN exhibited a better pharmacological response along with more predictable pharmacokinetic information, diminishing the risk of toxicity. Moreover, a nephroprotective effect against CsA related toxicity was observed in the histopathological evaluation when LN containing TweenÒ 80 was administered. Therefore, our preliminary findings suggest LN formulations would be a good alternative for CsA oral delivery, enhancing efficacy and reducing the risk of nephrotoxicity. Ó 2016 Elsevier B.V. All rights reserved.
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1. Introduction
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Although cyclosporine A (CsA) is widely used as an immunosuppressant, several concerns regarding its efficacy and toxicity balance have been raised in the last few decades [1]. Currently, the formulation for CsA oral administration is Sandimmune NeoralÒ, available on the market as preconcentrate microemulsion. This dosage form has been found to enhance drug bioavailability; however, interindividual variability and toxicity are still clinical issues to take into consideration in CsA therapy. Indeed, due to its unpredictable pharmacokinetic characteristics, therapeutic drug monitoring is mandatory in order to maintain CsA in the therapeutic range and therefore avoid undesirable side effects [2]. In addition, large quantities of some excipients used for the
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⇑ Corresponding author at: Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/Irunlarrea 1, E-31008 Pamplona, Spain. Tel.: +34 948 425 600x6519 (Office); fax: +34 948 425 649. E-mail address: [email protected] (M.J. Blanco-Prieto).
microemulsion preparation (i.e. surfactants and organic solvents) might be harmful for patients, leading to toxicity. The major clinical concern about CsA-induced toxicity is renal damage, characterized by a progressive kidney dysfunction associated with structural abnormalities, such as interstitial fibrosis, tubular atrophy and glomerular damage. Different factors are involved in this CsA-related nephrotoxicity, including molecular mechanisms, pharmacokinetics, pharmacogenetics [3] and the production of certain metabolites [4]. There is therefore an urgent need to develop suitable delivery systems for its oral administration that could improve patient compliance and safety. Although significant efforts have been made to design an ideal vehicle for CsA administration, most of the published research focuses on the enhancement of CsA oral bioavailability [5–12] and only a few papers are oriented toward evaluating the safety of these novel formulations [13–17]. Lipid nanoparticles (LN) have proved to enhance the bioavailability of different compounds as well as to prevent drug-related toxicity by the oral route in vivo [18,19]. Furthermore, CsA formulations based on LN and administered by the oral route have been
http://dx.doi.org/10.1016/j.ejpb.2016.01.011 0939-6411/Ó 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Guada et al., Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation, Eur. J. Pharm. Biopharm. (2016), http://dx.doi.org/10.1016/j.ejpb.2016.01.011
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demonstrated to improve CsA absorption in vivo as well as its immunosuppressive activity in vitro [20,21]. Therefore, the present study focused on the efficacy and safety of CsA LN formulations. The in vitro biocompatibility of the lipid nanovehicles was studied using the human colon adenocarcinoma (Caco-2) cell line and the in vivo pharmacodynamic effect and toxicity evaluations of the CsA LN were performed in Balb/c mice using Sandimmune NeoralÒ as reference formulation.
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2. Materials and methods
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2.1. Materials Ò
100 lL/well of a cell suspension in completed culture media (6. 25 104 cells/mL) was incubated in a 96-well culture microplate under standard cell growth conditions. After 24 h of incubation, the medium was replaced by completed fresh medium containing the treatments at increasing concentrations and cells were incubated for another 24 h under the same conditions. Then, treatments were removed and 200 lL of MTT dissolved in complete cellular media (0.5 mg/mL) was added to the wells and incubation was continued for 2 h. After MTT solution removal, a volume of 100 lL/well of DMSO was added to dissolve the blue formazan crystals. The absorbance at 540 nm was determined using a microplate reader (Labsystems iEMS Reader MF, Finland). Positive control of cytotoxicity was a 10% DMSO solution (100% cell inhibition) and cellular medium was used as negative control (0% cell inhibition). The analysis was performed in triplicate in at least three independent experiments. The cytotoxicity was calculated by the following equation:
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CsA and Tween 80 (Tw) were obtained from Roig Farma S.A. (Barcelona, Spain). PrecirolÒ ATO 5 was a gift from Gattefossé (Lyon, France). L-a-phosphatidylcholine from egg yolk (Lec), taurocholic acid sodium salt hydrate (TC), D-(+)-trehalose dihydrate, formic acid 98% for mass spectroscopy, chloroform (HPLC grade), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol and acetonitrile (HPLC gradient grade) were provided by Merck (Barcelona, Spain). Ammonium acetate (HPLC grade) was obtained from Scharlau (Sentmenat, Spain). Ascomycin was supplied by LC LaboratoriesÒ (Woburn, MA, USA). MEM cell culture media, heat-inactivated fetal bovine serum (FBS), Glutamax, MEM non-essential amino acids and penicillin/streptomycin antibiotics were obtained from GibcoÒ by Life Technologies (Barcelona, Spain). Sandimmune NeoralÒ (hereafter referred to as NeoralÒ) was from Novartis Pharmaceutical, S.A. (Barcelona, Spain). All other reagents employed throughout the experiments were of analytical grade.
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2.2. Preparation and characterization of lipid nanoparticles
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For this study, two LN formulations, differing in the surfactant composition, were formulated using the hot homogenization followed by ultrasonication method, as previously described [20]. The lipid phase consisted of PrecirolÒ ATO 5 and CsA, while the aqueous phase consisted of either a 2% (w/v) solution of Tw or Lec:TC (3:1). Briefly, the pre-heated aqueous phase was poured into the melted lipid phase and sonicated with a MicrosonTM ultrasonic cell disruptor (NY, USA). The resulting emulsion was removed from the heat and cooled in an ice bath to lead particle formation by lipid solidification. The nanoparticle dispersion was concentrated and washed by diafiltration using AmiconÒ Ultra-15 10,000 MWCO filters. LN were lyophilized using trehalose as cryoprotectant and characterized in terms of size, polydispersity index (PDI), zeta potential, morphology and encapsulation efficiency (EE) [20]. Unloaded nanoparticles were produced following the same protocol without CsA incorporation.
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2.3. In vitro studies
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2.3.1. Cell culture Caco-2 cells were supplied by American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely maintained in MEM supplemented with 20% (v/v) FBS, 1% (v/v) Glutamax, 1% (v/v) non-essential amino acids and 1% (v/v) penicillin/streptomycin in a controlled humidified atmosphere containing 5% CO2 at 37 °C. Cells were subcultured every 3–4 days when cell confluence was above 80% at a density of 1.2 104 cells/cm2.
2.4.3. Lymphocyte count The absolute lymphocyte count was used as pharmacodynamic marker for CsA. Peripheral blood samples were collected in EDTAK3 surface-coated tubes before the treatments (baseline) as well as 24 h after the 4th and 9th doses (on days 5 and 10 of the experiment). The cell count was performed by using a Roche hematology analyzer (Sysmex XT1800i, Roche Diagnostics S.L., Barcelona, Spain). The data are expressed as percentage of lymphocyte decrease compared to the baseline lymphocyte count of each animal.
2.3.2. Cytotoxicity assessment The cytotoxicity of the unloaded LN against Caco-2 cells was evaluated using the MTT assay for cell viability. For this purpose,
2.4.4. Cyclosporine A determination in whole blood samples The concentration of CsA in whole blood was quantified using a previously validated method based on ultra-high-performance
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Cytotoxicity ð%Þ ¼ ððS MÞ=ðD MÞÞ 100;
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where S is the absorbance obtained with the tested samples, M is the absorbance observed with cellular medium and D is the absorbance observed with 10% DMSO solution. The 50% inhibitory concentration (IC50) values were obtained from the dose–response curves using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA).
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2.4. In vivo studies
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2.4.1. Animals Female Balb/c mice (20 g) were purchased from Harlan Interfauna Ibérica (Barcelona, Spain). Animals were kept in cages under standard housing conditions with free access to food and water. Mice were exposed to alternating 12 h periods of light and darkness and were acclimatized for at least 5 days prior to the experiments. The experimental procedures were approved by the Animal Experimentation Ethics Committee of the University of Navarra (protocol number CEEA: 054-13).
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2.4.2. Experimental design Mice were randomly divided into 8 groups (n = 6). Two different doses of CsA LN formulations equivalent to 5 mg and 15 mg of drug per kg body weight were studied, using NeoralÒ as reference. Animals received for 15 days: (i) LN Lec:TC-CsA (5 mg/kg; approximately 17 mg of formulation), (ii) LN Lec:TC-CsA (15 mg/kg; approximately 50 mg of formulation), (iii) LN Tw-CsA (5 mg/kg; approximately 17 mg of formulation), (iv) LN Tw-CsA (15 mg/kg; approximately 50 mg of formulation), (v) NeoralÒ (5 mg/kg), (vi) NeoralÒ (15 mg/kg), (vii) unloaded LN (approximately 50 mg of formulation) and (viii) drinking water. All the treatments were dispersed in drinking water and administered orally once a day by gavage needle.
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liquid chromatography tandem mass spectrometry (UHPLC–MS/ MS) [21,22] at the same time points that were analyzed for lymphocyte count.
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2.4.5. Toxicity studies Clinical observations and body weights of the animals were recorded during the experiment. Twenty-four hours after the 15th administration, mice fasted overnight were sacrificed using a CO2 chamber. Peripheral blood samples were withdrawn, allowed to clot at room temperature for 30 min and centrifuged for 10 min at 9391g to obtain serum. Serum was analyzed for creatinine (CREA) and blood urea nitrogen (BUN) levels using a Roche semiautomatic analyzer (Cobas C311, Roche Diagnostics S.L., Barcelona, Spain) and Roche analytical kits. The organs, including kidneys, liver, spleen, thymus, stomach and intestines, were immediately isolated, weighed and fixed in 10% formalin for histopathological evaluation. Hematoxylin and eosin were used for staining. The relative weights of kidneys, liver, spleen and thymus are expressed as a percentage of body weight.
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2.5. Statistical analysis
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The data are expressed as mean values ± standard deviation. Statistical analysis was performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). Mann–Whitney U test was used to compare between different groups. P values below 0.05 were considered significant differences.
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3. Results
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3.1. Characterization of lipid nanoparticles
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Two different LN formulations differing in the stabilizing system were successfully produced. Table 1 summarizes the Precirol nanoparticles’ characteristics containing Lec:TC (referred as LN Lec:TC) and LNs containing Tw (referred as LN Tw). The LN formulations presented a narrow size distribution with PDI values below 0.209 and mean particle diameters between 118.1 ± 7.0 and 200.3 ± 5.2 nm, depending on the stabilizers. For both nanosystems negative surface charge and high entrapment efficiencies, around 93%, were obtained. The nanoparticles were morphologically characterized as individual particles with a well-defined spherical shape as observed in Fig. 1.
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3.2. Cytotoxicity studies
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The in vitro toxicity effect of the unloaded LN on Caco-2 cells evaluated by the MTT assay is presented in Fig. 2. As can be seen, precirol nanoparticles stabilized with Lec:TC showed a higher IC50 value compared to those stabilized with Tw (29.30 mg/mL and 4.11 mg/mL, respectively). Moreover, the cell damage was negligible when Caco-2 cells were exposed up to 11.76 mg/mL and 1.60 mg/mL of LN Lec:TC and LN Tw, respectively, obtaining cytotoxicity rates below 13%. Based on these results, unloaded LN containing Tw were selected for further in vivo experiments.
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Fig. 1. Morphological characterization of cyclosporine A loaded lipid nanoparticles, LN Lec:TC (A) and LN Tw (B), by transmission electron microscopy.
Fig. 2. Cytotoxicity effect of unloaded lipid nanoparticles against Caco-2 cells after 24 h of incubation. Results are represented by mean values ± standard deviation (n = 3). Black dashed lines represent the 50% inhibitory concentrations (IC50) of the formulations.
3.3. Pharmacodynamic evaluation
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The effect of CsA on peripheral blood lymphocytes after its oral administration in different delivery systems (the LN formulations and NeoralÒ) at doses of 5 and 15 mg/kg was studied. As shown in Fig. 3, the absolute lymphocyte count in the control groups (drinking water and unloaded LN) remained practically unchanged and there was no significant difference between them. Similar results were obtained for animals treated with the low CsA dose tested, wherein cell counts did not change regardless of the formulation (data not shown). Likewise, mice treated with the reference formulation (NeoralÒ) at the high CsA dose did not show any alteration in lymphocyte count compared to the control groups during the study period (Fig. 3). Interestingly, both LN formulations significantly changed the cell count. A decrease in lymphocyte count was reached after 10 days of treatment for the LN Lec:TC-CsA group. More relevant was the result found with the administration of LN Tw-CsA that entailed a significant reduction of lymphocytes on day 5 that remained lowered until day 10.
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Table 1 Characterization of the cyclosporine A loaded and unloaded lipid nanoparticles. Formulation
Size (nm)
PDI
Zeta potential (mV)
EE (%)
LN Lec:TC-CsA Unloaded LN Lec:TC
200.3 ± 5.2 199.7 ± 3.0
0.208 ± 0.011 0.209 ± 0.008
27.5 ± 0.8 29.6 ± 0.8
92.8 ± 3.3 –
LN Tw-CsA Unloaded LN Tw
119.9 ± 7.2 118.1 ± 7.0
0.163 ± 0.008 0.165 ± 0.013
14.4 ± 1.7 15.7 ± 1.4
94.1 ± 5.2 –
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Fig. 3. Phamacodynamic effect of cyclosporine A formulations after a daily oral dose administration equivalent to 15 mg/kg of drug at days 5 and 10 of the experiment. Results are represented by mean values ± standard deviation (n = 6). Statistical differences are represented by ⁄ = p < 0.05, ⁄⁄ = p < 0.01 compared to drinking water group; a = p < 0.05, a0 = p < 0.01 compared to unloaded lipid nanoparticles group; b = p < 0.01 compared to Sandimmune NeoralÒ.
Fig. 4. Cyclosporine A whole blood levels after a daily oral dose administration of formulations equivalent to 15 mg/kg of drug at days 5 and 10 of the experiment. Results are represented by mean values ± standard deviation (n = 6). Statistical differences are represented by ⁄⁄ = p < 0.01 compared to Sandimmune NeoralÒ at the same day; # = p < 0.01 compared to the same formulation at different days.
3.4. Cyclosporine A whole blood levels
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CsA blood concentration was monitored at days 5 and 10 of the experiment prior to the next dose. Whole blood levels of drug were not measurable in animals receiving 5 mg/kg at the days assessed regardless of the formulation. Therefore, these groups were excluded from the toxicity studies. Fig. 4 shows CsA blood concentrations obtained at days 5 and 10 in mice receiving daily 15 mg/kg of CsA. As can be observed, the LN Tw-CsA was able to reach higher concentrations of drug in an earlier stage compared to the reference formulation, whereas no differences in drug levels between the NeoralÒ and the LN Lec:TC-CsA groups were observed on day 5. Interestingly, in mice treated with LN Lec:TC-CsA and LN Tw-CsA, the drug concentrations did not significantly change over time, meaning that LN formulations provided stable levels of CsA throughout the days studied. This relevant fact was not observed in the NeoralÒ group, which showed a significant rise in CsA concentration at day 10 with respect to day 5.
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3.5. In vivo toxicity studies
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The behavior of the animals was normal throughout the whole duration of the experiment and no physical signs of toxicity were observed among the different groups. Body weights of treated mice were similar to those observed in the drinking water control group (data not shown). CREA and BUN levels of the CsA treated animals measured in serum were comparable to those of the controls (drinking water and unloaded LN), except for the LN Tw-CsA group where CREA values resulted in statistically higher when compared to levels of the drinking water control (Table 2). Nevertheless, this observation was not considered relevant since the CREA values obtained from LN Tw-CsA group remained within the reference range reported for mice [23]. No changes in the relative weights of kidneys and liver were found in CsA treated groups compared to the control groups (drinking water and unloaded LN groups). However, a significant increase in the lymphoid organs (thymus and spleen) was observed for mice receiving CsA (Table 3). Histological examinations of liver, spleen, thymus, stomach and intestines of animals exposed to the drug did not differ in tissue morphology of the organs of those mice receiving water (data not shown). On the contrary, significant changes in the histological structure of the kidneys were observed in all CsA treated groups (Fig. 5). These histological changes were characterized by an
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Table 2 Creatinine (CREA) and blood urea nitrogen (BUN) levels after 15-day treatment. Mean ± SD (n = 6).
*
Parameters
Drinking water
Unloaded LN
NeoralÒ
LN Lec:TC-CsA
LN Tw-CsA
CREA (mg/dL) BUN (mg/dL)
0.27 ± 0.03 57.5 ± 19.6
0.29 ± 0.05 70.6 ± 6.5
0.29 ± 0.02 58.8 ± 23.0
0.29 ± 0.02 71.5 ± 19.2
0.34 ± 0.03* 69.6 ± 38.6
p < 0.05 vs control group (drinking water).
Table 3 The relative organ weights (%) at the end of the experiment. Mean ± SD (n = 6).
* ** a b
Organs
Drinking water
Unloaded LN
NeoralÒ
LN Lec:TC-CsA
LN Tw-CsA
Thymus Spleen Kidneys Liver
0.183 ± 0.055 0.416 ± 0.041 1.329 ± 0.091 4.379 ± 0.347
0.181 ± 0.026 0.429 ± 0.030 1.439 ± 0.056 4.071 ± 0.204
0.268 ± 0.042*,a,**,b 0.766 ± 0.110**,a,**,b 1.381 ± 0.061 4.514 ± 0.291
0.236 ± 0.043*,b 0.923 ± 0.189 **,a,**,b 1.368 ± 0.089 4.603 ± 0.387
0.310 ± 0.059**,a,**,b 0.918 ± 0.268 **,a,**,b 1.359 ± 0.085 4.538 ± 0.446
p < 0.05. p < 0.01 vs control groups. Drinking water. Unloaded LN.
Please cite this article in press as: M. Guada et al., Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation, Eur. J. Pharm. Biopharm. (2016), http://dx.doi.org/10.1016/j.ejpb.2016.01.011
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Fig. 5. Kidney histology of mice receiving the following: water (control) (A), Sandimmune NeoralÒ (B), LN Lec:TC-CsA (C) and LN Tw-CsA (D) at the end of the experiment (hematoxylin and eosin stain; X200). Images show no histopathological changes, with normal structure for the glomerulus and renal tubules (A), severe tubulonephrosis with marked eosinophilia of the cytoplasm of the tubular cells and obvious loss of their nucleus (arrows) (B and C), and slight tubulonephrosis with a minor vacuolization of the cytoplasm of the tubular cells and discrete loss of their nucleus (arrows) (D).
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increase in glomerular hypercellularity and a severe tubulonephrosis in the convoluted tubules of the kidneys. This renal injury was significantly less evident in animals treated with LN Tw-CsA, which was defined as moderate compared to the severe tubule damage in the other CsA treated groups.
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4. Discussion
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The main objective of the present investigation was to evaluate the pharmacodynamic effect of CsA incorporated in LN as well as to assess the safety of these delivery systems in mice. For this purpose, two CsA LN formulations were prepared, LN Lec:TC and LN Tw. The safety of novel delivery systems is a fundamental aspect to consider during their development. In this work, we assessed the cytotoxicity of unloaded LN using Caco-2 cells as in vitro model. Due to the ability of Caco-2 cells to mimic the intestinal epithelium, they have been widely used to evaluate the cytotoxic effect of vehicles designed for the oral route as well as a screening model to reduce the number of animals for in vivo studies [24]. The IC50 data obtained indicate that LN containing Lec:TC were more biocompatible than those containing Tw. This result can be explained by the physiological nature of these surfactants. Nevertheless, both nanosystems exhibited negligible cytotoxicity against Caco-2 cells, indicating their in vitro biocompatibility as suitable vehicles for oral administration. Next, the pharmacodynamic effect of CsA orally administered in different delivery systems was investigated in vivo using the
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lymphocyte count as a biological marker. The reduction in the number of lymphocytes in peripheral blood has been experimentally used as an indicator of immunosuppression [25–27]. CsA blocks the activation of lymphocyte entailing the inhibition of cytokine production (mainly IL-2) essential for proliferation, differentiation and maturation of T-helper cells, which in turn are involved in the maturation of B cells [28]. In order to evaluate the effect of the CsA LN on the lymphocyte count, the treatments were daily administered to healthy mice for 15 days at two different doses (5 and 15 mg/kg) which lay within the human therapeutic range. The low dose corresponds to that used for the treatment of autoimmune disorders while the high dose is the maximum amount prescribed in transplantation. During the treatments, the lymphocyte count was unaltered at the low dose, regardless of the formulation administered. Therefore, it seems that CsA at dose of 5 mg/kg was suboptimal to produce a pharmacodynamic response in this species. Besides, non-quantifiable CsA blood levels were obtained during the study, possibly due to the fast metabolization or elimination of the drug from the bloodstream. It has been reported that much higher doses than those used for humans are needed in other species to reach CsA therapeutic levels by the oral route [29,30]. Furthermore, a lack of response was also observed in animals receiving NeoralÒ at the highest dose studied. Interestingly, both of the CsA LN formulations were able to decrease the lymphocyte count, reaching an earlier effect with LN containing Tw that was maintained up to 10 days. This observation may be attributed to the CsA concentration encountered in blood on day 5, which was significantly higher when LN Tw-CsA was given. Also, the absorption pathway could be involved in the
Please cite this article in press as: M. Guada et al., Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation, Eur. J. Pharm. Biopharm. (2016), http://dx.doi.org/10.1016/j.ejpb.2016.01.011
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pharmacological activity since no correlation between CsA levels and effect was observed for the LN Lec:TC-CsA formulation and NeoralÒ. When LN are orally administered, the particles are transported by the M-cells through the mesenteric lymphatic system before going through the general circulation [31] and probably they accumulate in lymphoid organs causing a prompt response. A better in vitro inhibitory effect on T-cell proliferation of negative surface charged CsA delivery system has been reported compared to neutral and positive ones, which was attributed to the higher affinity of the negatively charge carriers for these cells [32]. This is another explanation for the favorable pharmacological effect observed with the LN formulations compared to the reference formulation, which has a surface charge near neutrality [11]. Moreover, although thymus and spleen of CsA treated animals were histologically unaltered, they presented higher relative weights compared to control animals. This fact was considered a sign of immunosuppression as these are specialized lymphoid organs associated with the immune response. Along with this, lymphoid organs are rich in cyclophilin, which is the main protein involved in the mechanism of T-cell inhibition produced by CsA exposure [33]. Regarding CsA blood levels, drug concentrations obtained on day 5 in mice daily treated with LN formulations at 15 mg/kg of CsA remained similar to those achieved on day 10, indicating more predictable pharmacokinetic information that might facilitate the dose adjustment. As mentioned, nanoparticles can be taken up from the bloodstream by phagocytes and accumulated in the organs of the mononuclear phagocyte system such as the spleen and liver [34]. Targeting the drug to the spleen, as the major lymphoid organ, might contribute to the immunosuppressive effect. This could be the reason why CsA LN yield to an early response with more predictable drug blood levels than the reference formulation. When NeoralÒ was administered, the CsA blood levels significantly rose over time, increasing the risk of toxicity if the drug is not carefully monitored, as is well known in human clinical practice. Considering that LN promote drug transport to the general circulation by the intestinal lymphatic system, by avoiding the P-glycoprotein efflux and the presystemic metabolism [35], more consistent and reproducible bioavailability of the drug is expected. In this regard, CsA LN could offer the advantage of diminishing inter-individual variability in drug absorption, which is one of the issues presented by this immunosuppressant when it is orally administered in marketed formulations. However, further studies are needed to confirm this hypothesis. Nephrotoxicity is the major side effect of CsA and is characterized by functional and structural abnormalities, CREA and BUN levels being indicators of renal function. Therefore, these parameters are carefully monitored in patients receiving CsA to prevent renal damage. In the present study, no alteration in CREA and BUN concentrations was observed across the CsA treated groups. However, histological alterations of the kidneys were observed, which were more severe in the groups treated with NeoralÒ and LN containing Lec:TC (Fig. 5). The renal injury was characterized by a severe tubulonephrosis along with abnormalities in the convoluted tubules and glomerular hypercellularity. Interestingly, these observations were less apparent in mice receiving LN containing Tw, suggesting the protective action of this delivery system against CsA-related renal damage. One hypothesis that explains this result might be associated with the way in which the drug reaches the renal system. Due to the fact that Lec:TC are degraded faster by digestive enzymes and the LN containing these surfactants lose their physical properties at gastric and intestinal pHs [21], larger quantities of drug are released than that from LN containing Tw. As a consequence, most of the drugs reach the targeted tissues in its free form, as is the case with liquid lipid systems (i.e. microemulsion). Indeed, comparable renal damage outcomes were
obtained in the mice treated with LN Lec:TC-CsA and NeoralÒ. On the other hand, LN containing Tw have been shown to be more resistant to the gastrointestinal environment (pH changes, digestive enzymes, etc.) attributed to the surfactant (Tw), which is degraded more slowly due to the stearic hindrance, leading to the improved CsA bioavailability [21]. In this context, probably part of the drug reaches the target in its entrapped form, which minimizes the formation of metabolites associated with nephrotoxicity so that the renal injury is reduced. All in all, the nephroprotective behavior obtained with LN TwCsA along with the benefits observed with both LN systems in terms of the pharmacodynamic effect and the lower fluctuations of CsA blood levels (Figs. 3 and 4, respectively) leads us to suggest that these CsA delivery systems are a promising strategy to overcome toxicity issues in long-term treatments with this immunosuppressant.
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5. Conclusion
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The CsA LN formulations developed exhibited relevant advantages over the reference formulation, NeoralÒ. First, the decrease in lymphocyte count as an indicator of immunosuppression obtained with LN was not observed after NeoralÒ administration. In addition, lower fluctuations in CsA blood levels were obtained, leading to more predictable pharmacokinetic information, and therefore facilitating dose adjustments to reduce the risk of toxicity. Moreover, LN contain Tw diminished CsA-related nephrotoxicity despite their enhanced bioavailability, making it possible to extend the therapeutic window.
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Acknowledgment
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This work has been carried out in the framework of the COST Action TD1004. M. Guada is grateful to ‘‘Asociación de Amigos de la Universidad de Navarra” for the fellowship grant.
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References
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Contents lists available at ScienceDirect
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Research Paper
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Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation
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Melissa Guada a,b, Hugo Lana a, Ana Gloria Gil c,d, Maria del Carmen Dios-Viéitez a, Maria J. Blanco-Prieto a,b,⇑ a
Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/Irunlarrea 1, E-31008 Pamplona, Spain Instituto de Investigación Sanitaria de Navarra, IdiSNA, C/Irunlarrea 3, E-31008 Pamplona, Spain Department of Pharmacology and Toxicology, University of Navarra, E-31008 Pamplona, Spain d Drug Development Unit, University of Navarra (DDUNAV), E-31008 Pamplona, Spain b c
a r t i c l e
i n f o
Article history: Received 18 November 2015 Revised 18 January 2016 Accepted in revised form 19 January 2016 Available online xxxx Keywords: Cyclosporine A Lipid nanoparticles Immunosuppression T-lymphocyte Pharmacodynamics Pharmacokinetics Nephrotoxicity Oral route Caco-2 cells Biocompatibility
a b s t r a c t The pharmacodynamic effect and the safety of cyclosporine A lipid nanoparticles (CsA LN) for oral administration were investigated using Sandimmune NeoralÒ as reference. First, the biocompatibility of the unloaded LN on Caco-2 cells was demonstrated. The pharmacodynamic response and blood levels of CsA were studied in Balb/c mice after 5 and 10 days of daily oral administration equivalent to 5 and 15 mg/kg of CsA in different formulations. The in vivo nephrotoxicity after 15 days of treatment at the high dose was also evaluated. The results showed a significant decrease in lymphocyte count (indicator of immunosuppression) for the CsA LN groups which was not observed with Sandimmune NeoralÒ. CsA blood levels remained constant over the time after treatment with LN, whereas a proportional increase in drug blood concentration was observed with Sandimmune NeoralÒ. Therefore, CsA LN exhibited a better pharmacological response along with more predictable pharmacokinetic information, diminishing the risk of toxicity. Moreover, a nephroprotective effect against CsA related toxicity was observed in the histopathological evaluation when LN containing TweenÒ 80 was administered. Therefore, our preliminary findings suggest LN formulations would be a good alternative for CsA oral delivery, enhancing efficacy and reducing the risk of nephrotoxicity. Ó 2016 Elsevier B.V. All rights reserved.
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1. Introduction
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Although cyclosporine A (CsA) is widely used as an immunosuppressant, several concerns regarding its efficacy and toxicity balance have been raised in the last few decades [1]. Currently, the formulation for CsA oral administration is Sandimmune NeoralÒ, available on the market as preconcentrate microemulsion. This dosage form has been found to enhance drug bioavailability; however, interindividual variability and toxicity are still clinical issues to take into consideration in CsA therapy. Indeed, due to its unpredictable pharmacokinetic characteristics, therapeutic drug monitoring is mandatory in order to maintain CsA in the therapeutic range and therefore avoid undesirable side effects [2]. In addition, large quantities of some excipients used for the
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⇑ Corresponding author at: Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, C/Irunlarrea 1, E-31008 Pamplona, Spain. Tel.: +34 948 425 600x6519 (Office); fax: +34 948 425 649. E-mail address: [email protected] (M.J. Blanco-Prieto).
microemulsion preparation (i.e. surfactants and organic solvents) might be harmful for patients, leading to toxicity. The major clinical concern about CsA-induced toxicity is renal damage, characterized by a progressive kidney dysfunction associated with structural abnormalities, such as interstitial fibrosis, tubular atrophy and glomerular damage. Different factors are involved in this CsA-related nephrotoxicity, including molecular mechanisms, pharmacokinetics, pharmacogenetics [3] and the production of certain metabolites [4]. There is therefore an urgent need to develop suitable delivery systems for its oral administration that could improve patient compliance and safety. Although significant efforts have been made to design an ideal vehicle for CsA administration, most of the published research focuses on the enhancement of CsA oral bioavailability [5–12] and only a few papers are oriented toward evaluating the safety of these novel formulations [13–17]. Lipid nanoparticles (LN) have proved to enhance the bioavailability of different compounds as well as to prevent drug-related toxicity by the oral route in vivo [18,19]. Furthermore, CsA formulations based on LN and administered by the oral route have been
http://dx.doi.org/10.1016/j.ejpb.2016.01.011 0939-6411/Ó 2016 Elsevier B.V. All rights reserved.
Please cite this article in press as: M. Guada et al., Cyclosporine A lipid nanoparticles for oral administration: Pharmacodynamics and safety evaluation, Eur. J. Pharm. Biopharm. (2016), http://dx.doi.org/10.1016/j.ejpb.2016.01.011
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demonstrated to improve CsA absorption in vivo as well as its immunosuppressive activity in vitro [20,21]. Therefore, the present study focused on the efficacy and safety of CsA LN formulations. The in vitro biocompatibility of the lipid nanovehicles was studied using the human colon adenocarcinoma (Caco-2) cell line and the in vivo pharmacodynamic effect and toxicity evaluations of the CsA LN were performed in Balb/c mice using Sandimmune NeoralÒ as reference formulation.
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2. Materials and methods
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2.1. Materials Ò
100 lL/well of a cell suspension in completed culture media (6. 25 104 cells/mL) was incubated in a 96-well culture microplate under standard cell growth conditions. After 24 h of incubation, the medium was replaced by completed fresh medium containing the treatments at increasing concentrations and cells were incubated for another 24 h under the same conditions. Then, treatments were removed and 200 lL of MTT dissolved in complete cellular media (0.5 mg/mL) was added to the wells and incubation was continued for 2 h. After MTT solution removal, a volume of 100 lL/well of DMSO was added to dissolve the blue formazan crystals. The absorbance at 540 nm was determined using a microplate reader (Labsystems iEMS Reader MF, Finland). Positive control of cytotoxicity was a 10% DMSO solution (100% cell inhibition) and cellular medium was used as negative control (0% cell inhibition). The analysis was performed in triplicate in at least three independent experiments. The cytotoxicity was calculated by the following equation:
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CsA and Tween 80 (Tw) were obtained from Roig Farma S.A. (Barcelona, Spain). PrecirolÒ ATO 5 was a gift from Gattefossé (Lyon, France). L-a-phosphatidylcholine from egg yolk (Lec), taurocholic acid sodium salt hydrate (TC), D-(+)-trehalose dihydrate, formic acid 98% for mass spectroscopy, chloroform (HPLC grade), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol and acetonitrile (HPLC gradient grade) were provided by Merck (Barcelona, Spain). Ammonium acetate (HPLC grade) was obtained from Scharlau (Sentmenat, Spain). Ascomycin was supplied by LC LaboratoriesÒ (Woburn, MA, USA). MEM cell culture media, heat-inactivated fetal bovine serum (FBS), Glutamax, MEM non-essential amino acids and penicillin/streptomycin antibiotics were obtained from GibcoÒ by Life Technologies (Barcelona, Spain). Sandimmune NeoralÒ (hereafter referred to as NeoralÒ) was from Novartis Pharmaceutical, S.A. (Barcelona, Spain). All other reagents employed throughout the experiments were of analytical grade.
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2.2. Preparation and characterization of lipid nanoparticles
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For this study, two LN formulations, differing in the surfactant composition, were formulated using the hot homogenization followed by ultrasonication method, as previously described [20]. The lipid phase consisted of PrecirolÒ ATO 5 and CsA, while the aqueous phase consisted of either a 2% (w/v) solution of Tw or Lec:TC (3:1). Briefly, the pre-heated aqueous phase was poured into the melted lipid phase and sonicated with a MicrosonTM ultrasonic cell disruptor (NY, USA). The resulting emulsion was removed from the heat and cooled in an ice bath to lead particle formation by lipid solidification. The nanoparticle dispersion was concentrated and washed by diafiltration using AmiconÒ Ultra-15 10,000 MWCO filters. LN were lyophilized using trehalose as cryoprotectant and characterized in terms of size, polydispersity index (PDI), zeta potential, morphology and encapsulation efficiency (EE) [20]. Unloaded nanoparticles were produced following the same protocol without CsA incorporation.
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2.3. In vitro studies
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2.3.1. Cell culture Caco-2 cells were supplied by American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely maintained in MEM supplemented with 20% (v/v) FBS, 1% (v/v) Glutamax, 1% (v/v) non-essential amino acids and 1% (v/v) penicillin/streptomycin in a controlled humidified atmosphere containing 5% CO2 at 37 °C. Cells were subcultured every 3–4 days when cell confluence was above 80% at a density of 1.2 104 cells/cm2.
2.4.3. Lymphocyte count The absolute lymphocyte count was used as pharmacodynamic marker for CsA. Peripheral blood samples were collected in EDTAK3 surface-coated tubes before the treatments (baseline) as well as 24 h after the 4th and 9th doses (on days 5 and 10 of the experiment). The cell count was performed by using a Roche hematology analyzer (Sysmex XT1800i, Roche Diagnostics S.L., Barcelona, Spain). The data are expressed as percentage of lymphocyte decrease compared to the baseline lymphocyte count of each animal.
2.3.2. Cytotoxicity assessment The cytotoxicity of the unloaded LN against Caco-2 cells was evaluated using the MTT assay for cell viability. For this purpose,
2.4.4. Cyclosporine A determination in whole blood samples The concentration of CsA in whole blood was quantified using a previously validated method based on ultra-high-performance
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Cytotoxicity ð%Þ ¼ ððS MÞ=ðD MÞÞ 100;
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where S is the absorbance obtained with the tested samples, M is the absorbance observed with cellular medium and D is the absorbance observed with 10% DMSO solution. The 50% inhibitory concentration (IC50) values were obtained from the dose–response curves using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA).
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2.4. In vivo studies
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2.4.1. Animals Female Balb/c mice (20 g) were purchased from Harlan Interfauna Ibérica (Barcelona, Spain). Animals were kept in cages under standard housing conditions with free access to food and water. Mice were exposed to alternating 12 h periods of light and darkness and were acclimatized for at least 5 days prior to the experiments. The experimental procedures were approved by the Animal Experimentation Ethics Committee of the University of Navarra (protocol number CEEA: 054-13).
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2.4.2. Experimental design Mice were randomly divided into 8 groups (n = 6). Two different doses of CsA LN formulations equivalent to 5 mg and 15 mg of drug per kg body weight were studied, using NeoralÒ as reference. Animals received for 15 days: (i) LN Lec:TC-CsA (5 mg/kg; approximately 17 mg of formulation), (ii) LN Lec:TC-CsA (15 mg/kg; approximately 50 mg of formulation), (iii) LN Tw-CsA (5 mg/kg; approximately 17 mg of formulation), (iv) LN Tw-CsA (15 mg/kg; approximately 50 mg of formulation), (v) NeoralÒ (5 mg/kg), (vi) NeoralÒ (15 mg/kg), (vii) unloaded LN (approximately 50 mg of formulation) and (viii) drinking water. All the treatments were dispersed in drinking water and administered orally once a day by gavage needle.
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liquid chromatography tandem mass spectrometry (UHPLC–MS/ MS) [21,22] at the same time points that were analyzed for lymphocyte count.
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2.4.5. Toxicity studies Clinical observations and body weights of the animals were recorded during the experiment. Twenty-four hours after the 15th administration, mice fasted overnight were sacrificed using a CO2 chamber. Peripheral blood samples were withdrawn, allowed to clot at room temperature for 30 min and centrifuged for 10 min at 9391g to obtain serum. Serum was analyzed for creatinine (CREA) and blood urea nitrogen (BUN) levels using a Roche semiautomatic analyzer (Cobas C311, Roche Diagnostics S.L., Barcelona, Spain) and Roche analytical kits. The organs, including kidneys, liver, spleen, thymus, stomach and intestines, were immediately isolated, weighed and fixed in 10% formalin for histopathological evaluation. Hematoxylin and eosin were used for staining. The relative weights of kidneys, liver, spleen and thymus are expressed as a percentage of body weight.
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The data are expressed as mean values ± standard deviation. Statistical analysis was performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego, CA, USA). Mann–Whitney U test was used to compare between different groups. P values below 0.05 were considered significant differences.
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3. Results
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3.1. Characterization of lipid nanoparticles
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Two different LN formulations differing in the stabilizing system were successfully produced. Table 1 summarizes the Precirol nanoparticles’ characteristics containing Lec:TC (referred as LN Lec:TC) and LNs containing Tw (referred as LN Tw). The LN formulations presented a narrow size distribution with PDI values below 0.209 and mean particle diameters between 118.1 ± 7.0 and 200.3 ± 5.2 nm, depending on the stabilizers. For both nanosystems negative surface charge and high entrapment efficiencies, around 93%, were obtained. The nanoparticles were morphologically characterized as individual particles with a well-defined spherical shape as observed in Fig. 1.
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The in vitro toxicity effect of the unloaded LN on Caco-2 cells evaluated by the MTT assay is presented in Fig. 2. As can be seen, precirol nanoparticles stabilized with Lec:TC showed a higher IC50 value compared to those stabilized with Tw (29.30 mg/mL and 4.11 mg/mL, respectively). Moreover, the cell damage was negligible when Caco-2 cells were exposed up to 11.76 mg/mL and 1.60 mg/mL of LN Lec:TC and LN Tw, respectively, obtaining cytotoxicity rates below 13%. Based on these results, unloaded LN containing Tw were selected for further in vivo experiments.
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Fig. 1. Morphological characterization of cyclosporine A loaded lipid nanoparticles, LN Lec:TC (A) and LN Tw (B), by transmission electron microscopy.
Fig. 2. Cytotoxicity effect of unloaded lipid nanoparticles against Caco-2 cells after 24 h of incubation. Results are represented by mean values ± standard deviation (n = 3). Black dashed lines represent the 50% inhibitory concentrations (IC50) of the formulations.
3.3. Pharmacodynamic evaluation
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The effect of CsA on peripheral blood lymphocytes after its oral administration in different delivery systems (the LN formulations and NeoralÒ) at doses of 5 and 15 mg/kg was studied. As shown in Fig. 3, the absolute lymphocyte count in the control groups (drinking water and unloaded LN) remained practically unchanged and there was no significant difference between them. Similar results were obtained for animals treated with the low CsA dose tested, wherein cell counts did not change regardless of the formulation (data not shown). Likewise, mice treated with the reference formulation (NeoralÒ) at the high CsA dose did not show any alteration in lymphocyte count compared to the control groups during the study period (Fig. 3). Interestingly, both LN formulations significantly changed the cell count. A decrease in lymphocyte count was reached after 10 days of treatment for the LN Lec:TC-CsA group. More relevant was the result found with the administration of LN Tw-CsA that entailed a significant reduction of lymphocytes on day 5 that remained lowered until day 10.
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Table 1 Characterization of the cyclosporine A loaded and unloaded lipid nanoparticles. Formulation
Size (nm)
PDI
Zeta potential (mV)
EE (%)
LN Lec:TC-CsA Unloaded LN Lec:TC
200.3 ± 5.2 199.7 ± 3.0
0.208 ± 0.011 0.209 ± 0.008
27.5 ± 0.8 29.6 ± 0.8
92.8 ± 3.3 –
LN Tw-CsA Unloaded LN Tw
119.9 ± 7.2 118.1 ± 7.0
0.163 ± 0.008 0.165 ± 0.013
14.4 ± 1.7 15.7 ± 1.4
94.1 ± 5.2 –
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Fig. 3. Phamacodynamic effect of cyclosporine A formulations after a daily oral dose administration equivalent to 15 mg/kg of drug at days 5 and 10 of the experiment. Results are represented by mean values ± standard deviation (n = 6). Statistical differences are represented by ⁄ = p < 0.05, ⁄⁄ = p < 0.01 compared to drinking water group; a = p < 0.05, a0 = p < 0.01 compared to unloaded lipid nanoparticles group; b = p < 0.01 compared to Sandimmune NeoralÒ.
Fig. 4. Cyclosporine A whole blood levels after a daily oral dose administration of formulations equivalent to 15 mg/kg of drug at days 5 and 10 of the experiment. Results are represented by mean values ± standard deviation (n = 6). Statistical differences are represented by ⁄⁄ = p < 0.01 compared to Sandimmune NeoralÒ at the same day; # = p < 0.01 compared to the same formulation at different days.
3.4. Cyclosporine A whole blood levels
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CsA blood concentration was monitored at days 5 and 10 of the experiment prior to the next dose. Whole blood levels of drug were not measurable in animals receiving 5 mg/kg at the days assessed regardless of the formulation. Therefore, these groups were excluded from the toxicity studies. Fig. 4 shows CsA blood concentrations obtained at days 5 and 10 in mice receiving daily 15 mg/kg of CsA. As can be observed, the LN Tw-CsA was able to reach higher concentrations of drug in an earlier stage compared to the reference formulation, whereas no differences in drug levels between the NeoralÒ and the LN Lec:TC-CsA groups were observed on day 5. Interestingly, in mice treated with LN Lec:TC-CsA and LN Tw-CsA, the drug concentrations did not significantly change over time, meaning that LN formulations provided stable levels of CsA throughout the days studied. This relevant fact was not observed in the NeoralÒ group, which showed a significant rise in CsA concentration at day 10 with respect to day 5.
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3.5. In vivo toxicity studies
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The behavior of the animals was normal throughout the whole duration of the experiment and no physical signs of toxicity were observed among the different groups. Body weights of treated mice were similar to those observed in the drinking water control group (data not shown). CREA and BUN levels of the CsA treated animals measured in serum were comparable to those of the controls (drinking water and unloaded LN), except for the LN Tw-CsA group where CREA values resulted in statistically higher when compared to levels of the drinking water control (Table 2). Nevertheless, this observation was not considered relevant since the CREA values obtained from LN Tw-CsA group remained within the reference range reported for mice [23]. No changes in the relative weights of kidneys and liver were found in CsA treated groups compared to the control groups (drinking water and unloaded LN groups). However, a significant increase in the lymphoid organs (thymus and spleen) was observed for mice receiving CsA (Table 3). Histological examinations of liver, spleen, thymus, stomach and intestines of animals exposed to the drug did not differ in tissue morphology of the organs of those mice receiving water (data not shown). On the contrary, significant changes in the histological structure of the kidneys were observed in all CsA treated groups (Fig. 5). These histological changes were characterized by an
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Table 2 Creatinine (CREA) and blood urea nitrogen (BUN) levels after 15-day treatment. Mean ± SD (n = 6).
*
Parameters
Drinking water
Unloaded LN
NeoralÒ
LN Lec:TC-CsA
LN Tw-CsA
CREA (mg/dL) BUN (mg/dL)
0.27 ± 0.03 57.5 ± 19.6
0.29 ± 0.05 70.6 ± 6.5
0.29 ± 0.02 58.8 ± 23.0
0.29 ± 0.02 71.5 ± 19.2
0.34 ± 0.03* 69.6 ± 38.6
p < 0.05 vs control group (drinking water).
Table 3 The relative organ weights (%) at the end of the experiment. Mean ± SD (n = 6).
* ** a b
Organs
Drinking water
Unloaded LN
NeoralÒ
LN Lec:TC-CsA
LN Tw-CsA
Thymus Spleen Kidneys Liver
0.183 ± 0.055 0.416 ± 0.041 1.329 ± 0.091 4.379 ± 0.347
0.181 ± 0.026 0.429 ± 0.030 1.439 ± 0.056 4.071 ± 0.204
0.268 ± 0.042*,a,**,b 0.766 ± 0.110**,a,**,b 1.381 ± 0.061 4.514 ± 0.291
0.236 ± 0.043*,b 0.923 ± 0.189 **,a,**,b 1.368 ± 0.089 4.603 ± 0.387
0.310 ± 0.059**,a,**,b 0.918 ± 0.268 **,a,**,b 1.359 ± 0.085 4.538 ± 0.446
p < 0.05. p < 0.01 vs control groups. Drinking water. Unloaded LN.
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Fig. 5. Kidney histology of mice receiving the following: water (control) (A), Sandimmune NeoralÒ (B), LN Lec:TC-CsA (C) and LN Tw-CsA (D) at the end of the experiment (hematoxylin and eosin stain; X200). Images show no histopathological changes, with normal structure for the glomerulus and renal tubules (A), severe tubulonephrosis with marked eosinophilia of the cytoplasm of the tubular cells and obvious loss of their nucleus (arrows) (B and C), and slight tubulonephrosis with a minor vacuolization of the cytoplasm of the tubular cells and discrete loss of their nucleus (arrows) (D).
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increase in glomerular hypercellularity and a severe tubulonephrosis in the convoluted tubules of the kidneys. This renal injury was significantly less evident in animals treated with LN Tw-CsA, which was defined as moderate compared to the severe tubule damage in the other CsA treated groups.
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4. Discussion
317
The main objective of the present investigation was to evaluate the pharmacodynamic effect of CsA incorporated in LN as well as to assess the safety of these delivery systems in mice. For this purpose, two CsA LN formulations were prepared, LN Lec:TC and LN Tw. The safety of novel delivery systems is a fundamental aspect to consider during their development. In this work, we assessed the cytotoxicity of unloaded LN using Caco-2 cells as in vitro model. Due to the ability of Caco-2 cells to mimic the intestinal epithelium, they have been widely used to evaluate the cytotoxic effect of vehicles designed for the oral route as well as a screening model to reduce the number of animals for in vivo studies [24]. The IC50 data obtained indicate that LN containing Lec:TC were more biocompatible than those containing Tw. This result can be explained by the physiological nature of these surfactants. Nevertheless, both nanosystems exhibited negligible cytotoxicity against Caco-2 cells, indicating their in vitro biocompatibility as suitable vehicles for oral administration. Next, the pharmacodynamic effect of CsA orally administered in different delivery systems was investigated in vivo using the
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lymphocyte count as a biological marker. The reduction in the number of lymphocytes in peripheral blood has been experimentally used as an indicator of immunosuppression [25–27]. CsA blocks the activation of lymphocyte entailing the inhibition of cytokine production (mainly IL-2) essential for proliferation, differentiation and maturation of T-helper cells, which in turn are involved in the maturation of B cells [28]. In order to evaluate the effect of the CsA LN on the lymphocyte count, the treatments were daily administered to healthy mice for 15 days at two different doses (5 and 15 mg/kg) which lay within the human therapeutic range. The low dose corresponds to that used for the treatment of autoimmune disorders while the high dose is the maximum amount prescribed in transplantation. During the treatments, the lymphocyte count was unaltered at the low dose, regardless of the formulation administered. Therefore, it seems that CsA at dose of 5 mg/kg was suboptimal to produce a pharmacodynamic response in this species. Besides, non-quantifiable CsA blood levels were obtained during the study, possibly due to the fast metabolization or elimination of the drug from the bloodstream. It has been reported that much higher doses than those used for humans are needed in other species to reach CsA therapeutic levels by the oral route [29,30]. Furthermore, a lack of response was also observed in animals receiving NeoralÒ at the highest dose studied. Interestingly, both of the CsA LN formulations were able to decrease the lymphocyte count, reaching an earlier effect with LN containing Tw that was maintained up to 10 days. This observation may be attributed to the CsA concentration encountered in blood on day 5, which was significantly higher when LN Tw-CsA was given. Also, the absorption pathway could be involved in the
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pharmacological activity since no correlation between CsA levels and effect was observed for the LN Lec:TC-CsA formulation and NeoralÒ. When LN are orally administered, the particles are transported by the M-cells through the mesenteric lymphatic system before going through the general circulation [31] and probably they accumulate in lymphoid organs causing a prompt response. A better in vitro inhibitory effect on T-cell proliferation of negative surface charged CsA delivery system has been reported compared to neutral and positive ones, which was attributed to the higher affinity of the negatively charge carriers for these cells [32]. This is another explanation for the favorable pharmacological effect observed with the LN formulations compared to the reference formulation, which has a surface charge near neutrality [11]. Moreover, although thymus and spleen of CsA treated animals were histologically unaltered, they presented higher relative weights compared to control animals. This fact was considered a sign of immunosuppression as these are specialized lymphoid organs associated with the immune response. Along with this, lymphoid organs are rich in cyclophilin, which is the main protein involved in the mechanism of T-cell inhibition produced by CsA exposure [33]. Regarding CsA blood levels, drug concentrations obtained on day 5 in mice daily treated with LN formulations at 15 mg/kg of CsA remained similar to those achieved on day 10, indicating more predictable pharmacokinetic information that might facilitate the dose adjustment. As mentioned, nanoparticles can be taken up from the bloodstream by phagocytes and accumulated in the organs of the mononuclear phagocyte system such as the spleen and liver [34]. Targeting the drug to the spleen, as the major lymphoid organ, might contribute to the immunosuppressive effect. This could be the reason why CsA LN yield to an early response with more predictable drug blood levels than the reference formulation. When NeoralÒ was administered, the CsA blood levels significantly rose over time, increasing the risk of toxicity if the drug is not carefully monitored, as is well known in human clinical practice. Considering that LN promote drug transport to the general circulation by the intestinal lymphatic system, by avoiding the P-glycoprotein efflux and the presystemic metabolism [35], more consistent and reproducible bioavailability of the drug is expected. In this regard, CsA LN could offer the advantage of diminishing inter-individual variability in drug absorption, which is one of the issues presented by this immunosuppressant when it is orally administered in marketed formulations. However, further studies are needed to confirm this hypothesis. Nephrotoxicity is the major side effect of CsA and is characterized by functional and structural abnormalities, CREA and BUN levels being indicators of renal function. Therefore, these parameters are carefully monitored in patients receiving CsA to prevent renal damage. In the present study, no alteration in CREA and BUN concentrations was observed across the CsA treated groups. However, histological alterations of the kidneys were observed, which were more severe in the groups treated with NeoralÒ and LN containing Lec:TC (Fig. 5). The renal injury was characterized by a severe tubulonephrosis along with abnormalities in the convoluted tubules and glomerular hypercellularity. Interestingly, these observations were less apparent in mice receiving LN containing Tw, suggesting the protective action of this delivery system against CsA-related renal damage. One hypothesis that explains this result might be associated with the way in which the drug reaches the renal system. Due to the fact that Lec:TC are degraded faster by digestive enzymes and the LN containing these surfactants lose their physical properties at gastric and intestinal pHs [21], larger quantities of drug are released than that from LN containing Tw. As a consequence, most of the drugs reach the targeted tissues in its free form, as is the case with liquid lipid systems (i.e. microemulsion). Indeed, comparable renal damage outcomes were
obtained in the mice treated with LN Lec:TC-CsA and NeoralÒ. On the other hand, LN containing Tw have been shown to be more resistant to the gastrointestinal environment (pH changes, digestive enzymes, etc.) attributed to the surfactant (Tw), which is degraded more slowly due to the stearic hindrance, leading to the improved CsA bioavailability [21]. In this context, probably part of the drug reaches the target in its entrapped form, which minimizes the formation of metabolites associated with nephrotoxicity so that the renal injury is reduced. All in all, the nephroprotective behavior obtained with LN TwCsA along with the benefits observed with both LN systems in terms of the pharmacodynamic effect and the lower fluctuations of CsA blood levels (Figs. 3 and 4, respectively) leads us to suggest that these CsA delivery systems are a promising strategy to overcome toxicity issues in long-term treatments with this immunosuppressant.
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5. Conclusion
448
The CsA LN formulations developed exhibited relevant advantages over the reference formulation, NeoralÒ. First, the decrease in lymphocyte count as an indicator of immunosuppression obtained with LN was not observed after NeoralÒ administration. In addition, lower fluctuations in CsA blood levels were obtained, leading to more predictable pharmacokinetic information, and therefore facilitating dose adjustments to reduce the risk of toxicity. Moreover, LN contain Tw diminished CsA-related nephrotoxicity despite their enhanced bioavailability, making it possible to extend the therapeutic window.
449
Acknowledgment
459
This work has been carried out in the framework of the COST Action TD1004. M. Guada is grateful to ‘‘Asociación de Amigos de la Universidad de Navarra” for the fellowship grant.
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