Comparative Biodistribution and Pharmacokinetic Analysis of

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Article pubs.acs.org/molecularpharmaceutics

Comparative Biodistribution and Pharmacokinetic Analysis of Cyclosporine‑A in the Brain upon Intranasal or Intravenous Administration in an Oil-in-Water Nanoemulsion Formulation Sunita Yadav,†,‡ Florence Gattacceca,§ Riccardo Panicucci,‡ and Mansoor M. Amiji*,† †

Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University, Boston, Massachusetts 02115, United States Novartis Institute of Biomedical Research, Cambridge, Massachusetts 02142, United States § IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Montpellier University, Montpellier F-34298, France ‡

ABSTRACT: The main objective of this study was to evaluate comparative biodistribution and pharmacokinetics of cyclosporine-A (CsA) following intranasal (IN) administration versus intravenous (IV) administration in Sprague−Dawley rats using an oil-in-water nanoemulsion delivery system. CsA, a hydrophobic peptide that is also a substrate for P-glycoprotein, is a well-known immunosuppressive agent. In the brain, CsA has been shown to be a potent anti-inflammatory and neuroprotective agent. CsA nanoemulsions (CsA-NE) and solution formulations (CsA-S) were prepared using an ultrasonication method and were characterized for drug content, encapsulation efficiency, globule size, and zeta potential. We compared the uptake of CsA-NE and CsA-S in brain regions and peripheral organs following IN and IV administration using LC-MS/MS based bioanalytical method. CsA-NE IN resulted in the highest accumulation compared to that with any other treatment and route of administration; this was consistent for all three regions of brain that were evaluated (olfactory bulbs, mid brain, and hind brain). The brain/blood exposure ratios of 4.49, 0.01, 0.33, and 0.03 for CsA-NE (IN), CsA-NE (IV), CsA-S (IN), and CsA-S (IV), respectively, indicated that CsA-NE is capable of direct nose-to-brain transport, bypassing the blood− brain barrier. Furthermore, CsA-NE administration reduces nontarget organ exposure. These studies show that IN delivery of CsA-NE is an effective way of brain targeting compared to that of other treatment strategies. This approach not only enhances the brain concentration of the peptide but also significantly limits peripheral exposure and the potential for off-target toxicity. KEYWORDS: cyclosporine-A, intranasal administration, intravenous administration, oil-in-water nanoemulsion, brain delivery, pharmacokinetics

1. INTRODUCTION The incidence of brain disorders, and especially chronic agerelated neurodegenerative diseases, is increasing rapidly in the United States and around the world. Diseases of the central nervous system (CNS), such as Alzheimer’s and Parkinson’s diseases, require specific delivery of drugs into the brain for effective treatment. Disease-modifying treatment options are extremely challenging due to the blood brain−barrier’s (BBB) ability to limit entry of small, nonpolar compounds.1 Cyclosporine-A (CsA) is an 11 amino acid peptide originally isolated from the fungus Tolypocladium inflatum, that was initially discovered as an immunosuppressive agent by Borel and his team.2 Although the protective effects of CsA were first discovered in T-lymphocytes, brain has an approximate 20-fold greater tropism for CsA compared to that of T-cells.3 The benefits of oral CsA as a neuroprotective agent are achieved only with a very high dose (i.e., >10 mg/kg) and chronic administration of the drug. At such a high dosage and upon chronic administration, systemic CsA levels produce limiting © XXXX American Chemical Society

negative side effects, such as immune suppression, hepatotoxicity, and nephrotoxicity. As such, the use of oral or systemically administered CsA as a potential neurotherapeutic has not been considered so far. Delivery of CsA to the brain has also been attempted through other local delivery strategies, such as intracerebroventricular or intracranial delivery with a mini-pump or catheter infusion or by bolus injection.4 These routes of administration are highly invasive and will not be practical for drugs that need to be given on a chronic basis. The discovery of these obstacles in applying CsA as a therapeutic imposed by various protective mechanisms in brain, such as tight junctions of the BBB and the P-glycoprotein efflux transporter, has increased interest in developing alternative noninvasive strategies to overcome these Received: December 15, 2014 Revised: March 13, 2015 Accepted: March 18, 2015

A

DOI: 10.1021/mp5008376 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

2. MATERIALS AND METHODS 2.1. Materials. High omega-3 fatty acid-containing extra pure flax-seed oil was kindly provided by Jedwards International (Quincy, MA). Lipoid E80 was purchased from Lipoid GMBH (Ludwigshafen, Germany). Tween 80 and stearylamine were purchased from Sigma Chemicals, Inc. (St. Louis, MO). The Strata-X-CW 33 μm polymeric weak cation 30 mg/mL solid phase extraction (SPE) columns were purchased from Phenomenex, Inc. (Torrance, CA). Solvents were purchased from Fisher Scientific (Fair Lawn, NJ). The LC-MS/MS analysis of CsA extracted from biological samples was carried out using an AB SCIEX’s API-5000 triple quad instrument at PhenoLogix, a division of Phenomenex, Inc. (Torrance, CA). Ascomycin, used as an internal standard, was purchased from Sigma-Aldrich (St. Louis, MO). CsA was purchased from Tocris Biosciences (Minneapolis, MN). 2.2. Preparation and Characterization of Nanoemulsion Formulations. The oil-in-water nanoemulsion formulations of CsA for IN and IV administrations were prepared by an ultrasonication method as described previously.15 Briefly, a prewarmed oil phase (1 mL) consisting of flax-seed oil and 125 mg of CsA dissolved in ethanol was gradually added to the prewarmed aqueous phase (4 mL) containing 120 mg of egg phosphatidylcholine (Lipoid E80), 10 mg of polysorbate 80 (Tween 80), and 10 mg of stearylamine. The resultant mixture was stirred for 2 min using a Silverson homogenizer (model no. L4RT-A, Silverson Machines, Dartmouth, MA) at 6000 rpm and ultrasonicated for 10 min using a Vibra Cell VC 505 probe sonicator (Sonics and Material Inc., Newtown, CT) at 22% amplitude and 50% duty cycle. Blank nanoemulsions were prepared similarly without the addition of CsA. The morphology of the oil droplets in the nanoemulsion formulations was visualized by transmission electron microscopy (TEM) analysis. The nanoemulsion was placed on Formvar-coated copper grids (EM Sciences, Hatfield, PA, USA) and negatively stained at room temperature with 50 μL of 1.5% (w/v) phosphotungstic dye for 10 min. Excess liquid was drained off with Whatman filter paper, and the grid containing the dry film of nanoemulsion sample was observed with an EOL 100-X transmission electron microscope (Peabody, MA). CsA aqueous solution was prepared as a control using a similar composition as the nanoemulsions except for the inclusion of flax-seed oil. The aqueous suspension was prepared by mixing the CsA in ethanol first and adding it to the aqueous phase containing egg phosphatidylcholine (Lipoid E80), Tween 80, and stearylamine at the same proportions as for the nanoemulsion formulations above. Ethanol in the solution was evaporated by bubbling nitrogen gas. Formulations were stored at 4 °C until use. Blank CsA-encapsulated nanoemulsion formulation and CsA solution were characterized for particle size and surface charge, with use of dynamic light scattering, on Brookhaven Instrument’s 90 Plus ZetaPALS particle size analyzer (Holtsville, NY) at a 90° fixed angle and at 25 °C. The formulation was analyzed for the extent of drug loading (i.e., the amount of peptide incorporated in the nanoemulsion) and encapsulation efficiency (i.e., the amount of peptide present in the internal or oil phase of the nanoemulsion) by high-performance liquid chromatography (HPLC). For loading, CsA-nanoemulsion was diluted with 100% methanol, and 50 μL of the dissolved nanoemulsion was injected on HPLC. Mobile phase A, consisting of 1% trifluoroacetic acid (TFA) in water,

hurdles for utilizing the potential of CsA for various brain disorders. Over the past several years, the intranasal (IN) route of therapeutic administration has received significant attention as a potential noninvasive alternative approach for the delivery of biological therapeutics to the brain,5−7 utilizing pathways along olfactory and trigeminal nerves innervating the nasal passages. While the exact mechanism of drug transport via intranasal to CNS is not entirely understood, an accumulating body of evidence demonstrates that one or multiple of the following pathways might be involved: pathway involving nerves in nasal passages to the brain, vascular pathways, CSF, or lymphatic system distribution.8 Although the IN route affords potential of direct delivery to brain, the drug needs to pass the nasal epithelial membrane barrier, which suffers from challenges including enzymatic degradation, tight junctions, and mucociliary clearance, for the delivery of biological therapeutics.9 Indeed, the IN bioavailability of large molecular weight drugs (e.g., peptides, proteins) transported directly from nose to brain tends to be low (∼1−2% maximum). Important factors in enhancing the bioavailability of therapeutics for CNS diseases include alterations in the physicochemical properties of the therapeutic agents, improving nasal residence time, mucosal and epithelial permeability, efficiency of transport, and subsequent specificity of therapeutic delivery in the brain upon IN administration.10 Encapsulation of the active agent in a nanoparticle-based delivery vehicle can serve as a very powerful strategic approach to affect nose-tobrain transport. For example, when ovalbumin or glial-cell derived neurotrophic factor was encapsulated in cationic liposomes, there was an increase in stability of these proteins as well as improved efficiency of delivery in the brain as compared to that with the same dose administered in aqueous solution.11 Over the past several years, we have shown that oil-in-water nanoemulsions made with omega-3 polyunsaturated fatty acid (PUFA) rich edible oils can significantly influence BBB penetration and availability of small molecules (e.g., paclitaxel and saquinavir) as well as peptides (e.g., lipid-modified DALDA opioid analgesic peptide) upon systemic administration in rodent models. We hypothesized that encapsulation of these molecules in the nanoemulsion would enhance circulation halflife by reducing rapid clearance from the systemic circulation and that the presence of omega-3 PUFA would facilitate BBB permeability.12−14 In this study, we have investigated comparative biodistribution, pharmacokinetics, and bioavailability of CsA when administered by either an intranasal (IN) or intravenous (IV) route in Sprague−Dawley rats in flax-seed oil-containing nanoemulsion formulations. Our hypothesis was that IN administration would not only enhance CsA availability in the brain but also restrict systemic exposure and toxicity. Two important experimental questions were considered in the study. First, does IN CsA administered in flax-seed oil nanoemulsions achieve high enough concentrations in the brain for potential therapeutic utility as compared to that using an IV administered formulation? Second, in comparison with IV dosing, can IN administered CsA limit systemic exposure to liver, kidney, and other organs and potentially offer a safer therapeutic strategy? As such, IN administered CsA formulated in the nanoemulsion could be a repurposed therapeutic, specifically for the treatment of neuroinflammation associated with neurodegenerative diseases. B

DOI: 10.1021/mp5008376 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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bicarbonate (pH 8.6) and using ascomycin as an internal standard. Columns were washed with 50 mM ammonium bicarbonate, followed by washing with 50% aqueous methanol. After speed drying the columns, analyte was eluted with 2 volumes of methanol. Samples were dried using speed vacuum, reconstituted with buffer, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described below. 2.7. Extraction of CsA from Whole Blood Samples. Aliquots of whole blood (200 μL) were combined with 50 μL of internal standard (200 ng/mL ascomycin in 50:50 MeOH/ H2O) and 200 μL of 2.5% ZnSO4 in distilled water. After briefly vortexing samples, 300 μL of 100% methanol was added, and samples were again vortexed. The samples were centrifuged at 14 000 rpm for 5 min to remove cell debris, and the entire supernatant was loaded onto the cation exchange SPE column. The SPE method for whole blood samples was similar to the brain samples, except equilibration was performed with distilled water and washing was performed using water first followed by 50% methanol. Samples were dried using a high speed vacuum, reconstituted with buffer, and analyzed with LC-MS/MS. 2.8. Extraction of CsA from Peripheral Tissues. Liver, spleen, kidney, heart, and lung tissues were homogenized in saline, and 200 μL aliquots were used to perform SPE using a similar process as that used for the whole blood sample preparation. Samples were eluted with 100% methanol twice followed by drying using solvent evaporator before analyzing with LC-MS/MS. 2.9. LC-MS/MS Analysis of CsA. A CsA stock solution of 1 mg/mL was prepared in methanol. For a standard curve, brain, blood, and tissue samples of untreated animals were individually combined with 50 μL of different concentration of CsA. Ascomycin at 200 ng/mL in 50:50 methanol was used as an internal standard. Calibration and test samples were processed through an SPE column and finally analyzed using an AB SCIEX’s API-5000 LC-MS/MS triple quad instrument at Phenomenex (Torrence, CA). Standard curves were prepared using the peak areas obtained by monitoring the mass transitions m/z from 1220.1 to 1202.9. The regression analyses for all standard curves showed correlation coefficient (r) values of greater than 0.99. The API-5000 LC-MS/MS instrument provided a linear standard curve in the CsA concentration range of 0.1−50 ng/mL for brain tissues samples and 1−200 ng/mL for whole blood samples, and for other peripheral tissues, a linearity range of 16−4000 ng/mL was used for quantitation. From the n = 4 animals for each time point and per group, outlier concentrations were identified as being 10 times higher or lower than the geometric mean of the remaining three concentrations. Outliers were removed for data presentation and analysis. 2.10. Quantitative Pharmacokinetic Analysis. Noncompartmental pharmacokinetic analysis of CsA concentrations in brain, whole blood, and in different tissues as a function of time was carried out using Certara’s Phoenix WinNonlin, version 1.3, software (St. Louis, MO). Area-under-the-curve from time zero to the last collection time (i.e., AUC0−last) and the associated standard error of the mean (SEM) were calculated using the linear trapezoidal method. Brain targeting efficiency following CsA administration in nanoemulsion versus aqueous solution and upon IN versus IV administration was calculated as the mean brain to blood exposure ratio over the study time period.

and mobile phase B, as 1% TFA in acetonitrile, were pumped through an Agilent Zorbax 300SB-C18 column (C18, particle size 3.5 μm, 4.6 mm × 100 mm) at a flow rate of 1 mL/min. The gradient was 30% B to 100% B in 8 min, and peptide elution was monitored at a wavelength of 215 nm. For encapsulation efficiency, formulations were first diluted 100 times; of this, 0.5 mL was transferred to poly(vinylidene difluoride) (PVDF) ultrafree centrifugal filter units having 0.1 μm pore size (UFC40VV25, Millipore, Bedford, MA) and centrifuged at 5000g for 15 min at 4 °C. The encapsulated drug remains in the donor chamber, and the aqueous phase moves through the filter into the sample recovery chamber. The aqueous phase was injected on the HPLC, and the concentration of the peptide in the aqueous phase was estimated; encapsulation efficiency was calculated based on mass balance. 2.3. Experimental Animal Model. Adult female Sprague− Dawley rats, weighing between 190 and 230 g, were purchased from Charles River Laboratories (Cambridge, MA) and housed under a 12 h light/dark cycle with food and water provided ad libitum. The experimental animals were cared for in accordance with institutional guidelines, and they were allowed to acclimate for at least 48 h prior to initiation of any experiments. All the experiments with Sprague−Dawley rats described in this publication were approved by the Northeastern University’s Institutional Animal Care and Use Committee. 2.4. Intranasal and Intravenous Formulation Administration. For the IN administration, the rats (n = 4) were first anesthetized with a mixture of ketamine/xylazine, at a dose of 80 and 20 mg/kg, respectively, and were placed in a supine position with their nose at an upright 90° angle. A total of 45− 50 μL/rat (5 μL/nostril) of the nanoemulsion and 80−85 μL/ rat of CsA solution was administered to rats using a micropette (Eppendorf P-20), with a hold time of 2 min between each dose. The drop was placed at the opening, allowing the animal to snort the drop into the nasal cavity. The total CsA dose administered to each rat was 5 mg/kg. For the IV administration, rats (n = 4) were briefly anesthetized using isoflurane inhalation, and 500 μL of the diluted nanoemulsion or CsA solution formulations was administered slowly through the caudal vein of the rat tail using a 1 mL syringe. Similar to the IN administration, the total CsA dose administered to each rat was 5 mg/kg. 2.5. Blood, Brain, and Other Tissue Harvesting. Experimental animals were euthanized at predetermined time points of 30 min and 1, 2, 4, and 6 h postdosing, and blood was collected using a 3 mL syringe through cardiac puncture. Whole brain, liver, heart, kidney, spleen, and lung were quickly isolated and were washed with ice cold 1× sterile phosphate buffer saline, pH 7.4. Samples were stored at −80 °C until further analysis. 2.6. Extraction of CsA from Brain Tissue Samples. The frozen whole brain from each animal was dissected into three parts, olfactory bulb, mid brain, and hind brain regions, to study uptake from rostral to caudal regions of the tissue. The separate tissue samples were rinsed with sterile saline to remove surface blood and then homogenized in saline using a tissue homogenizer. Two-hundred microliters of the tissue homogenate was used for the extraction using Strata-X-CW 33 μm polymeric weak cation exchange column. Columns were preconditioned with 100% methanol and equilibrated with 50 mM ammonium bicarbonate, and samples were loaded on the column after premixing with 3 volumes of 50 mM ammonium C

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Molecular Pharmaceutics Table 1. Physicochemical Characterization of CsA Formulationsa

a

formulations

hydrodynamic diameter (nm)

PDI

zeta potential (mV)

% encapsulation

CsA loading conc. (mg/mL)

blank nanoemulsion CsA-nanoemulsion

260 ± 20 272 ± 12

0.38 ± 0.1 0.3 ± 0.09

−41 ± 8 57 ± 10

88 ± 13

25

Each value represents the mean standard deviation (SD); n = 3.

Figure 1. Transmission electron microscopy (TEM) of the nanoemulsion. The oil droplets of the nanoemulsion sample were spherical, and their size was in the range of 100−220 nm. Scale bar is 100 nm.

2.11. Statistical Analysis. For each control and test group, a total of four animals were used per group and time point. Quantitative results were compared using ANOVA followed by Bonferroni’s post-test. The difference was considered significant for p < 0.05.

NE-CsA has a positive surface charge, whereas the CsA solution was found to be negative due to a lack of any surface morphology (Table 1). Furthermore, TEM of the NE-CsA formulation confirmed its spherical shape and a size range of 100−220 nm (Figure 1). 3.2. CsA Blood Kinetics Following IN and IV Administration of Solution and Nanoemulsion Formulations. For both formulations, IN administration of CsA resulted in significantly lower concentrations in the blood at all assessed time points compared to that from IV administration of CsA (Figure 2a,b). CsA blood levels were significantly lower when comparing CsA-NE delivered IN to CsA-S delivered IV at 4 h (p < 0.01) and when comparing CsA-S IN to CsA-S IV at 1 h (p < 0.05), 2 h (p < 0.05), and 4 h (p < 0.01) time points. After IN administration of both formulations (Figure 2a), slow steady blood levels of CsA were obtained, with no clear decreasing trend over the study period. For CsA-NE, blood concentrations even increased from 45 ng/mL at 30 min to 114 ng/mL 6 h postadministration. For CsA delivered as a solution (CsA-S), after a marked decrease between 30 and 60 min, CsA

3. RESULTS 3.1. Nanoemulsion Formulation and Characterization. The final composition that showed the best particle size distribution for nanoemulsion was found to be 2.4% w/v lipoid E80, 0.2% w/v Tween 80, 0.2% w/v stearylamine, and 20% w/v flax-seed oil. CsA nanoemulsion formulations prepared by the sonication method resulted in a uniform milky white emulsion with a total concentration of 25 mg/mL and an 88 ± 13% encapsulation efficiency obtained from HPLC. The maximum loading of CsA in the solution was found to be 12 mg/mL, with 64 ± 19% encapsulation. Particle size analysis for NE-CsA revealed that they were smaller in diameter (i.e., average = 272 nm) compared to that of particles in CsA solution (average = 366 nm). Surface charge (zeta potential) analysis showed that D

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Figure 2. Mean (±SD) blood concentration−time curves of cyclosporine-A (CsA) in Sprague−Dawley rats after intranasal (A; IN) or intravenous (B; IV) administration of CsA-nanoemulsion (CsA-NE) or CsA-solution (CsA-S) at a dose of 5 mg/kg. Data represents the mean of n = 4, with *p < 0.05 or **p < 0.01 showing significantly different groups − CsA NE IV to CsA-S IV (p < 0.01); CsA-S IN and CsA IV 1 h, 2 h (p < 0.05); CsA IN and CsA IV 4 h (p < 0.01).

Figure 3. Area-under-the-curve (AUC) values calculated from mean cyclosporine-A (CsA) concentration in blood and different regions of brain after administration of CsA-nanoemulsion (CsA-NE) or CsA-solution (CsA-S) via the intranasal (IN) or intravenous (IV) route. Data represents the mean (±SD) of n = 3−4, with *p < 0.05 or **p < 0.01 showing significantly different groups compared to various control groups. OB, olfactory bulb; MB, mid brain; HB, hind brain.

reduced blood exposure over 6 h when compared to S, from 25 055 to 19 347 ng/mL·min by the IN route. Differences in AUClast were significant (p < 0.01) when comparing either CsA-NE IN or CsA-S IN to CsA-S IV. Altogether, IN delivery and NE formulation of CsA both contributed to decreased blood CsA concentrations. Blood PK was also explored. Due to a huge interindividual variability, no typical PK profile could be obtained: for example, after IV administration of CsA-NE or CsA-S, the kinetics started with an increase in concentration, which is theoretically impossible. Consequently, no usual PK parameters (clearance, mean residence time, or distribution volume) were calculated, and the conclusions were drawn from global exposure data, as determined by AUClast calculation.

blood concentrations remained stable throughout the study period. Differences observed between CsA-NE and CsA-S for CsA blood levels at each time point after IN delivery were not statistically significant. IV administration resulted in higher blood concentrations for CsA-S than for CsA-NE (Figure 2b). However, concentrations markedly decreased between 240 and 360 min for both formulations, but the decrease was slower for CsA-NE, leading to a higher CsA blood concentration for CsANE at the last, 6 h, time point. This suggests a longer residence time of CsA when delivered as nanoemulsions, after IV administration. Global exposure over time was assessed by calculating AUClast as described. Lower exposure was confirmed for IN versus IV administration, with AUClast being 7-fold lower for NE and 10-fold lower for S (Figure 3). NE delivery also E

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Figure 4. Mean (±SD) concentration−time curves of cyclosporine-A (CsA) in different regions of rat brain after intranasal (IN) or intravenous (IV) administration of CsA-nanoemulsion (CsA-NE) or CsA-solution (CsA-S) at a dose of 5 mg/kg.

Figure 5. Mean ng/g brain concentration−time plot of cyclosporine-A (CsA) in rats after intranasal (IN) or intravenous (IV) administration of CsAnanoemulsion (CsA-NE) or CsA-solution (CsA-S) at a dose of 5 mg/kg.

3.3. CsA Brain Distribution Following IN and IV Administration of Solution and Nanoemulsion Formulations. Following intranasal administration of CsA-NE, the maximum concentrations obtained in the olfactory bulb, mid brain, and hind brain were similar, in the range of 200−300 ng/ g of tissue (Figure 4). However, the kinetics was different in the three brain parts. The peak concentration was obtained in olfactory bulbs after 1 h, versus 4 h in mid brain, suggesting a rostral to caudal gradient over time. The trend in the hind brain was less clear, probably due to the huge interindividual variability. Although higher concentrations in each part of the

brain appeared to be reached using IN administration via nanoemulsions (CsA-NE IN), when compared with other conditions, the differences were not statistically significant due to high variability. Hence, whole brain concentrations were calculated based on the sum of amount of CsA in the three regions of the brain divided by the sum of the weights of the three brain subparts (Figure 5). As expected, NE-CsA delivered intranasally induced higher brain uptake compared to that from other strategies at all of the time points examined except for 30 min, when CsA-S IN displayed the highest brain concentration (Figure 4). However, F

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Figure 6. Brain-to-blood concentration ratio of cyclosporine-A (CsA) after administration of CsA-nanoemulsion (CsA-NE) or CsA-solution (CsAS) via the intranasal (IN) or intravenous (IV) route. Data represents the mean (±SD) of n = 3−4, with *p < 0.05 or *p < 0.01 compared to various control groups.

Figure 7. Comparison of brain targeting efficiency of intranasal (IN) and intravenous (IV) routes of delivery for both cyclosporine-A (CsA)nanoemulsion and CsA-solution. Data represents the mean (±SD) of n = 3−4, with *p < 0.05 or *p < 0.01 compared to various control groups.

observed interindividual variability in the concentrations measured, especially in brain subparts. Apart from the natural physiological interindividual variability, this variability might be introduced due to inaccurate IN delivery of the dose due to small volumes, nonhomogeneous withdrawal of biological samples (especially brain subparts), and the difficulty of accurately quantifying drug in a small sample, particularly in olfactory bulbs, which weigh around 100−200 mg. This issue made it challenging to obtain statistical significance, in some cases, when comparing groups. However, when comparing global exposure or ratios over time, it was possible to obtain statistically significant results, clearly demonstrating the benefit of NE IN administration of CsA over other strategies.

no significant differences could be obtained. Brain CsA concentrations after CsA-NE IN delivery increased from 29.0 ng/g at 30 min to 365 ng/g at 240 min and then decreased to 23.6 ng/g at 360 min, suggesting a maximum brain exposure between 120 and 360 min. When comparing global exposure over time between delivery conditions using the AUClast parameter (Figure 3), the higher exposure of each brain subpart after CsA-NE IN delivery was confirmed and was significant (p < 0.05) for CsA-NE IN versus CsA-NE IV in olfactory bulbs and for CsA-NE IN versus the three other conditions in mid brain (p < 0.05). Furthermore, whole brain AUClast was significantly higher for CsA-NE IN when compared to that from all other strategies (p < 0.01). We G

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Figure 8. Comparison of cyclosporine-A (CsA) distribution 4 h postadministration in peripheral tissues from IN and IV routes of delivery for both CsA-nanoemulsion and CsA-solution. Data represents the mean (±SD) of n = 4, with *p < 0.05 or **p < 0.01 showing significantly different groups compared to control groups.

3.4. CsA Brain Targeting Following IN versus IV Administration of Solution or Nanoemulsion Formulations. Intranasal administration of CsA-NE resulted in a high brain-to-blood concentration ratio, increasing from 1.03 to more than 10 from 30 to 240 min (Figure 6). Other strategies did not allow brain targeting; the brain-to-blood ratio remained under 1 at all time points, with the highest value of 0.713 obtained for CsA-S IN at 120 min. At 360 min, the brain-toblood ratio was very low for all strategies (≤0.103) due to a decrease in brain concentration, whereas the blood concentration remained stable. Considering the global brain targeting efficiency over 6 h, evaluated as the ratio AUCbrain/AUCblood, CsA-NE IN performed significantly better than other strategies (p < 0.001), with brain being 4.49 times more exposed than blood, whereas other strategies lead to higher blood rather than brain exposure (ratio ≤ 0.33) (Figure 7). 3.5. CsA Biodistribution in Highly Perfused Tissues Following IN versus IV Administration of Solution or Nanoemulsion Formulations. Examination of peripheral tissues for the distribution of CsA was performed to determine if the brain targeting obtained with CsA-NE IN was brainspecific or if nontargeted organs were also more exposed to the drug. IN administration resulted in overall considerably lower exposure of peripheral organs to CsA when compared to that from IV administration (Figure 8). The CsA-NE IN formulation further reduced nontargeted organ exposure, specifically for liver and kidney, compared to that with CsANE IV. Interestingly, the NE formulation induced lower concentrations versus solution in liver, kidney, and spleen after IV administration, whereas lung and heart appeared to have increased exposure.

which not only plays a key role in brain function specifically in cognitive and behavioral functions in the CNS, but also serves as a biocompatible excipient. Also, omega-3 fatty acids are known to be critical modulators of neuronal function and play a key role in regulation of neuroinflammatory and oxidative stress-mediated mechanisms in the normal CNS during aging and chronic neurological diseases.16 As volume is one of the biggest limitations for intranasal dosing, we were able to achieve as high as 25−30 mg/mL loading for CsA. Our findings from PK and distribution studies of CsA reveal that a higher quantity of CsA can be effectively delivered to the different regions of the brain by intranasal administration of CsA formulated as nanoemulsion. Overall, higher brain uptake of CsA was achieved when it was delivered intranasally compared to that when delivered intravenously. This is likely due to the fact that CsA, being a Pgp substrate, is unable to cross the BBB and when it is delivered through the intranasal route it is able to bypass the BBB. As nanoemulsion formulations showed further improved CsA brain delivery when compared to that using a solution, it can be assumed that nanoemulsions provide an advantage due to the longer residence time in the nasal mucosa and also can bypass efflux from the different Pgp transporters that are present in nasal mucosa. To the best of our knowledge, no studies have so far looked into the distribution of a therapeutic drug in subregions of the brain; most studies have been focused on the whole brain concentration, which might be misleading, as drug can get trapped in particular regions of the brain and an efficacious level might not be reached in the target region. In this study, we attempted to measure distribution in three regions of the brain using a sensitive LC-MS method for quantitative analysis and found that mid brain region, which is a potential site for many neuroinflammatory disseases, showed statistically significant higher exposure with NE-CsA delivered IN. The higher uptake observed in the mid brain region suggests that one of the routes of uptake for nanoparticles was through the olfactory epithelium and redistribution to the mid brain region through CSF.19 However, one cannot rule out uptake through the trigeminal pathway, as one portion of the trigeminal neural pathway enters the brain through the

4. DISCUSSION This study compared the intranasal route of delivery to the intravenous route of administration and compared a nanoemulsion formulation to a solution for attaining the targeted delivery of CsA to the brain. We developed CsA-encapsulated nanoemulsions composed of naturally occurring lipids and flaxseed oil. We used flax-seed oil (rich in omega-3 fatty acid) H

DOI: 10.1021/mp5008376 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

a consequence, mucin and the nanoemulsion, carrying an overall positive charge on the surface, demonstrate a strong electrostatic interaction in solution and hence the potential for a longer residence time. A further advantage of using small nanoparticulate systems is that they provide a larger surface area for the interaction between the peptide and the epithelial cells and hence uptake through the nasal mucosa will be enhanced. One of the biggest advantages that emerged from this study was the reduced systemic exposure, resulting in turn in reduced peripheral tissue exposure, due to CsA-NE administered intranasally. As discussed in the introduction section, to be able use CsA as a neuroprotective agent, there is a need for specific brain targeting. Delivery of nanoemulsions through the intranasal route resulted in the lowest exposure in highly perfused tissues such as the liver, kidney, and spleen; one could expect CsA nanoemulsions to have more specific effects in the brain and less nontarget toxicity. Multiple studies have been performed to evaluate the effect of CsA as a neuroprotective agent. 22−24 It has been demonstrated that the protective effects of CsA may be enhanced by mechanical disruption of the brain parenchyma.25,26 Another approach involves the administration of endogenous ligands or their analogues to increase the permeability of the BBB by activation of receptors on the endothelial cells that comprise it.27 Selective B2 bradykinin receptor agonist, cereport, has been shown to transiently increase the permeability of the BBB.28 Most of these studies were performed with high doses or with chronic use of CsA, and, to our surprise, none of the studies looked into the therapeutic levels reached via the peripheral route to examine whether a correlation exists between the levels and the pharmacodynamic effect. Moreover, such a high dosage and chronic injection of CsA are reported to produce negative side effects such as nephrotoxicity and hepatotoxicity, among others. In one study, Schinkel et al. studied the tissue distribution of CsA in double mdr1a knockout mice and compared the results to normal mice.29 The tissues levels of CsA in MDR-1a (+/+) and (−/−) mice 4 h after intravenous injection of CsA (1 mg/ kg) were found to be 10.5 ± 0.7 and 178 ± 10 ng/g, respectively. The levels obtained in whole brain after CsA NE delivery via the intranasal route at the 4 h time point were around 400 ng/g at 5 mg/kg, which is well above the reported levels in normal mice and close to the levels in double knockout mice. Furthermore, when comparing the levels obtained in our study to the EC50 values, we found that we are above the EC50 of CsA. It is been reported that EC50 values of CsA for IL2 inhibition in humans are around 277 ng/mL (including outliers) and 276 ng/mL (excluding outliers).30 In another paper, the EC50 for cyclosporine is reported to be 200 ng/mL based on calcineurin phosphatase activity.31 The 50% inhibitory concentration (IC50) of CsA for the cellular synthesis of various cytokines ranged from 20 to 60 ng/mL.32 These comparisons show that, although these are not brain target EC50 values, we could potentially expect therapeutic effects at the levels that we obtained in the brain using CsA NE delivered intranasally and potentially limit any unwanted side effects due to systemic exposure.

cribriform plate alongside the olfactory pathway, as elucidated by Frey et al.8,17 It has been shown that direct delivery through the olfactory and trigeminal nerve pathways is one of the major components of intranasal delivery, as evidenced by the fact that fluorescent tracers were found to be associated with olfactory nerves as they traverse the cribriform plate18 and drug concentrations in the olfactory bulbs and trigeminal nerves are generally among the highest CNS concentrations observed.17 This was experimentally shown in an IGF-1 study in which rapid delivery to multiple areas of the CNS along the olfactory and trigeminal pathways was achieved upon IN delivery.17 Involvement of multiple pathways makes it difficult to distinguish whether a higher distribution in rostral brain regions at later time points is due to the olfactory or trigeminal route. Although we cannot rule out drug transport through the CSF, CSF is not the only contributing factor for brain transport, as we observed higher amounts in mid brain regions after 1 h and it is known that the turnover rate for CSF in rats is much shorter, approximately 1 h.17 When addressing blood kinetics, we observed an unexpected pharmacokinetic profile after IV administration, with an increase in concentration instead of the expected decrease, until 240 min. Due to the high variability in the concentrations measured, there was actually no significant difference between concentrations obtained at different time points except between NE-IV at 60 and 240 min (p = 0.03). A similar unexpected profile was obtained in albino rats by Nakarani et al.,21 with an increase in blood concentrations in the first hours of the kinetics after IV administration of Sandimmune or a nanosuspension. Regarding the elimination half-life of CsA in rats, which varies between 2 and 20 h depending on the study design,20,21 our 6 h study did not allow us to characterize the blood pharmacokinetic profile of CsA: the concentrations do not vary much between 0 and 6 h and the variation with time is in the same range as the measurement’s variability. Therefore, we assumed that the evolution of the concentration with time is not significant and cannot be analyzed using a pharmacokinetic model but that the experiments are still informative with regard to global exposure over time. Hence, these global exposure data (AUC) allowed us to find significant differences and to conclude that the brain targeting efficiency is improved using the NE-IN strategy. We found that intranasal delivery induced lower exposure in blood compared to that from intravenous administration at all time points investigated with both CsAnanoemulsions and CsA-solution. This is a great advantage due to the fact that lower blood exposure will lead to lower offtarget effects. For a molecule with a potentially narrow therapeutic window, such as CsA, widespread immunosuppression could be avoided and a more specific brain targeted effect could be expected. Our other hypothesis for the targeted delivery of CsA was that encapsulation of the peptide in a nanoparticulate system will lead to a higher residence time in the nasal mucosa and hence would provide an opportunity for sustained brain availability. Our data showed brain uptake at later time points using the nanoemulsion formulation when dosed intranasally compared to that from an equivalent dose of CsA in solution. This could also be due to the fact that the positive charges on the nanoemulsion can bind strongly to negatively charged materials such as cell surfaces and mucus. It is known that mucus contains mucins that have different chemical compositions, but some contain significant proportions of sialic acid. At physiological pH, sialic acid carries a net negative charge and, as

5. CONCLUSIONS Comparative biodistribution, pharmacokinetic, and brain targeting efficiency upon IN and IV CsA administration in flax-seed oil-containing nanoemulsions and aqueous solution I

DOI: 10.1021/mp5008376 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

(7) Frey, W. H. Neurologic agents for nasal administration to the brain. Patents EP 0504263 B1, 1997. (8) Dhuria, S. V.; Hanson, L. R.; Frey, W. H., II Intranasal delivery to the central nervous system: mechanisms and experimental considerations. J. Pharm. Sci. 2010, 99, 1654−1673. (9) Graff, C. L.; Pollack, G. M. Functional evidence for Pglycoprotein at the nose−brain barrier. Pharm. Res. 2005, 22, 86−93. (10) Vyas, T. K.; Tiwari, S. B.; Amiji, M. M. Formulation and physiological factors influencing CNS delivery upon intranasal administration. Crit. Rev. Ther. Drug Carrier Syst. 2006, 23, 319−347. (11) Migliore, M. M.; Vyas, T. K.; Campbell, R. B.; Amiji, M. M.; Waszczak, B. L. Brain delivery of proteins by the intranasal route of administration: a comparison of cationic liposomes versus aqueous solution formulations. J. Pharm. Sci. 2010, 99, 1745−1761. (12) Vyas, T. K.; Shahiwala, A.; Amiji, M. M. Improved oral bioavailability and brain transport of Saquinavir upon administration in novel nanoemulsion formulations. Int. J. Pharm. Sci. 2008, 347, 93− 101. (13) Shah, L.; Gattacceca, F.; Amiji, M. CNS delivery and pharmacokinetic evaluations of DALDA analgesic peptide analog administered in nano-sized oil-in-water emulsion formulation. Pharm. Res. 2014, 31, 1315−1324. (14) Tiwari, S. B.; Amiji, M. M. Improved oral delivery of paclitaxel following administration in nanoemulsion formulations. J. Nanosci. Nanotechnol. 2006, 6, 3215−3221. (15) Sandip, T.; Yi-Meng, T.; Mansoor, A. Preparation and in vitro characterization of multifunctional nanoemulsions for simultaneous MR imaging and targeted drug dleivery. J. Biomed. Nanotechnol. 2006, 2, 217−224. (16) Farroqui, A. Recent development on the neurochemistry of docosanoids. Lipid Mediators and Their Metabolism in the Brain; Springer: New York, 2011; Chapter 2, Vol. 49. (17) Thorne, R. G.; Pronk, G. J.; Padmanabhan, V.; Frey, W. H., II Delivery of insulin-like growth factor-I to the rat brain and spinal cord along olfactory and trigeminal pathways following intranasal administration. Neuroscience 2004, 127, 481−496. (18) Jansson, B.; Bjork, E. Visualization of in vivo olfactory uptake and transfer using fluorescein dextran. J. Drug Targeting 2002, 10, 379−386. (19) Davson, H.; Segal, M. B. The return of the cerebrospinal fluid to the blood: the drainage mechanism. Physiology of the CSF and BloodBrain Barriers; CRC Press: Boca Raton, FL, 1996. (20) Guan, P.; Lu, Y.; Qi, J.; Niu, M.; Lian, R.; Hu, F.; Wu, W. Enhanced oral bioavailability of cyclosporine A by liposomes containing a bile salt. Int. J. Nanomed. 2011, 6, 965−974. (21) Nakarani, M.; Patel, P.; Patel, J.; Patel, P.; Murthy, R. S.; Vaghani, S. S. Cyclosporine A-nanosuspension: formulation, characterization and in vivo comparison with a marketed formulation. Sci. Pharm. 2010, 78, 345−361. (22) Osman, M. M.; Lulic, D.; Glover, L.; Stahl, C. E.; Lau, T.; van, L. H.; Borlongan, C. V. Cyclosporine-A as a neuroprotective agent against stroke: its translation from laboratory research to clinical application. Neuropeptides 2011, 45, 359−368. (23) Borlongan, C. V.; Stahl, C. E.; Keep, M. F.; Elmér, E.; Watanabe, S. Cyclosporine-A enhances choline acetyltransferase immunoreactivity in the septal region of adult rats. Neurosci. Lett. 2000, 279, 73−76. (24) Kumar, P.; Kumar, A. Neuroprotective effect of cyclosporine and FK506 against 3-nitropropionic acid induced cognitive dysfunction and glutathione redox in rat: possible role of nitric oxide. Neurosci. Res. 2009, 63, 302−314. (25) Borlongan, C. V.; Fujisaki, T.; Watanabe, S. Chronic cyclosporine-A injection in rats with damaged blood−brain barrier does not impair retention of passive avoidance. Neurosci. Res. 1998, 32, 195−200. (26) Hayashi, T.; Kaneko, Y.; Yu, S.; Bae, E.; Stahl, C. E.; Kawase, T.; van Loveren, H.; Sanberg, P. R.; Borlongan, C. V. Quantitative analyses of matrix metalloproteinase activity after traumatic brain injury in adult rats. Brain Res. 2009, 1280, 172−177.

formulations were examined in Sprague−Dawley rats. This study demonstrates that CsA, as a PgP substrate, was not very efficiently transported into the brain through the BBB upon IV administration even in nanoemulsion formulations. IN administration of CsA, especially in a nanoemulsion formulation, performed significantly better than that using an aqueous solution. In addition, the IN administered nanoemulsion limited systemic exposure of CsA to blood and peripheral tissues, thus possessing significant potential to reduce off-target effects. In this study, we have demonstrated significant advantages of using CsA nanoemulsions upon IN administration for targeting the brain, which opens the possibility of using this peptide as a potential neuroprotective and therapeutic agent in the treatment of neuroinflammation. There is yet another advantage of decorating these nanoparticles with various targeting molecules to aim specifically at the olfactory epithelium to further enhance the nose-to-brain delivery and avoid uptake through the respiratory epithelium and eventually limit access into the vascular system. Although the formulations prepared constituted naturally occurring lipids and flax-seed oil, further evaluation of the safety of the delivery vehicle will need to be performed. The results suggests that IN delivery of nanoemulsion systems can act as a viable targeting strategy for enhanced delivery of potent biological therapeutics to multiple regions in the brain and hence provides an important therapeutic opportunity for CNS diseases.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (617) 373-3137; Fax: (617) 373-8886; E-mail: m.amiji@ neu.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was partially supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health through grant R21-NS066984. We also deeply appreciate the assistance of various individuals at Phenomenex, Inc. in Torrence, CA, in the development of cyclosporine-A LC/MS/MS analytical methods used for this study and subsequent discussions we had regarding the analysis and results. We would like to thank Srujan Kumar for his assistance with in vivo studies. We also deeply appreciate the assistance of Dr. Lara Milane with the critical review of the manuscript.

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REFERENCES

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DOI: 10.1021/mp5008376 Mol. Pharmaceutics XXXX, XXX, XXX−XXX