Dual release behavior of atorvastatin and alpha-lipoic acid from PLGA microspheres for the combination therapy in peripheral nerve injury

Accepted Manuscript Dual release behavior of atorvastatin and alpha-lipoic acid from PLGA microspheres for the combination therapy in peripheral nerve injury Hakan Eroğlu, Assoc. Prof., Mohammad Karim Haidar, Emirhan Nemutlu, Şükrü Öztürk, Cem Bayram, Kezban Ulubayram, Levent Öner PII:

S1773-2247(17)30224-1

DOI:

10.1016/j.jddst.2017.04.028

Reference:

JDDST 366

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 16 March 2017 Revised Date:

22 April 2017

Accepted Date: 23 April 2017

Please cite this article as: H. Eroğlu, M.K. Haidar, E. Nemutlu, Şüü. Öztürk, C. Bayram, K. Ulubayram, L. Öner, Dual release behavior of atorvastatin and alpha-lipoic acid from PLGA microspheres for the combination therapy in peripheral nerve injury, Journal of Drug Delivery Science and Technology (2017), doi: 10.1016/j.jddst.2017.04.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Dual Release Behavior of Atorvastatin and Alpha-lipoic Acid from PLGA Microspheres for the Combination Therapy in Peripheral Nerve Injury Hakan Eroğlua*, Mohammad Karim Haidara, Emirhan Nemutlub, Şükrü Öztürkc, Cem Bayramd, Kezban Ulubayramc,e, Levent Önera

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Hacettepe University Faculty of Pharmacy, Department of Pharmaceutical Technology, 06100, Sıhhiye, Ankara, Turkey b Hacettepe University Faculty of Pharmacy, Department of Analytical Chemistry, 06100, Sıhhiye, Ankara, Turkey c Hacettepe University, Institute for Graduate Studies in Science and Engineering, Bioengineering Division, 06100, Beytepe, Ankara, Turkey d Hacettepe University, Advanced Technologies Application and Research Center, 06800, Beytepe, Ankara, Turkey e Hacettepe University, Faculty of Pharmacy, Department of Basic Pharmaceutical Sciences, 06100, Sıhhiye, Ankara, Turkey

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*Corresponding Author Assoc. Prof. Hakan Eroğlu Hacettepe University Faculty of Pharmacy Department of Pharmaceutical Technology Adnan Saygun Street, 06100 Sıhhiye-Ankara/Turkey

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Phone: 00 90 533 6520574 Fax: 00 90 312 310 0906 e-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract

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The major focus of this study was to prepare poly(lactic-co-glycolic) acid (PLGA) microspheres containing atorvastatin calcium (ATR) in combination with alpha-lipoic acid (ALA). PLGA microspheres will maintain dual-release for providing neuroprotective effects for peripheral nerve injury. For this purpose, microspheres were prepared by spray dryer with different drug:polymer ratios. Microsphere formulations were evaluated for particle size distribution, preparation and encapsulation efficiency, surface morphology, in-vitro release and dose dependent cytotoxicity test with L-929 and B-35 cells. TGA, DSC and FTIR analysis were performed for the investigation of physicochemical properties of the PLGA microspheres. Encapsulation efficiencies were calculated as >70% for ALA and >62% for ATR. FTIR results indicated that there were no interaction between the polymer and the active ingredients. A novel analytical method has been developed and fully validated, which would allow for quantification of ATR and ALA simultaneously. Release profiles showed that ALA is released within the first 17 hours and ATR release lasted for 17 days. Finally, results showed that there was no any toxicity associated with ALA and ATR containing PLGA formulations on both B-35 and L-929 cells. It was concluded that PLGA formulations with dual effects are promising systems for the treatment of peripheral nerve injury.

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Keywords: Combination therapy, Atorvastatin calcium, Alpha-lipoic acid, Microsphere, Peripheral nerve injury.

1. Introduction 2

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Peripheral nerve injury is a clinical situation, which is observed depending on various reasons and affects the life quality of the patients seriously. Traumatic injuries as well as metabolic, toxic and vascular reasons might be the causes for peripheral nerve injury [1]. The major causes of trauma are laceration, focal contusion (armed injuries) traction injuries, compression injuries, drug injection injuries and electrical injuries. Especially peripheral nerve injury observed in arm and foot injuries affect the daily life of the patients in a higher degree. It is very clearly stated in the classification of peripheral nerve injuries that prognosis includes a time period of starting from weeks up to months. The surgery is almost necessary; however clinical outcomes that do not end positively is observed in most cases with incomplete recovery [2, 3]. The primary injury effect of compression in peripheral nerve injury models starts just from the edge of the compressing material and spreads towards the healthy surrounding tissue. The primary injury is defined as the effect stimulated by compression and secondary effect is the injury caused by the edema and microvascular damage, which is induced by attenuation of the microcirculation. In fact, what underlie the secondary injury are the biochemical events happening just after trauma. When the blood flow is disturbed after the trauma, cellular functions are destructed, intracellular/extracellular edema is formed and finally cell death occurs [4]. In the lack of oxygen, very little amount of energy is formed, intracellular energy depots are used and therefore the ionic gradient between intracellular and intercellular environment is disrupted [4, 5]. Although the degree of ischemic injury is limited by the maintenance of blood recirculation, the degree of injury paradoxically increases depending on the interaction between blood and injured tissue, which is named as reperfusion injury [6].

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Despite all these clear facts, there is still no an optimum treatment perspective for nerve injuries. On the other hand, the perspective is focused on the symptomatic treatment of the secondary cascades of the nerve injuries [7]. It is a clear fact that we can do nothing on the primary injury (without surgery); so the perspectives for treatment are generally focused on preventing secondary injury or at least keeping the injury degree at a constant level. As a result, depending on both the long-term treatment necessity and loss of man-power, nerve injury treatment has a crucial effect on health economics of the countries. So, development of effective and safe formulations is of paramount importance. In the last decade, researchers have extensively focused of dual delivery of various active ingredients [8-10]. These combination products not only provide a better therapy, but also improve the patients’ compliance to the treatment. Another advantage of combination drug products is the simultaneous delivery of more than one active ingredient at the site of action. Depending on the different release characteristics and different mechanism pathways, maintenance of the active pharmaceutical ingredients (APIs) for a longer term at the target area and synergistic effects can also be achieved.

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The aim of this study is to formulate poly(lactide-co-glycolic acid) (PLGA) microspheres containing alpha-lipoic acid (ALA) and atorvastatin (ATR), depending on their antioxidant and neuroprotective effects, respectively. For the combination theraphy, ALA was chosen as one of the active pharmaceutical ingredients (APIs) since it is mainly involved as an essential cofactor in the mitochondrial alpha ketoacid dehydrogenease complexes [11-13]. Although depending on being an essential chemical for the normal oxidative metabolic pathways, the only mechanism was considered as the former mechanism. It was found that exogenous alpha lipoat (AL) is rapidly absorbed by the cells and reduced to dihydrolipoat. The power of this reduction comes from both NADH and NADPH. AL has the power of manipulating the NADH/NAD+ and NADPH/NADP+ ratios, which certainly results in governing effect of numerous cellular metabolism pathways. Briefly, ALA is considered to be a promising antioxidant agent against the mitochondrial dysfunctions, which are generally observed in neurodegenerative traumatic events [14]. In addition to that mechanism, it was shown that AL inhibits the mitochondrial calcium transport and assists to maintain the intracellular calcium balance [15, 16]. In addition to these former findings AL also scavengers hydroxyl radicals, hypochlorous acid, nitric oxide (NO), peroxynitrite, hydrogen peroxide and reduced oxygen radicals [17]. Packer et al also showed that in the rats that are fed with ALA and in which lipid peroxidation is induced, the accumulation of lipid peroxidation products showed 50% decrease, which may be considered as to show the neuroprotective effects of ALA. Therefore, ALA is expected to show above-mentioned effects in the early phase of neuronal injury and recovery mitochondrial functions as well as scavenger free radicals, which are main causes of secondary injury.

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The second API of this combination, which is ATR, is a well-known member of statins and generally used as cholesterol lowering agents in clinics. ATR has been shown to exert neuroprotective effects in many studies depending on their pleiotropic effects. In recent studies, the researchers have focused on the neuroproptective effects of statins in ischemic and traumatic brain injury [18-21], autoimmune disease [22, 23], Alzheimer’s disease [24] and spinal cord injury [25] studies. The underling effect of neuroprotective mechanism of statins have not clearly been identified yet, however the major explanations are based on pleiotropic effects that include antioxidative and anti-inflammatory mechanisms [26]. In addition to these explanations, pleiotropic effects also include isoprenylization, myelination, modulation of immune response and changes of oxidative and nitrosative stress levels [27]. It is notable that three major peripheral mechanisms also exist, which favor the neuroprotective effects of statins as: i-attenuation of oxidative stress, ii-regulation of vascular functions and iiimodulation of peripheral immune response [28]. In the present study, ALA and ATR containing microsphere formulations not only provide sustained delivery of the APIs, but also maintain attenuated dose at the site of action. Many research groups use poly(lactide-co-glycolic acid) (PLGA) microsphere formulations for delivery of numerous APIs such as clarithromycin delivery for bone 4

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regeneration [29], gefitinib for treatment of EGFR-positive metastatic non-small cell lung cancer [30], triamcinolone acetonide [31], isoperidone as antipsychotic [32], ropinirole for rotenone-induced parkinsonism [33] and norvancomycin for osteomyelitis [34] and ATR containing PLGA nanospheres for brain targeting in neurodegenerative diseases [35]. PLGA is a synthetic polymer that is also approved by Food and Drug Administration for the use in humans. Therefore, it is generally used in preparation of microsphere formulations due to its biodegradable and biocompatible properties.

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Within the light of these facts, we have prepared PLGA microsphere formulations that are intended to have a potential use in treatment of peripheral nerve injury. Implanted microspheres in traumatic area, depending on the sustained release characteristics of these formulations, will provide a long-term maintenance of these two APIs. Actually, combination of two APIs is proposed to have a dual effect at the traumatic area with their different mechanism of actions. As explained in the former paragraphs, one of the major pathways of neurodegeneration is the destruction of the intracellular calcium homeostasis followed by accumulation of excess amount of intracellular calcium that results in cellular death. Therefore the proposed mechanism for ALA will be i- the inhibition of mitochondrial calcium transport, ii-maintenance of intracellular homeostasis after neurodegeneration, iii-scavengering free radicals, which are formed after primary injury and iv-maintaining the functioning of mitochondria that is necessary for cell existance. In addition to that mechanism ATR will be decreasing the oxidative damage, regulate vascular functions and peripheral immune responses in the long-term recovery of neurodegeneration.

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In the literature, different researchers have described numerous methods for the quantification of ALA and ATR separately from various samples. Some examples for these methods are the quantification of ALA from skin samples by HPLC method [36], in human plasma by LC-MS/MS [37], in human plasma using HPLC coupled with electrochemical detection [38], in dietary supplements using a boron-doped diamond electrode [39], in biological specimens by HPLC with fluorescence detection [40], in plasma by LC-ESI-MS [41] and in rat plasma by LC-ESI-MS/MS [42]. In addition to that, ATR has been quantified either alone or in combination of other APIs by using different analytical methods such as in human plasma by LC-MS/MS [4345] and atorvastatin in combination with niacin in human plasma by LC-MS/MS [46]. In this study, we have also developed a novel sensitive HPLC method for the first time in literature to quantify ALA and ATR amounts from a single sample of pharmaceutical formulation simultaneously. In summary, for combination therapy in peripheral nerve injury, it was aimed to formulate PLGA microspheres containing ALA and ATR. PLGA microspheres were prepared by spray dryer and particle size distribution, encapsulation efficiency, surface morphology, in-vitro dual release characteristics and dose dependent cytotoxicity with L-929 and B-35 cells were investigated in details. TGA, DSC and 5

ACCEPTED MANUSCRIPT FTIR analyses were also performed for the investigation of physicochemical properties of PLGA microspheres. The performances of these formulations are discussed in detail for the combination therapy. 2. Materials and Methods

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2.1. Materials ATR and ALA were used as the APIs, which are encapsulated in PLGA microspheres. ATR (PubChem CID: 15378998) was a generous gift from Biofarma Pharmaceuticals (İstanbul, Turkey) and ALA (PubChem CID: 864) was purchased from Sigma Aldrich (St. Louis, USA). PLGA was from Boehringer Ingelheim (Ingelheim, Germany) and all other chemicals, which are used for preparation of microspheres and analysis, were of analytical grade and used without further purification. L-929 cell line was purchased from ATCC (ATCC® CCL-1™) and B-35 cell line were kindly donated by Prof. Dr. Ismet Deliloglu Gurhan from Ege University. MTT and all cell culture materials were purchased from Sigma Aldrich (St. Louis, USA) and Greiner Bio-One, respectively. Cell culture medium and supplements were from Biochrom (Merck, Germany). 2.2. Methods

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2.2.1. Simultaneous Quantification of ALA and ATR by Chromatography The HPLC system (HP1200, Hewlett Packard GmBH, Germany) was equipped with a quaternary pump, an auto sampler, an injector with a 100µL loop, a column oven, a UV detector and a HP ChemStation software (Rev.B.04.03). The chromatographic parameters, column type, pH (2.5 and 4.5), organic phase type (methanol, acetonitrile or mixture of them), ratio in mobile phase componenets and flow rate were tested for optimum separation conditions. The optimum set of condition for the simultaneous analysis of ATR and ALA was determined as follows: a reverse phase Inertsil ODS-3 C18 column (5µm; 150x4.6 mm i.d.) that is conditioned at 40°C; the mobile phase consisting of a mixture of 50mM pH 4.5 sodium dihydrogen phosphate buffer and acetonitrile (55:45, v/v); the mobile phase was pumped at a flow rate 1.0 mL/min.; total injection volume of the samples was set as 20 µL and finally detection wavelength was set as 212 nm for ATR and ALA. 2.2.2. Analytic method validation Analytic method validation of the developed method was evaluated according to ICH guideline requirements [47]. For that purpose linearity, accuracy, precision and specificity and stability studies were evaluated. 2.2.2.1. Linearity The working solutions for calibration were prepared from separate stock solutions of ATR and ALA at concentrations of 100 µg/mL. A calibration curve was constructed from blank sample and 7 non-zero samples covering the total range of 0.5 µg/mL up 6

ACCEPTED MANUSCRIPT to 25 µg/mL. Calibration curves were generated on 6 different batches and linearity was assessed by weighed (1/x2) least squares regression analysis. The acceptance criterion was set as the 2% coefficient of variation at same concentration of the 6 different batches.

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2.2.2.2. Specificity/Selectivity The chromatogram of standards containing 4 µg/mL ALA and ATR was compared the chromatograms of mobile phase and placebo samples (unloaded microsphere formulation) for the investigation of any possible interactions with the APIs in order to prove specificity of the HPLC method.

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2.2.2.3. Recovery Three concentrations at low, medium and high (2, 8 and 25 µg/mL) were set as the control points of the analytical method. Six different solutions at these three concentrations were prepared and analyzed with the HPLC method. The recoveries of the ATR and ALA were then calculated with the calibration curve and the results were evaluated over the coefficient of variation at these concentrations for six replicates.

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2.2.2.4. Accuracy and Precision Within batch accuracy and precision evaluations were performed by repeated analysis of solutions containing ATR and ALA having concentrations of 2, 8 and 25 µg/mL as low, medium and high concentrations. The reproducibility of the determined concentrations was evaluated over six different batches. The repeatability of the stock solutions was assessed by six replicates over the same batch. The results are expressed in terms of relative standard deviations for precision and bias for accuracy.

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2.2.2.5. Stability The short-term stability of ATR and ALA was examined by keeping the stock solutions at concentrations of low (2 µg/mL) and high (25 µg/mL) at 37±0.5°C for 8 days. For each concentration, three replicates were analyzed in one analytical batch. The concentrations of ATR and ALA after each storage period were related to its initial concentration as determined for the samples that were freshly prepared and possessed immediately. 2.2.2.6. Sensitivity The sensitivity of the analytical method has been evaluated by the determination of limit of detection (LOD) and limit of quantification (LOQ) parameters according to the EMA guideline on, which is on Validation of Analytical Procedures [47]. The LOD value was recorded, as the concentration of ATR and ALA with the signal to noise ratio of the HPLC chromatogram was 3:1. Similarly the LOQ value was recorded as the signal to noise ratio was 10:1.

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2.2.3. Simultaneous Thermogravimetric and Differential Scanning Calorimetry Analysis Simultaneous Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) were performed using a Q600 SDT system (TA Instruments) in order to verify the thermal stability of the samples. TGA and DSC curves were obtained in the temperature range from 50-550°C, using platinum crucibles with about 1 mg of samples, under nitrogen atmosphere (100 mL/min) and heating rate of 10°C/minutes

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2.2.4. Fourier Transform Infrared Spectroscopy-FTIR Fourier Transform Infrared (FTIR, Thermo Nicolet is50) analysis was conducted to verify the encapsulation of ATR and ALA inside the PLGA microspheres. The samples in contact with ATR crystal were scanned in the IR range from 650 - 4000 cm-1 and the background spectrum was subtracted from the IR signal.

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2.2.5. Formulation and Characterization of PLGA Microspheres PLGA microspheres containing ATR and ALA were prepared at three different drug:polymer ratios by using RG503H PLGA as 1:5, 1:10 and 1:20 in order to investigate the effect on encapsulation efficiency. For this purpose, certain amount of ATR and ALA were dissolved in 200 µL of methanol. Afterwards, this solution was added to PLGA containing dichloromethane (DCM) solution. Microspheres were prepared by using Büchi B-290 Spray Dryer. The conditions for spray drying were set as - inlet temperature: 90oC; -flow rate: 10 mL/min; -outlet temperature: 49oC and aspiration: 80%. The microspheres were collected from the collection vessel and kept at 4oC until further use. The polymer amount is increased while keeping the APIs amounts constant in order to investigate the ratio of polymer increase on encapsulation, surface morphology, particle size distribution and in-vitro release characteristics of the PLGA microspheres.

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2.2.5.1. Preparation Efficiency The preparation efficiencies of the microsphere formulations were determined by calculating the ratio of the weight of the microspheres to the sum of polymer and ATR/ALA amounts. The batches were prepared over three replicates and the resulting microspheres were collected in a single container. 2.2.5.2. Surface Morphology The surface characteristics of the microsphere formulations were investigated with Scanning Electron Microscopy (SEM, FEI, Quanta 400F, Holland). The samples were mounted on aluminum stubs and sputter-coated with gold–palladium (AuPd) under an argon atmosphere before analysis. 2.2.5.3. Mean Particle Size Distribution The mean particle size distribution for each formulation containing ATR and ALA was determined by Malvern Mastersizer S-2000. For this purpose, microspheres were 8

ACCEPTED MANUSCRIPT added into water containing Tween 80 (1%-w/v) and homogenously dispersed. All measurements were performed over 6 replicates. The statistical comparisons were done by using IBM SPSS Statistics 23.0 software with the one-way ANOVA. The p value was set as 0.05 and the post hoc comparisons were done by Dunnett T3 test.

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2.2.5.4. Encapsulation Efficiency 10 mg of microspheres have been accurately weighed and transferred into a vial containing 1 mL of DCM. The vial is vortexed for 2 minutes for the destruction of microsphere structures. Afterwards, DCM is evaporated until dryness and 1 mL of methanol is added into the vial for the solubilization of ATR and ALA. The vial is vortexed for 2 minutes and kept in the ultrasonic bath for an additional 2 minutes to recover the total amount of ATR and ALA existing in the samples. This solution is directly filtered through 0.45 µm PTFE syringe filter and analyzed with the developed HPLC method. The encapsulation efficiency was determined over 6 replicates and results were expressed with standard error (SE) of means.

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2.2.6. In-Vitro Release Studies In-vitro release tests were carried out in a horizontal shaker. 10 mg microspheres were accurately weighed and added into 30 mL of phosphate buffer (pH=7.4) in flasks. Flasks were fixed in a horizontal shaker water bath (37±0.5oC) at 50 rpm. At predetermined time intervals, 1 mL of sample was withdrawn and replaced with a fresh medium immediately for maintaining sink conditions. The sample was filtered through 0.45 µm cellulose acetate filters and analyzed by using the above-mentioned HPLC method. For each formulation, in-vitro release characteristics are investigated over 3 replicates and the release kinetics were analyzed by GraphPad software. 2.2.7. Cytotoxicity Studies

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2.2.7.1. Dose and Time Dependent Cytotoxicity of Drugs

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In order to evaluate the cytotoxicity of ATR and ALA, L-929 and B-35 cell lines were used as fibroblast and neuron cells, respectively. Four different dose of each drug (0.5, 1, 3 and 5 µg/mL) and two different culture time periods (24 and 48 hours) were set to define dose and time dependent cytotoxicity for APIs. For preparing of different concentrations of drugs, stock solution of each was prepared, sterilized with filter (0.22 µm pore size) and dilution series were done by using of cell culture growth medium, Dulbecco’s MEM (High glucose, Biochrom, Germany) supplemented with 10% Fetal Bovine Serum and 0.1% penicillin-streptomycin. 10% dimethyl sulfoxide (DMSO) solution containing DMEM and DMEM without drug were used positive and negative treatment, respectively. For evaluation of cytotoxicity of drugs, both L929 and B-35 cells were cultured (1.104 cells/well) into growth medium for 24 h and after 24 hours, drug treatment was performed. After specific time period, colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed as describe before [48]. Briefly, at each culture time, drug treated cells were incubated with 5 mg/mL of MTT reagent (Sigma-Aldrich, St. Louis, Missouri) 9

ACCEPTED MANUSCRIPT for 3.5 h. After that, DMSO was added to dissolve the formazan crystals and the absorbance of formazan was measured at 570 nm using an ELISA micro plate reader (VERSAmax, Molecular Devices, CA).

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2.2.7.2. Cytotoxicity of Drug Loaded Microspheres To determine cytotoxicity of the ATR and ALA loaded PLGA microspheres (drug:polymer ratio = 1:20, 1:10 and 1:5), briefly, they were sterilized under UV light for 45 min and homogenized into cell culture medium with vortex for 2 min. Then, sterile microspheres were applied with the concentration of 1 mg/mL on L-929 and B35 cell cultures (each well contain 1x104 cells) and cultured for 24 and 48 hours. After specific time culture period, colorimetric MTT assay was performed to investigate cytotoxicity of microspheres as mentioned previous section.

3. Results and Discussion

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2.2.8. Statistical Analysis All experiments were performed as 8 replicates (n=8) and data were reported as mean ± standard deviation. The statistical differences between groups were calculated using one-way ANOVA method with Graph Pad Prism 6.0 (GraphPad Software, Inc, San Diego, CA). If the affect was found to be statistically significant, post-hoc comparisons were evaluated by Tukey HSD (Honestly Significant Difference) test. Results were considered as statistically significance when p < 0.05.

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3.1. Analytical Method Validation for ALA and ATR

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3.1.1. Specificity The chromatographic conditions were found to be specific for the detection and quantification of ATR and ALA. There were no other interfering peaks in the chromatograms originating from neither the excipients used in the formulation nor the reagents and chemicals used in the analytical method (Figure 1).

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3.1.2. Linearity The linearity of the calibration curves for ATR and ALA has been experimentally evaluated over 7 non-zero samples that cover the range of 0.5 – 25 µg/mL. Nine different analytical conditions were investigated for the optimum separation of ALA and ATR. The optimum condition was selected in which the pH value of the mobile phase was 4.5 and consisting of NaH2PO4 buffer: acetonitrile at the ratio of 55:45 (v/v). The flow rate of the mobile phase was set as 1 mL/min and the detection was performed at 212 nm. Representative chromatogram consisting of 4 µg/mL ALA and ATR is shown Figure 1a and 1b, which also shows the selectivity of the HPLC method. According to this novel method, retention times of ALA and ATR were recorded as 3.375 and 4.730 minutes, respectively. [Figure 1]

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ACCEPTED MANUSCRIPT The studies were carried out over 6 replicates and the calibration curves are found to be linear with the equations given below for ATR and ALA, respectively within the concentration range of 0.5-25 µg/mL: for ALA

= 71.241 − 1.5006 2 = 0.9998

for ATR

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= 9.8517 + 0.0466 2 = 0.9996

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3.1.3. Recovery The recovery capability of the analytical method has been investigated over three concentrations, which were determined as low, medium and high for ALA and ATR concentrations separately (2, 8 and 25 µg/mL). The recovery of ALA and ATR were ranging between 98.55 and 101.30 with very low coefficient of variation ≤ 1.45% (Table 1).

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Table 1. Recovery values for ALA and ATR at concentrations of 2, 8 and 25 µg/mL ALA

Found Concentration (µg/mL)

Recovery %

CV %

Bias %

2 8 25

1.971±0.018 7.906±0.051 25.116±0.179

98.55 98.82 100.46

0.922 0.650 0.715

1.450 1.175 0.464

0.889 0.375 0.848

0.650 0.212 1.304

2 8 25

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Added Concentration (µg/mL) (n=6)

1.987±0.017 7.983±0.030 25.326±0.214

ATR

99.35 99.78 101.30

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SD: Standard Deviation; CV: Coefficient of Variation

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3.1.4. Accuracy and Precision Three concentrations of ALA and ATR were selected as low (2 µg/mL), medium (8 µg/mL) and high (25 µg/mL) concentrations of the calibration curve. The samples were analyzed by the HPLC method and the results are calculated by the mean, standard deviation (SD) and coefficient of variation (CV) as shown in Table 2. 3.1.5. Stability The stability of the samples was investigated for the short-term period of 8 days and the samples were subjected to same in-vitro release conditions. The results of the stability investigations revealed that there was no significant change in the concentration of the samples regarding the ALA and ATR concentrations as briefly summarized in Table 3

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ACCEPTED MANUSCRIPT Table 2. Repeatability and reproducibility results of ALA and ATR at concentrations of 2, 8, 25 µg/mL ALA

Reproducibility (Intra-Day)

2 8 25 2 8 25

Bias %

1.743 1.254 0.169

0.350 0.612 0.744

0.901 0.673 0.105

1.800 1.587 0.004

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2.007±0.035 8.049±0.101 24.814±0.042 ATR 1.964±0.017 8.127±0.054 24.999±0.026 ALA 1.976±0.026 8.124±0.094 24.725±0.181 ATR 2.037±0.029 7.911±0.097 25.046±0.103

CV%

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2 8 25

Average

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Repeatability (Inter-Day)

Concentration (µg/mL) (n=6) 2 8 25

1.315 1.157 0.732

1.200 1.550 1.100

1.382 1.226 0.411

1.850 1.112 0.184

SD: Standard Deviation; CV: Coefficient of Variation

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Table 3. Stability results of ALA and ATR samples (n=3) Day 0

Day 1

Day 5

Day 8

Mean

SD

CV %

2 8 25

1.934 7.973 25.391

1.974 8.073 24.924

1.961 8.013 25.164

0.024 0.078 0.206

1.249 0.979 0.819

2.045 8.048 25.275

1.985 7.957 24.944

1.988 1.949 7.922 8.083 25.266 25.075 ATR 2.006 1.993 7.972 7.976 25.093 25.115

2.007 7.989 25.275

0.026 0.040 0.135

1.344 0.503 0.540

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2 8 25

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Stability

ALA

Concentration (µg/mL) (n=3)

3.1.6. Sensitivity Regarding the sensitivity of the analytical method, LOD and LOQ values were calculated as 0.027 µg/mL - 0.084 µg/mL for ALA; and 0.011 µg/mL - 0.031 µg/mL for ATR, respectively 3.2. Characterization of PLGA Microspheres Containing ALA and ATR 3.2.1. Preparation Efficiency The sum of the weight of polymeric material (PLGA) and APIs (B) were taken into account for the calculation of preparation efficiency. The final efficiency is calculated 12

ACCEPTED MANUSCRIPT by the ratio of A/B*100, where A is the amount of microspheres. As a result, the preparation efficiency was calculated as 20.08%, 30.28%, and 31.66% for the microsphere batches having the drug:polymer ratio of 1:5, 1:10 and 1:20, respectively.

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3.2.2. Surface Morphology Morphology investigations were accomplished for the evaluation of surface characteristics of the microsphere formulations by using Scanning Electron Microscopy (SEM). For this purpose, both unloaded and loaded microspheres are subjected to the above-mentioned procedure and the results showed that both all microspheres were regular and spherical in shape. However, as the loading ratio increased, the structural morphology of the microspheres had slightly shifted from spherical structure (Figure 2). According to the SEM investigations, as the polymer amount is increased with respect to the drug, larger microspheres are formed with the spray drying method. The reason for this elevation is the increase in the viscosity of the solution, which is sprayed through the nozzle. As the viscosity is increased, droplets with larger dimensions are formed inside the nozzle and therefore larger microspheres are formed after immediate evaporation of the solvent in the droplets [49, 50]. [Figure 2]

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3.2.3. Particle Size Distribution The particle size distributions for microsphere formulations are determined as 1.457 ± 0.679 µm, 1.2162±0.654µm, 0.895 ± 0.397 µm and 0.797 ± 0.338 µm for formulations having 1:20, 1:10 and 1:5 drug:polymer ratio and unloaded microsphere formulations, respectively (mean ± geometric standard deviation). As a result of these measurements, it was clearly observed that particle size of the microspheres increased significantly as the polymer ratio is increased from 1:5 to1:20. According to the comparison results, there were no statistically significant difference between formulations having 1:5 and 1:10 drug polymer ratio (p=0.218). In similar, the difference in the particle size distributions of unloaded and 1:5 drug:polymer ratio formulations was insignificant with p value of 0.627. On the other hand, formulations having 1:10 and 1:20 drug:polymer ratios are found to be statistically different in terms of particle size distribution when compared to unloaded formulation (p=0.000). 3.2.4. Thermal Behavior of PLGA Microspheres Containing ALA and ATR Figure 3 shows TGA and DSC curves of ATR and ALA. TGA curve of ATR exhibits a multi-step decomposition in addition to mass loss of water in the range of 50–112 °C, with ∆m = 5 %. The atorvastatin was thermally stable up to 205 °C, with additional mass loss of 2 % only. A drastic stage of mass loss was observed in 205– 306 °C (∆m = 45 %), 306–407 °C (∆m = 13 %) and 407–525 °C (∆m = 16 %), 13

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respectively. Since ALA is a moderately hydrophobic compound, no remarkable water loss was observed until 162 °C (∆m < 2 %). Thermal decomposition completed between 162 and 235 °C in a single step (Figure 3). DSC curve of ATR exhibits endothermic events at 52, 86 and 111 °C, indicating the loss of water, which can also be seen in the TG curve. Then two endothermic events which can be attributed to a phase transition characteristic of ATR, observed at 164 °C, and 183 ° [51]. Pure ALA exhibits a sharp melting transition at 60 °C and thermally decomposes completely at 235 °C. [Figure 3]

[Figure 4]

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Each ALA and ATR containing microspheres present lower thermal stability compared to empty microspheres as indicated in Figure 4. Weight loss begins at lower temperatures in ALA and ATR containing samples where ALA completely decomposes around 190 – 235 °C. The initial decomposition temperature shifts to lower values as the (ALA+ATR):PLGA ratio increases from 1:20 to 1:5. For 1:20 formulations ∆m begins at 270 °C for empty PLGA microspheres whereas decomposition begins at 230, 216 and 192 °C for ALA and ATR incorporated samples in increasing order. In addition, ATR presence is much more obvious in PLGA with 20% active agent loaded samples exhibiting a decomposition slope after 350 °C, where PLGA polymer degrades completely, indicating that a remarkable ATR amount remains.

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Figure 5 shows the DSC curves of the PLGA samples. The sharpness of the characteristic endothermic peak of PLGA microspheres at around 350 °C reduced and shifted in other samples confirming the previous TGA data that shows ATR and ALA containing microspheres have lower thermal stability. The exothermic event at 280 °C is much more intense in above-mentioned microspheres with ALA and ATR, referring the thermal decomposition of both polymer and other components. The exothermic ATR decomposition peak can also be seen at 462 °C in DSC curves of API containing PLGA microspheres. In addition to that as the API concentration in PLGA microspheres increased, it was found that observed Tg was decreased, which is also in accordance with the previous literature findings [52]. [Figure 5]

3.2.5. FTIR Results Figure 6 presents the IR spectra of ATR, ALA and PLGA samples. ATR exhibits characteristic peaks at 1651 cm-1 (C=O stretching), 1313 cm-1 (C-N stretching), 1216 cm-1 (C-F stretching) and 1657 cm-1 (C=C bending). ALA spectrum also exhibits the C=O stretching at 1700 cm-1 and C-H stretching at 1466 cm-1. Each PLGA sample spectra seems to be identical to each other, indicating the encapsulation of both ALA 14

ACCEPTED MANUSCRIPT and ATR was carried out successfully since ATR-FTIR technique collects IR data directly from the microsphere surface. The obtained IR signals from PLGA samples can be referred to C=O stretching (1750 cm-1), aliphatic deformation and wagging vibrations (1450 – 1380 cm-1 region) and C-O ester stretching at 1100 – 1200 cm-1 region.

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[Figure 6] 3.3. Encapsulation Efficiency and Dual Release Behavior of ALA and ATR from PLGA Microspheres

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ATR and ALA contents of the microsphere formulations were investigated over 6 replicates by using the novel analytical method. According to the results, the encapsulation efficiencies were calculated for ALA as 82.471 ± 3.456, 81.235± 4.562, 70.252 ± 3.609 % and for ATR as 69.622 ±4.889, 62.470 ± 6.832, 64.131 ±7.237 % for the formulations having drug:polymer ratio of 1:5, 1:10 and 1:20, respectively.

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In-vitro release experiments were carried over 3 replicates in the pH 7.4 sodium phosphate buffer medium containing flasks. At each predetermined time intervals, the sample, which is withdrawn, has been replaced with fresh medium in order to maintain sink conditions. The release profiles of the formulations have revealed that as the drug:polymer ratio decreases from 1:5 to 1:20, the release time for ALA also decreases from 17 hours to 4 hours. Similar to that finding for ATR, the release time for formulation having drug:polymer ratio of 1:5 was recorded as 401 hours (≈17 days). However, it was only 303 hours (≈13 days) for the microsphere formulations having 1:20 drug:polymer ratio. The total release profiles for all formulations are shown in Figure 7a and 7b for ALA and ATR, respectively. According to the analysis of the release kinetics of ATR and ALA from PLGA microspheres, release kinetics for ATR was zero order with r2 values of 0.9796, 0.9915 and 0.9431 for formulations of 1:5, 1:10 and 1:20 respectively. On the other hand since release of ALA was completed rapidly, only for the formulation of 1:5 drug:polymer ratio could be calculated as first order release kinetics with r2 value of 0.8120. The number of points on the release profile was insufficient for calculating the release kinetics for 1:10 and 1:20 drug:polymer ratio formulations. A second important result that we observed for in-vitro release studies is the different release characteristics of ALA and ATR. In all cases ALA is more rapidly released with respect to ATR. In fact this was a desired characteristics, which is also beneficial for the in-vivo experiments. As well known, neuroregeneration is a long process that needs to recover completely. According to our plan, the dual effect of the APIs, which are encapsulated in the microspheres will be more effective in such kind of a release trend. At the initial stage ALA will show its effect through antioxidant activity as mentioned before. Afterwards, the level of ATR will be at a certain level, which will also continue after the release of ALA is completed. Therefore, approximately more 15

ACCEPTED MANUSCRIPT than 183 hours, a certain amount of ATR will be available in the target area with the microsphere formulations having 1:5 drug:polymer ratio. As the encapsulated amount in the microspheres decreased from 1:5 formulation to 1:20, the release of both APIs are completed in shorter time intervals. This dual release is expected to show a satisfactory performance in the acute and chronic phased of neuronal injury, depending on different mechanism of actions of ALA and ATR.

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[Figure 7]

3.4. Effect of Drugs and Microsphere Formulations on Cell Viability

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MTT assay was performed to determine cytotoxicity of ATR and ALA and drug loaded PLGA formulations on fibroblast (Figure 8) and neuron cells (Figure 9) at different concentrations and time intervals. The toxicity of PLGA microspheres has long been investigated and the particles found to be nontoxic. However, type of drug and their dose may exert some cytotoxic effects. [Figure 8]

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For ALA, cell viability of L-929 was found about 100% for 24 and 48 hours at all concentrations and there was no significant difference between tested concentration at both time intervals (p>0.05) (Figure 8A). B-35 viability was about over 70% for 24 hours (Figure 9A). It was noticed that neurons cells viability increased from 75 % to 108 % with increasing ALA concentration for 48 hours, which is statistically significant (p<0.05) (Figure 9A). So, ALA has a positive effect on B-35 proliferation. Results concluded that there was no any toxicity associated with ALA on both B-35 and L-929 cells by increasing culture time and drug concentration.

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For ATR, cell viability was about 100 % for both cell types at 48 hours (Figure 8B and 9B). B-35 viability was over 80% for 24 hours and over 100 % for 48 hours, indicating no statistically significance difference between all tested concentrations (p>0.05). Drug concentrations did not affect viability of both types of cells. As a result, it can be concluded that, viability of both cells did not change with increasing drug concentration and it was slightly increased over time (Figure 8B and Figure 9B). In order to evaluate the cytotoxicity of ALA and ATR loaded PLGA microspheres, L929 and B-35 cells were cultured with 1mg/mL microspheres for 24 and 48 hours. After incubation, colorimetric MTT assay was performed for investigation of cell cytotoxicity. 100% viability was accepted for both cells at time zero. For all PLGA microsphere formulations, there was no significant difference in cell viability on both fibroblast and neurons with increasing culture time (Figure 8C and 9C). On the other hand, for all drug:polymer ratios cell viability was above toxic level, even with a high PLGA or drug content. Based on these results, it can be summarized that, drug loaded

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4. Conclusion In this study, we have succeeded to formulate PLGA microspheres containing ALA and ATR for the first time. Microspheres are planned to be used in the treatment of peripheral nerve injury models depending on their sustained release characteristics and ease of dosing at the site of action. According to the in-vitro release results, for all formulations we have obtained different release trends for ALA and ATR. In the early phase in addition to the relatively fast release of ALA, ATR is also released at lower concentrations. Therefore, dual effect of this combination formulation is expected to maintain more effective treatment in nerve injury. In addition to that, a novel HPLC method, which is capable of quantifying ALA and ATR simultaneously has been developed for the first time in literature. This novel method has also been validated through parameters of linearity, accuracy, precision, stability, sensitivity and selectivity. As a result, it can be concluded that PLGA microspheres containing ALA and ATR as combination can be suitable candidates for a new treatment option in peripheral nerve injury. After this method development and formulation characterization study, further in-vivo investigations on experimental peripheral nerve injury models are needed for evaluating the efficiency of these formulations. According to our experimental results, we believe that PLGA microspheres with the drug:polymer ratio of 1:5 will be the most suitable formulation with relatively fast release of ALA (showing rapid effect at the site of action) and sustained release of ATR for long term maintenance of the API at the site of action for a better neuroprotective effect.

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Acknowledgements The authors would like to thank TÜBİTAK for funding the study with the project number 115S202.

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Figure 2. Scanning Electron Microscopy images of the PLGA microsphere formulations having drug:polymer ratios of A-unloaded; B-1:5; C-1:10 and D-1:20 at the magnification ratio of 10,000x. Figure 3. TGA and DSC chromatograms of A. Atorvastatin calcium, B. Alpha-lipoic acid.

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Figure 4. TGA of microsphere formulations. Figure 5. DSC thermograms of microsphere formulations.

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Figure 6. FTIR spectrum of ALA, ATR and microsphere formulations. Figure 7. In-vitro release profiles of the microsphere formulations for A. Alpha-lipoic acid B. Atorvastatin calcium.

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Figure 8. Cytotoxicity of ALA (A), ATR (B) and ALA and ATR loaded PLGA microspheres with different drug concentration and drug:polymer ratios (C) on fibroblast (L-929) cells. All data were presented as mean ± standard deviation, and * considered as statistically significant for p<0.05. ATR: Atorvastatin calcium, ALA: Alpha-lipoic acid (100% viability was accepted for both cells at time zero).

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Figure 9. Cytotoxicity of ALA (A), ATR (B) and drug loaded micro particles (C) on neurons (B-35) cells with respected to drug concentration and drug:polymer ratios. All data were presented as mean ± standard deviation, and * considered as statistically significant for p<0.05. ATR: Atorvastatin calcium, ALA: Alpha-lipoic acid (100% viability was accepted for both cells at time zero).

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