Intercalation of olanzapine into CaAl and NiAl Layered Double Hydroxides for dissolution rate improvement: Synthesis, characterization and in vitro toxicity

Accepted Manuscript Intercalation of olanzapine into CaAl and NiAl Layered Double Hydroxides for dissolution rate improvement: Synthesis, characterization and in vitro toxicity João G. Pontes-Neto, Magaly A.M. Lyra, Mônica F.L.R. Soares, Luíse L. Chaves, José L. Soares-Sobrinho PII:

S1773-2247(19)30477-0

DOI:

https://doi.org/10.1016/j.jddst.2019.05.034

Reference:

JDDST 1085

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 4 April 2019 Revised Date:

17 May 2019

Accepted Date: 20 May 2019

Please cite this article as: Joã.G. Pontes-Neto, M.A.M. Lyra, Mô.F.L.R. Soares, Luí.L. Chaves, José.L. Soares-Sobrinho, Intercalation of olanzapine into CaAl and NiAl Layered Double Hydroxides for dissolution rate improvement: Synthesis, characterization and in vitro toxicity, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.05.034. 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

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“Intercalation of Olanzapine Into CaAl and NiAl Layered Double Hydroxides For

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Dissolution Rate Improvement: Synthesis, Characterization and in vitro Toxicity”

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João G. Pontes-Netoa, Magaly A. M. Lyraa, Mônica F. L. R. Soaresa, Luíse L. Chavesa, José L. SoaresSobrinhoa* a

Núcleo de Controle de Qualidade de Medicamentos e Correlatos, Universidade Federal de Pernambuco.

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Postal address: Universidade Federal de Pernambuco, Centro de Ciências da Saúde, Departamento de Ciências Farmacêuticas. Av. Artur de Sá, S/N. Cidade Universitária. CEP: 50740-520. Recife – PE, Brazil.

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*Corresponding author: [email protected]. Phone Number: +55 (81) 21267515

ABSTRACT

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The aim of this work was to develop and characterize different Layered Double Hydroxides (LDHs) loaded with of Olanzapine (OLZ), CaAl:OLZ and NiAl:OLZ, by thermogravimetry (TG), differential scanning calorimetry (DSC), Fourier Transform Infrared spectroscopy (FT-IR) and X-ray diffraction (XRD), dissolution rate improvement, inhibition of AAPH-induced hemolysis and toxicity prospections with Artemia salina assay. No peaks of OLZ were found on diffractograms of both systems (5%) indicating amorphization of OLZ. This behavior was decreased with the increasing of drug. FT-IR spectra showed decreasing, displacements, suppressions and enlargements of bands in specific regions suggesting OLZ intercalation in both HDLs. No OLZ endothermic events was found in DSC curves indicating a decrease in the drug crystallinity, excepting for samples with 30 % of OLZ. The dissolution profile evidenced that in 45 minutes CaAl:OLZ 5% and NiAl:OLZ 5% presented the maximum drug solubilization of 89.08 and 61.66 %, respectively. In the inhibition of AAPHinduced hemolysis, both systems, showed significant inhibition (p ≤ 0.05) compared to the negative control, and Artemia salina assay evidenced that both HDLs (5%) did not cause the significant death of the specimens compared to OLZ in 24h. The exposed results reinforced the proposed use of LDHs as functional excipients.

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Adsorption; Intercalation.

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Keywords:

Solubility

Improvement;

Drug

Delivery;

Inorganic

Carrier;

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1 INTRODUCTION The pharmaceutical industry still faces a challenge in the development of novel

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strategies to increase the dissolution rate and bioavailability of poor water-soluble drugs

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orally administered. Among other factors, this is due to the growing number of New

39

Chemical Entities (NCEs), where more than 40% are lipophilic and present low aqueous

40

solubility [1]. It is also reported that 70% of potential drug candidates are discarded

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before reaching pharmaceutical technology labs, because of such limitations [2]. These

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drugs are generally classified as class II, according to the Biopharmaceutical

43

Classification System (BCS), where the dissolution process in the gastrointestinal tract

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represents a limiting step, decreasing its rate and degree of absorption, which results in

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a low bioavailability [3,4].

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In order to avoid such problems, several approaches have been explored, such as

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the particle size reduction [5], modification of the drug crystalline structure [1], solid

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dispersions [3,6], formation of salts [7], among others, aiming to increase the

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dissolution rate of these drugs. However, high energy levels are applied in most

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methods currently used, and it is known that certain materials does not maintain its

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stability after such processes [8], leading to agglomeration and crystal growth due to the

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high surface energy and size distribution [9]. In addition, it is possible to observe, in

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most conventional aqueous solubility enhancement strategies, that the large increase in

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the dissolution rate is rapidly followed by the drug recrystallization into a more stable

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form, but less soluble, which limits its absorption [5,8].

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The amorphous form of drugs represents a high energy state that exhibits greater

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aqueous solubility and dissolution rate, thus resulting in an increased bioavailability [1].

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In this context, the Layered Double Hydroxides (LDHs) represent a promising

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alternative due to their great capacity of adsorption and intercalation of biomolecules,

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allowing the stabilization of the amorphous form of a, previously, crystalline drug [10–

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12].

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The LDHs belong to a class of clay minerals composed by di and trivalent metal

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cations, arranged in superposed layers with two-dimensional organized structure and

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flexible pores, presenting anionic chemical entities in their interlayer domains [13,14].

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They can be found in nature or synthetized with a simple and low-cost route. In addition

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to the LDHs application as solubility promoters, it is also used in modified drug

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delivery systems, for its promising storage and release of bioactive molecules

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intercalated in their interlayer spaces or adsorbed on their surface [15,16].

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Among the 20 most frequent prescriptions in the US in the last decade,

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olanzapine (OLZ) is an atypical or second-generation antipsychotic, used in the

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treatment of psychic disorders, with no extrapyramidal side effects and sedation,

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commonly

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thienobenzodiazepine chemically known as 2-methyl-4-(4-methylpiperazin-1-yl)-10H-

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thieno [2,3- b] [1,5]-benzodiazepine or C17H20N4S, consists in a yellow powder

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practically insoluble in water (12 - 44 mg/L), slightly soluble in acetonitrile, ethyl

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acetate and freely soluble in chloroform. According to the BCS, OLZ belongs to the

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class II, because of its low aqueous solubility and high permeability [17–20]. Despite

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OLZ mechanism of action is not completely understood, it is already known that it is

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related to the 5-HT2A (serotonin) and D2 (dopamine) receptors and antioxidant activity,

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which contributes to the decrease in side effects observed in other drugs of the same

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therapeutic class [20–22].

in

first

generation

antipsychotics.

This

drug,

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observed

As the scientific community observes great advances in the potential

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applications of inorganic materials, more knowledge is needed regarding their basic

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physicochemical characteristics such as surface, shape and dosage, which, among other

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factors, influence the toxicology of the inorganic material. To ensure safe use, it is

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necessary to understand such physicochemical characteristics, its interactions with the

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drug, the in vitro and in vivo behavior to anticipate possible adverse effects [23].

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Therefore, the main goal of this work was the physicochemical characterization of the

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two drug delivery systems based on calcium/aluminum and nickel/aluminum LDHs

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(CaAl-LDH and NiAl-LDH respectively) loaded with OLZ, the evaluation of OLZ

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dissolution rate improvement and their in vitro tests performances towards toxicity

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prospections.

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2 MATERIAL AND METHODS

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2.1 Material

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Aluminum nitrate nonahydrate [Al(NO3)3•9H2O], calcium nitrate tetrahydrate

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[Ca(NO3)2•4H2O], nickel nitrate hexahydrate [Ni(NO3)2•6H2O] and sodium hydroxide

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PA were all purchased from Sigma-Aldrich®. Throughout the synthesis process was

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used deionized water and all the other solvents used were had analytical grade. The

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OLZ used was purchased from Sansh BioTech pvt. Ltd, (New Delhi, India). 3

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2.2 Synthesis of CaAl and NiAl-based Layered Double Hydroxides (LDH) The LDH synthesis process was performed by the conventional co-precipitation

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method [16]. For the CaAl-LDH synthesis, a mixed solution of Ca(NO3)2•4H2O and

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Al(NO3)3•9H2O and another alkaline solution of sodium hydroxide (pH 10) were

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simultaneously added by dripping (1 mL/min) to a round bottom flask with sodium

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hydroxide solution (pH 10 ± 0,5) under constant stirring and nitrogen atmosphere. For

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the NiAl-LDH synthesis, the same procedure described above was performed, using

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Ni(NO3)2•6H2O as the source of divalent metal cation, instead of the calcium nitrate.

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Deionized water was used throughout the process. After the precipitate formation and

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the end of dripping, the synthesis product was cooled under room temperature, washed

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three times with deionized water by centrifugation for 10 minutes (3600 rpm), to

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separate the precipitate from the water. The CaAl-LDH and NiAl-LDH were dried in

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oven (50° C) overnight.

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2.3 Obtention of LDH:OLZ systems

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LDH:OLZ systems were obtained by the solvent technique [24]. Briefly, OLZ

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was completely dissolved in acetone and, subsequently, CaAl-LDH or NiAl-LDH was

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added, resulting the CaAl:OLZ and NiAl:OLZ systems in different drug concentrations.

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The suspension was maintained in orbital shaker table (109/1TC, Ethik Technology®) (1

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h) for solvent evaporation. The solvent residue was vacuum dried (MOD 302, TekSet®)

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at 60° C (2 h). Physical Mixtures (PMs) of both LDHs and OLZ were obtained (PM

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CaAl:OLZ and PM NiAl:OLZ) with the lowest and highest drug concentration, and

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also, unloaded LDH and free OLZ were submitted to the same solvent technique for

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comparative purposes.

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2.4 Characterization

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2.4.1 Thermal Analysis

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The systems with different drug concentrations, unloaded LDHs, free OLZ and

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the respective PMs were characterized by thermal analysis. The thermogravimetric

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curves (TG) and the first derivative of the TG curve (DTG) were obtained from

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Shimadzu® thermobalance, model DTG-60H, under nitrogen atmosphere at 50 mL.min-

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in each loaded sample, the respective amount of unloaded LDHs, 5 mg (± 0.2) free OLZ

flow rate, and heating rate of 10° C.min-1. The equivalent amount of 5 mg (± 0.2) OLZ

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and the PMs were analyzed between 25 to 500° C in aluminum sample port, after the

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instrument calibration with calcium oxalate monohydrate sample. The analyses were

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performed in triplicated. The thermoanalytical data were analyzed using the software

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TA-60WS, version 2.20 (Shimadzu®). The differential scanning calorimetry (DSC) curves of the samples with the same

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concentration mentioned above, were obtained in DSC Q200 (Shimadzu®) Sweep

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Calorimeter under similar conditions of flow and heating rate as TG analysis. Samples

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were analyzed between 25 to 300° C placed in aluminum samples port hermetically

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sealed. The determinations were performed in triplicate. Indium and zinc were used to

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calibrate the temperature scale and the enthalpy response.

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2.4.2 Infrared absorption spectroscopy

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The infrared spectra were obtained using PerkinElmer® (Spectrum 400)

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equipment with attenuated total reflectance (ATR) device (Pike Technologies

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Spectroscopic Creativity) with zinc selenate crystal. The samples to be analyzed were

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transferred directly into the ATR device compartment. The micrographs were obtained

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with an average of 10 scans, 4 cm-1 resolution and range from 650 to 4000 cm-1.

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2.4.3 X-Ray diffraction

X-ray powder diffraction of the samples was performed using Shimadzu® XRD-

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700, with CuKα (1.5406 Å) radiation, equipped with copper anode. A thin layer of the

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samples powder was prepared on a glass support and analyzed between the range of 2 to

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60°, at a rate of 0.02°/s.

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2.5 In vitro drug release profile

The evaluation of the increased solubilization rate of OLZ was performed by the

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in vitro dissolution technique under sink conditions [25]. In this method, aliquots of free

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OLZ, CaAl:OLZ and NiAl:OLZ systems and their PMs were added in colorless

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capsules containing equivalent concentration of OLZ (5 mg). The dissolution tests were

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performed on the Varian® 7010 VK dissolver at 37° C (± 0.5 ° C) using 900 mL of

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phosphate buffer pH 6.8, previously aerated, as the dissolution medium, paddle

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apparatus under stirring (75 rpm). The drug quantification assay was performed in

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UV/Vis (λ = 260 nm) spectrophotometer at intervals of 0, 5, 10, 15, 20, 30, 45, 60, 120

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and 180 minutes.

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2.6 Inhibition of AAPH-induced hemolysis in rat erythrocytes The preparation of erythrocytes was performed with Wistar rats weighing

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between 200-220 g. The animals were anesthetized with ketamine and then blood

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collection was performed by retro orbital plexus rupture. The collected blood samples

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were centrifuged (2000 rpm) for 5 minutes. The supernatant was discarded and the

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erythrocytes were washed three times in phosphate buffered saline (PBS) pH 7.4. The

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erythrocytes were stored at 4° C, to be used within 6 hours [26].

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In the reaction tube, 300 µL of 10 % erythrocyte suspension in PBS (pH 7.4)

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was added to 100 µL of PBS (pH 7.4) containing the samples under study. Then, 200 µL

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of AAPH (200 µM) was added in PBS (pH 7.4). The reaction mixture was incubated at

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37° C (2 h), the volume was filled to 3 mL and centrifuged (1500 rpm) (5 min). The

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supernatant was used to determine hemolysis by spectrophotometer (λ = 540 nm) [27]

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and the results were expressed as percentage of hemolysis inhibition.

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2.7 In vitro toxicity

The In vitro toxicity by the Artemia salina assay [28] was performed with the

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developed systems who showed the most promising physicochemical characteristics and

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drug release profile. The microcrustaceans were kept in water (48 h) until their larvae

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hatch. The specimens of A. salina were distributed in 10 different tubes (10 specimens

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in each tube), containing the CaAl:OLZ 5%, NiAl:OLZ 5%, or their isolated

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components with the equivalent concentration present in the systems. The negative

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control was performed with the vehicle (1:1 sea water and mineral water), the positive

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control with potassium dichromate (K2Cr2O7) at concentrations of 50, 100 and 200

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µg/mL. Dead specimens were counted after 24 and 48 hours of experiment.

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3 RESULTS AND DISCUSSION

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3.1 Obtention of LDH:OLZ systems

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The systems were obtained in three different drug concentrations with the CaAl-

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LDH (CaAl:OLZ 5%, CaAl:OLZ 20%, CaAl:OLZ 30% w/w), the NiAl-LDH

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(NiAl:OLZ 5%, NiAl:OLZ 20%, NiAl:OLZ 30% w/w) and the physical mixtures

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corresponding to the lowest and highest drug concentration in the systems (PM

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CaAl:OLZ 5%, PM CaAl:OLZ 30%, PM NiAl:OLZ 5%, PM NiAl:OLZ 30% w/w)

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were prepared for comparison purposes. The association between LDH and drugs is

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widely reported, promoting better results towards drug performance and treatment 6

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efficiency in many diseases, such as in the anticancer therapy [29–31], inflammation

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[32,33], bacterial infections [34,35], neurodegenerative diseases [36,37], and others

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related to cholesterol [38], vitamins [39], etc. In these cases, LDH can either improve

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drug activity or act, synergistically, for itself contributing to the efficacy.

207 3.1 X-Ray diffraction

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X-Ray diffraction analysis were performed aiming to investigate the crystalline

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state of OLZ and the obtained LDH systems. The values of the interplanar distance

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(dhkl) were calculated through the Bragg’s equation, where the experimentally obtained

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θ values were used, as represented in the equation below:

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n . λ = 2dhkl . Senθ

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In this equation, n represents the reflection order of the peak (n = 1), λ

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corresponds to the X-rays wave-length used in the analysis (λ = 1.5406 Å), d represents

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the basal spacing of the hkl plane and θ is the experimentally determined Bragg angle.

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The calculation of the c-axis was performed using the d values of planes d003 and d006 (c

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= d003 + 2d006), while axis a was calculated with the d values of plane d110 (a = 2d110)

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[16].

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The crystalline nature of OLZ can be verified (Figure ) by characteristic

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diffraction patterns, with a prominent peak at 8.84º (2θ) and peaks of lower intensity

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between 10.5º – 23.9º (2θ), also identified [25]. In the literature, more than 25

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Olanzapine polymorphs have been reported, and the crystallinity pattern presented by

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the drug used in this study is characteristic of the form I [41,42].

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The diffraction patterns observed below 30° (2θ) for both unloaded LDHs,

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CaAl-LDH (Figure 1A) and NiAl-LDH (Figure 1B), are characteristic of hydrotalcite

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type compounds, where it was possible to evidence their crystallinity patterns by peaks

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at 2θ = 10.30º (d002) and 20.58º (d004) for CaAl-LDH [16,43,44] and 2θ = 11.24º (d003),

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23.0º (d006) for the NiAl-LDH [45,46], also reported in literature (14).

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Observing the basal spacing values of the unloaded CaAl-LDH and the

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CaAl:OLZ systems (table 1), it was noticed that there was a discrete, but growing,

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increase in the distance between the layers as the drug concentration increased. This was

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not observed in the PMs, which presented constant values and very close to the d

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parameter presented by the unloaded CaAl-LDH, similar to the reports in literature [47].

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It suggests that the drug intercalation occurred by ion exchange, where the nitrate anions

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(3.8 Å) [16], originally present in the interlayer space, were replaced by the OLZ, which 7

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space due to the drug intercalation. It is noticed that after the systems obtention, there

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was also an increase in the c-axis, corroborating the values of d presented by these

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systems. For values of 2θ around 30°, there are peaks derived from non-basal

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reflections, related to the internal structure of each layer. The crystallographic parameter

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a, related to the distance between two cations in the same layer, showed a value of 6.04

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Å.

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Table 1: Values of the 2θ (degrees) and interplanar distance (dhkl) of the binary systems based on CaAl-

CaAl-LDH

dhkl

CaAl:OLZ

CaAl:OLZ

CaAl:OLZ

5%

20%

30%

2θ

d (Å)

2θ

d (Å)

2θ

d (Å)

2θ

(002)

10,30

8,58

10,06

8,79

10,02

8,82

(004)

20,58

4,31

20,54

4,32

20,58

(110)

29,52

3,02

29,64

3,01

29,50

c (Å)

17,20

17,41

a (Å)

6,04

6,02

PM

CaAl:OLZ

CaAl:OLZ

5%

30%

d (Å)

2θ

d (Å)

2θ

d (Å)

10,00

8,84

10,28

8,60

10,28

8,60

4,31

20,58

4,31

19,98

4,44

19,98

4,44

3,02

29,48

3,02

29,42

3,03

29,42

3,03

17,44

17,46

17,48

17,48

6,04

6,04

6,06

6,06

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PM

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LDH and Olanzapine obtained from the X-ray diffraction.

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The interplanar distance values of the unloaded NiAl-LDH are shown in table 2,

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where the value of d in the plane d003 is 7.66 Å, similar to that found in literature, as

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well as its crystallinity parameter c, which is calculated differently (c = 3d003) [48–50].

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When comparing the values of d, in the same plane for binary systems and PMs, it was

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not possible to observe the basal spacing increasing. It suggests that either there was no

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OLZ intercalation by ion exchange with the nitrate anions or the intercalation did not

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promote structural variations in LDH. Also, it is possible that the OLZ formed an

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amorphous coating absorbed on the LDH surface, since the high absorption capacity is a

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well know characteristic for this material [47,51–54]. The d006 plane also did not show a

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considerable increase in the values of d for the binary systems and the PMs.

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Table 2: Values of the 2θ (degrees) and interplanar distance (dhkl) of the binary systems based on NiAlLDH and Olanzapine obtained from the X-ray diffraction.

dhkl

LDH-NiAl

NiAl:OLZ

NiAl:OLZ

NiAl:OLZ

5%

20%

30%

PM

PM

NiAl:OLZ

NiAl:OLZ

5%

30%

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d (Å)

2θ

d (Å)

2θ

d (Å)

2θ

d (Å)

2θ

d (Å)

2θ

d (Å)

(003)

11,54

7,66

11,72

7,55

11,62

7,61

11,58

7,64

11,64

7,60

11,64

7,60

(006)

23,00

3,82

23,18

3,83

23,08

3,85

23,10

3,85

23,10

3,85

23,06

3,85

(009)

35,42

2,53

35,58

2,52

35,46

2,53

35,52

2,52

35,52

2,52

35,28

2,54

c (Å)

22,98

22,65

22,83

22,92

22,80

22,80

a (Å)

5,06

5,04

5,06

5,04

5,04

5,08

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261 In figure 1A, we can observed the overlapping of the diffractograms generated

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by the CaAl:OLZ binary systems loaded with different drug concentrations, the

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respective PMs and the unloaded LDH. It can be noted that the diffractogram generated

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by CaAl:OLZ 5% shows only the crystalline patterns related to the unloaded CaAl-

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LDH. The highest intensity peak of free OLZ at 8.84º (2θ), which represents the largest

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number of repetitions of the same crystalline plane, is not present in this binary system,

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but is notable in the PM CaAl:OLZ 5%. Even though there is a smaller amount of the

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drug in the analysis, this low concentration did not prevent the OLZ from being detected

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in the PM. In addition to the peak

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diffractogram, other peaks between 14.84º and 24.0º (2θ) related to the drug were

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identified, which also were not observed in the binary system. This fact suggests that

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the solvent method used to form the CaAl:OLZ binary systems was efficient in

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promoting the drug amorphization.

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Figure 1

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at 8.84° (2θ) present in PM CaAl:OLZ 5%

Still in the same figure, in the CaAl:OLZ 20% diffractogram, it is already

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possible to note the presence of a small, but notable, peak related to the OLZ at 8.78º

280

(2θ), although the others reflections presented in OLZ pattern still remain absent. This

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suggests that, as the drug concentration increases, the CaAl-LDH begins to lose its

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ability to stabilize the OLZ amorphous form, which may lead to crystalline drug

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precipitation. Similarly, the diffraction pattern of CaAl:OLZ 30% present the same peak

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at 8.70° (2θ), but in higher intensity, which is related to the number of repetitions of this

285

same refraction plane. This diffractogram also showed another OLZ peak at 17.06º (2θ).

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However, the PM CaAl:OLZ 30% presented peaks between 10.74º and 24.02º (2θ) with

287

considerably greater intensity, besides the peaks mentioned previously in the binary

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system with same drug concentration.

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that, in the same way as the system discussed above, NiAl:OLZ 5% presented only the

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reflection patterns for the unloaded NiAl-LDH, while the PM NiAl:OLZ 5% presented

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the same OLZ peak at 8.90º (2θ). This shows that, despite the presence of impurities in

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NiAl-LDH, (see section 3.4), this system was still effective in stabilizing the amorphous

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form of the drug. The NiAl:OLZ 20% diffractogram shows the presence of drug peaks

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with values of 2θ between 8.96º and 24.18º. Also, in the NiAl:OLZ 30% diffractogram,

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no significant differences were observed when compared to the PM NiAl:OLZ 30%.

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These facts indicate that NiAl-LDH was not as efficient to decrease OLZ crystallinity as

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CaAl-LDH.

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3.3 Infrared absorption spectroscopy

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The Fourier transform infrared spectroscopy of both unloaded CaAl (Figure 2A)

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and NiAl-LDH (Figure 2B) present characteristic bands of hydrotalcite type compounds

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reported in the literature [16,46]. It is possible to observe broad bands between 3700

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and 3400 cm-1, related to the axial deformation of O—H bonds present in the layers’

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hydroxyls and water molecules adsorbed on the material, as well as the angular

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deformation of water molecules intercalated in the interlayer space, in the frequency

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between 1650 and 1620 cm-1. The nitrate anions vibrations in the interlayer space and

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the bands related to the interactions between metals and the oxygen of the hydroxyls,

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can be noted between 1410 - 1400 cm-1 and below 1000 cm-1, respectively. The bands

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between 1340 and 1360 cm-1 may correspond to the presence of carbonate ions, due to

311

the absorption of CO2 from the atmosphere during the synthesis process [16,43,44].

EP

TE D

M AN U

301

The OLZ spectrum shows the characteristic stretch of the only N—H bond

313

present in this molecule. This broad band and its transition to smaller wavelengths, as

314

well as the C=N band below 1600 cm-1, evidences the participation of these atoms in the

315

formation of hydrogen bonds of OLZ form I. Between 1600 and 1500 cm-1, the bands

316

are also associated with the double C=C bonds and the angular deformations of the C—

317

H and N—H bonds. [41,55,56]. The main differences between the OLZ polymorphs are

318

in the 600 to 1600 cm-1 region, where it was possible to identify characteristic peaks of

319

the form I, as the band present in 1517 cm-1, corroborating with the X-ray diffraction

320

data, presented earlier. The observed bands of OLZ spectrum are in accordance with

321

previous studies reported in the literature [55].

AC C

312

322 10

ACCEPTED MANUSCRIPT 323

Figure 2

324 In figure 2A, we can observe the overlapped vibrational spectra of the

326

CaAl:OLZ systems in different concentrations, the physical mixtures and its isolated

327

components, for comparative purposes. Comparing the CaAl:OLZ 5% system with its

328

isolated components, it shows lower intensity bands related to the axial deformation of

329

O—H bonds, present in the LDH layer and in the water molecules adsorbed on the

330

material surface, besides the displacement of the band in 3444 cm-1. In 3215 cm-1, the

331

band related to the OLZ N—H bond is not present in the CaAl:OLZ 5%, but can be

332

noted in the PM with the same drug concentration. In the following region, there was

333

considerable detachment and suppression of bands related to the C—H bonds vibration

334

of the benzene and thiophene ring, which was not observed in the PM.

SC

RI PT

325

Observing the CaAl:OLZ systems, in 1648 cm-1, it is notable a decrease in the

336

intensity of the characteristic band of O—H bonds of the water molecules, already

337

observed in the unloaded CaAl-LDH, that indicated the presence of water molecules in

338

the interlayer space. Also, the CaAl:OLZ 5% system presents no bands in the region

339

related to the C=N and C=C bonds vibrations, highlighted in gray, which was possible

340

to be note in the PM. It indicates a drug interaction between drug and delivery system,

341

that prevents the free vibration of the thiophene and diazepine rings. This hypothesis

342

supported by the bands suppression in 1009, 964 and 744 cm-1, related to characteristic

343

benzene, piperazine, diazepine and thiophene ring deformations.

TE D

M AN U

335

Such variations indicate that hydration water molecules may have given place to

345

the OLZ as it has, gradually, precipitated and adsorbed on the material surface,

346

promoting physical interactions. This justifies the suppression, intensity decrease or

347

displacement of certain OLZ bands, which prove that there was, in fact, interaction

348

between drug and carrier. In addition, changes below 1100 cm-1 suggest an interaction

349

with the material that involved the entire drug molecule.

AC C

350

EP

344

The NiAl:OLZ systems were also efficient in promoting interactions with OLZ.

351

In figure 2B, it can be observed that the NiAl:OLZ 5% system promoted similar

352

interactions compared to those mentioned in the CaAl:OLZ systems. In the frequency

353

between 3700 and 3100 cm-1, it is possible to observe the intensity decrease of the band

354

related to the O—H bonds axial deformation, and the suppression of the band related to

355

the OLZ N—H bond. One difference, when comparing with the CaAl:OLZ systems,

356

was observed in the region between 3000 and 2750 cm-1, highlighted in gray, which is a 11

ACCEPTED MANUSCRIPT suppression of the bands related to the C—H bonds, that were still evident in the PM

358

with the same drug concentration. This fact suggests that there was a greater interaction

359

with the OLZ carbon skeleton in this system. The bands in the lower frequency regions

360

also showed similar patterns to the previous system, suppressing the bands related to the

361

C=N, C=C, C—N and C—C bonds, as well as benzene ring breathing and piperazine,

362

diazepine and thiophene ring twists.

RI PT

357

It is known that events such as suppression, intensity decrease, displacement and

364

band enlargement, that were observed in similar regions in both systems (CaAl:OLZ

365

and NiAl:OLZ) in all concentrations, may be related to the formation of hydrogen

366

bonds. However, corroboration with the X-ray diffraction, as the drug concentration was

367

increased, the drug interactions with the respective LDH decreased gradually, as can be

368

noted with increasing of intensity and number of bands present. However, comparing

369

the CaAl:OLZ 30% and NiAl:OLZ 30% systems and their respective PM, it is possible

370

to notice a great difference in the intensity of these bands when compared with the free

371

drug, proving that, although it is probably saturated, the material still promotes

372

considerable interaction.

373 3.4 Thermal Analysis

TE D

374

M AN U

SC

363

The DSC and TG curves showed the thermal behavior of the free OLZ, loaded

376

and unloaded LDHs and their PM (Figure 3). The free OLZ melting point (Figure 3A)

377

consists of a well-defined endothermic event, typical of crystalline compounds, with

378

melting range between 193.59 (Tonset) and 196.73° C (Tendset), peak in 194.89° C, in

379

agreement with the literature [25,42,57]. It is possible to observe that there is no loss of

380

mass during this event and that, soon after its ending, the fusion is followed by the drug

381

degradation onset [42]. According to the literature, this melting point is the only

382

identifiable event in the DSC curves for the OLZ form I [57], a result that agrees with

383

the results discussed in the X-ray diffraction and Infrared analyzes. The derivative of the

384

TG curve shows the degradation event occurring approximately between 280 to 350 °

385

C, with its peak at 325.26 ° C.

AC C

EP

375

386

TG, DTG and DSC curves of CaAl-LDH and NiAl-LDH, respectively, are

387

represented in Figure 3B and 3C respectively, where it is noted that all the endothermic

388

events presented in the DSC curves are associated with mass loss in TG curves,

389

characteristic events of layer compounds with water molecules and hydroxyls in its

12

ACCEPTED MANUSCRIPT 390

structure [43]. In general, two main endothermic events were evidenced during the

391

compounds decomposition and their loss of mass. The first TG curve event of CaAl-LDH (Figure 3B), with Tonset at 66.21° C and

393

Tendset at 139.46° C, represents the mass loss (-10.821 %) resulting from the removal of

394

physically adsorbed water molecules on the layers surfaces and interlayer space. This

395

phenomenon was also observed in the TG curve of NiAl-LDH (Figure 3C), with Tonset at

396

82.51 ° C and Tendset at 144.96° C, losing 3.834% of its mass. These events can be

397

identified separately by the DSC curve of both LDHs in this same temperature range,

398

where the first endothermic event corresponds to the release of water adsorbed on the

399

surface, and the second event related to the water present in the interlayer space [43].

SC

RI PT

392

In the second event, the TG curve of CaAl-LDH presented Tonset at 215.40° C

401

and Tendset at 272.65° C, with 11.21 % of mass loss, while the NiAl-LDH curve had

402

Tonset at 285.66° C and Tendset at 319.37° C, with 14.913 % of mass loss. This event is

403

associated, in both cases, with the layer dehydroxylation and the reduction of interlayer

404

nitrate to nitrite [16].

M AN U

400

405

Table 3: Temperatures and enthalpy of the endothermic melting event of free Olanzapine, load and unloaded CaAl-LDH and NiAl-LDH, and their PM. Sample

Tonset DSC (°C)

Tpeak DSC (°C)

Tendset DSC (°C)

193,59

194,89

196,73

CaAl:OLZ 5%

-

-

-

CaAl:OLZ 20%

187,27

192,44

195,22

CaAl:OLZ 30%

191,64

194,41

196,50

PM CaAl:OLZ 5%

192,82

194,67

196,30

PM CaAl:OLZ 30%

193,70

195,05

196,64

NiAl:OLZ 5%

193,45

194,97

197,98

NiAl:OLZ 20%

193,66

194,90

196,47

NiAl:OLZ 30%

193,52

194,92

196,79

PM NiAl:OLZ 5%

193,83

194,93

199,23

PM NiAl:OLZ 30%

193,95

196,13

199,37

AC C

EP

OLZ

TE D

406 407

408 409 410

Figure 3

411 412 13

ACCEPTED MANUSCRIPT 413

Figure 4

414 Table 3 gather values of onset, peak and endset temperature of OLZ melting

416

event for CaAl:OLZ and NiAL:OLZ systems. Figure 4A shows the DSC curves of

417

CaAl:OLZ systems, free OLZ, unloaded CaAl-LDH, and the PMs for comparative

418

purposes. It is easily observed that the CaAl:OLZ 5% system presents only the water

419

loss and dehydroxylation events related to CaAl-LDH, absent the characteristic OLZ

420

melting point. Therefore, it is the exact overlap of the unloaded CaAl-LDH DSC curve.

421

In the PM CaAl:OLZ 5% curve, it was already possible to notice an endothermic event

422

with peak at 194.67° C, similar to the free OLZ, indicating that the PM did not show

423

any interaction with the drug that would change its thermal behavior significantly.

SC

RI PT

415

In the CaAl:OLZ 20% system thermograms (Figure 4A), it is already possible to

425

note the OLZ melting point with significant alterations (Tonset at 187.27° C and peak at

426

192.44° C), different from the free drug, which showed Tonset at 193.59° C and Tpeak at

427

194.89° C. The drug melt onset decreased 6.32° C, while de melting peak decreased

428

2.45° C. In the same way, there was a decrease on Tonset (191.64° C) for CaAl:OLZ 30%

429

system, which was not observed in the physical mixture. However, there was no

430

significant change in the Tpeak.

TE D

M AN U

424

The reduction of the drug melting point to lower temperatures can be explained

432

by the interaction between the LDHs and OLZ during the heating process of each

433

analysis. The CaAl:OLZ 5% system curve shows complete disappearance of the

434

endothermic peak corresponding to the OLZ solid-liquid transition. Such event,

435

reductions in the peak size, or even the enlargements, indicate that there was a decrease

436

in the drug crystallinity, corroborating with the results of X-ray diffraction presented

437

previously [58,59].

AC C

438

EP

431

Figure 4B shows the DSC curves of NiAl:OLZ systems, as well as their isolated

439

components, and respective PMs for comparative purposes. It can be noted that, in the

440

NiAl:OLZ 5% system, it is possible to observe the endothermic event related to the

441

OLZ melting. In contrast to the CaAl:OLZ systems, there were no significant changes in

442

Tonset or Tpeak. This indicates that NiAl-LDH was not as efficient as the CaAl-LDH in

443

the stabilization of the drug amorphous form.

444 445

3.5 In vitro Dissolution Profile

14

ACCEPTED MANUSCRIPT 446

In Figure 5A represents the dissolution profiles of free OLZ, the CaAl:OLZ

447

systems and the physical mixtures. In the first 30 minutes, it was observed that the free

448

OLZ soluble concentration was below the limit of detection of the analytical

449

methodology employed. The CaAl:OLZ 5% system, in 5 minutes, provided 18.0% of

450

soluble OLZ

451

compared to the free drug.

RI PT

released and 62.05% in 30 minutes. Considerable difference when

452 453

Figure 5

454

At 45 minutes of dissolution, only 3.49% of free OLZ was dissolved, while

456

CaAl:OLZ 5% presents 63.65% of soluble drug released. This value represents an

457

increase of 1823.78% in dissolution rate. The binary systems with 20 and 30% OLZ

458

started to promoted solubilization at 10 minutes, while the physical mixtures with 5 and

459

30% of drug loaded started at 30 minutes, as well as the free OLZ. At the end of the

460

dissolution, the CaAl:OLZ 5%, 20% and 30% systems promoted a total drug release of

461

89.08, 76.94 and 58.53% respectively. The physical mixtures with 5 and 30% of OLZ

462

presented maximum solubilization of 39.53 and 42.58%, respectively.

M AN U

SC

455

In Figure 5B, we can analyze the dissolution profiles of free OLZ, the NiAl:OLZ

464

systems, and the physical mixtures. Similarly to the systems containing NiAl:OLZ 5%

465

system also initiated the dissolution of the drug at 5 minutes of dissolution, presenting

466

19,11 % of drug release OLZ, while the PM NiAl:OLZ 5% only started the at 45

467

minutes, with 11.06 % of drug released. At the same point, while the free drug had 3.49

468

% of its total amount dissolved, the NiAl:OLZ 5% system promoted a drug release of

469

37.35 %, which represents an increase of 1070.02% in the rate of dissolution.

EP

The NiAl:OLZ 20% system started the drug release at 20 minutes (Figure 5B),

AC C

470

TE D

463

471

different from the system with 30 % drug loaded (10 min). At 180 min, both released

472

about 55 % of the drug, showing no significant differences. At the end of the

473

experiment, the NiAl:OLZ with 5, 20 and 30 % drug loaded provided drug release of of

474

61.66, 55.46 and 55.34 % of OLZ, respectively, whereas the physical mixtures with 5

475

and 30 % released 39,87 and 37.20 % of the drug, respectively, similar to the physical

476

mixtures with CaAl-LDH.

477

Knowing that the OLZ is a weak base (pKa = 7.5 ± 0.5), its dissolution is

478

favored under acidic conditions (22). However, even with the dissolution being

479

performed at pH 6.8, the CaAl:OLZ 5% system released almost 90% of the drug at 180 15

ACCEPTED MANUSCRIPT 480

minutes. In another dissolution study with the OLZ complexed in cyclodextrin, aiming

481

the drug solubility improvement [25], the authors achieved a maximum drug release

482

about 35% at 30 minutes. This value did not increase until the end of the experiment in

483

60 minutes. In a brief comparison, the CaAl:OLZ 5% system was more efficient than

484

the system proposed by the mentioned authors in increasing dissolution rate of OLZ In another study, solid dispersions of Olanzapine and two different carriers,

486

pregelatinized starch and sodium starch glycolate, also succeeded in increasing the

487

dissolution rate of the drug, when compared to the free OLZ. At 60 minutes, the authors

488

achieved maximum drug release about 70% in the most efficient proportion of the

489

formulation components. This result is similar to the one presented in this work,

490

mentioned in the previously [58].

SC

RI PT

485

It is expected that the systems containing 30% of drug load will promote a lower

492

dissolution rate, when compared to the systems with 5% OLZ, since the more

493

concentrated systems presents a higher degree of crystalline drug and less interaction

494

with the carrier [25,60]. The fact that the physical mixtures of both LDHs showed no

495

increase in the dissolution rate, also corroborates the results presented by the X-ray

496

diffraction and the thermal analysis, since these formulations showed overlapping of

497

crystalline patterns and drug melting point. The same applies to the infrared spectra,

498

where the physical mixtures showed overlapping bands related to the free OLZ.

TE D

M AN U

491

499 500

3.6 Hemolysis Inhibition Induced by AAPH The hemolysis inhibition of the erythrocyte is a great assay to investigate the

502

damage of free radical-induced membrane oxidation [61]. In this test, AAPH causes

503

hemolysis by the oxidation of lipids that make up the cell membrane, causing the loss of

504

the membrane integrity, leading to cell death [62]. In Figure 6, we analyze the

505

percentage of AAPH-induced hemolysis inhibition in erythrocytes incubated with the

506

CaAl:OLZ 5%, NiAl:OLZ 5%, CaAl-LDH, NiAl-LDH and free OLZ. The results were

507

77.48 ± 1.08, 64.04 ± 1.04, 77.24 ± 0.51, 66.92 ± 0.88 and 74.21 ± 0.65%, respectively,

508

when compared to the negative control (100% hemolysis). The trolox standard, in

509

concentrations of 50, 100 and 200 µg/mL, obtained a maximum reduction of 82.46 ±

510

0.74% of hemolysis.

AC C

EP

501

511 512

Figure 6

513 16

ACCEPTED MANUSCRIPT According to the results of the hemolysis inhibition, the EC50 values for the

515

CaAl:OLZ 5%, NiAl:OLZ 5% and free OLZ were 36.55, 21.04 and 23.32 µg/mL,

516

respectively, with 95% confidence interval ranging from 30.71 to 43.51, 10.67 to 41.51

517

and 19.05 to 28.54, respectively. The unloaded LDHs showed EC50 of 19.31 µg/mL for

518

CaAl-LDH and 18.67 µg/mL for NiAl-LDH with 95% confidence intervals ranging

519

from 13.74 to 27.24 and 19.05 to 28.54, respectively.

RI PT

514

Note, in Figure 6, that both systems and their free components showed

521

considerable hemolysis inhibition. Comparing the results with trolox, it is possible to

522

observe that the free OLZ presented greater hemolysis inhibition in concentrations of 50

523

and 100 µg/mL. No increase in the drug hemolysis inhibition capacity was observed

524

when associated with either CaAl-LDH or NiAl-LDH. However, both systems,

525

CaAl:OLZ 5% and NiAl:OLZ 5%, and the unloaded LDHs showed a statistically

526

significant hemolysis inhibition (p ≤ 0.05) when compared to the negative control.

M AN U

SC

520

Erythrocytes have been shown as a good choice in the study of patients with

528

psychiatric disorders. In a pioneering research with a large number of patients

529

diagnosed with schizophrenia, the researchers noted that under stable feeding conditions

530

or peroxidative conditions in the brain, the distribution of essential polyunsaturated fatty

531

acids in the red blood cell membranes reflects in the distribution in the central nervous

532

system [63]. This study reveals that such fatty acids are present in lower concentrations

533

in patients treated with Haloperidol, Clozapine, Olanzapine or Risperidone, when

534

compared to the control group. This is mainly due to the increase of lipid peroxidation

535

in the erythrocyte membrane, as evidenced by the increase of thiobarbituric acid

536

reactive substances levels, promoting hemolysis. The association between OLZ and

537

CaAl-LDH promotes a gain in the lipid peroxidation inhibition, when compared to free

538

OLZ, as proved in another work published by our research group [20]. Therefore,

539

avoiding hemolytic reactions is of great importance, since other antipsychotics, such as

540

quetiapine,

541

thrombocytopenia purpura [64].

AC C

EP

TE D

527

are

also

associated

with

blood

disorders

such

as

thrombotic

542 543

3.7 In vitro toxicity assay

544

The in vitro toxicity of the systems and their individual components was

545

evaluated by the A. salina test, which is considered useful for a preliminary general

546

evaluation, and there is good correlation with some cytotoxic assays with human cells

547

[65]. Thus, were evaluated the CaAl:OLZ 5%, NiAl:OLZ 5%, free OLZ and unloaded 17

ACCEPTED MANUSCRIPT 548

CaAl-LDH and NiAl-LDH in concentrations equivalent to those present in the systems

549

(Figure 7). After 24h (Figure 7A) of experiment, in the first count of A. salina specimens, it

551

was possible to observe that there was no death in the negative control, as well as in the

552

tubes containing the CaAl:OLZ 5% and unloaded LDHs, as shown in figure 13. The

553

NiAl:OLZ 5% presented a mortality rate of 1.11 ± 1.92 % at the equivalent drug

554

concentration of 100 µg/mL, but was not statistically significant (p ≤ 0.05) when

555

compared to the negative control with 0% mortality rate (Figure 7A). At the equivalent

556

concentration of OLZ (200 µg/mL), the NiAl:OLZ 5% system presented mortality rate

557

of 3.33 ± 0.0 %. In the case of the free OLZ, there was a mortality rate of 38.88 ± 12.61,

558

62.22 ± 1.92 and 84.44 ± 7.69% at drug concentrations of 50, 100 and 200 µg/mL,

559

respectively. The positive control, performed with potassium dichromate, presented a

560

mortality rate of 55.55 ± 8.38, 90.0 ± 5.77 and 98.8 ± 1.92% at the concentrations of 50,

561

100 and 200 µg/mL, respectively. The NiAl:OLZ 5% presented a EC50 value of 128.1

562

µg/mL with a 95% confidence interval ranging from 17.55 to 934.9 µg/mL, whereas the

563

free OLZ showed EC50 = 89.16 µg/mL with a 95% confidence interval ranging from

564

29.47 to 269.8 µg/mL.

M AN U

SC

RI PT

550

It was observed that there was a statistically significant (p ≤ 0.05) decrease in

566

OLZ toxicity in the CaAl:OLZ 5% system, reducing its mortality rate to 0%, even

567

though it had the same drug concentration as the tubes with free OLZ. As for the

568

NiAl:OLZ 5%, there was a small, but significant (p ≤ 0.05), mortality rate in the most

569

concentrated sample (200 µg/mL equivalent of OLZ) when compared to the negative

570

control. The unloaded CaAl-LDH and NiAl-LDH showed no mortality rate against A.

571

salina specimens on the first day of counting.

573 574 575

EP

AC C

572

TE D

565

Figure 7

A second count was performed with 48 hours (Figure 7B) and it was possible to

576

observe an increase in the mortality rate in the samples of both binary systems, free

577

OLZ, and positive control with potassium dichromate. At the respective concentrations

578

of 50, 100 and 200 µg/mL, the CaAl:OLZ 5% presented a mortality rate of 2.22 ± 3.84,

579

2.22 ± 1.92 and 6.66 ± 0.0 % and EC50 of 190.0 µg/mL, respectively. In the same

580

concentrations sequence, NiAl:OLZ 5% presented 24.44 ± 8.38, 26.66 ± 6.66 and 55.55

581

± 11.70 % mortality rate and EC50 = 169.1 µg/mL. 18

ACCEPTED MANUSCRIPT The statistical analysis, in a 95% confidence interval, showed that there was no

583

significant difference between the mortality rates presented by the negative control and

584

the CaAl:OLZ 5% system in 48 hours of the experiment, while the free drug presents a

585

high mortality rate, with no statistically significant difference (p ≤ 0.05), when

586

compared to the positive control (K2Cr2O7) at concentrations of 100 and 200 µg/mL.

587

However, in the comparison between the negative control and the NiAl:OLZ 5%

588

system, there is a statistically significant mortality rate, but still considerably lower than

589

the rates reported by the free OLZ, presenting a decrease of 30.13, 30.37 and 56.81 % of

590

the drug mortality rate at concentrations equivalents of 50, 100 and 200 µg/mL,

591

respectively. It is important to point out that in the 24h and 48h counts the samples with

592

unloaded CaAl-LDH and NiAl-LDH showed no toxicity, keeping alive all the A. salina

593

specimens, as well as the negative control.

595

M AN U

594

SC

RI PT

582

4 CONCLUSION

The physical-chemical characterization demonstrated a directly proportional

597

relationship between the OLZ concentration in the binary systems, with both LDHs, and

598

its crystalline precipitation. The systems with lower OLZ concentrations presented a

599

greater interaction with the carriers, that were able to stabilize the amorphous

600

conformation of the drug. Therefore, the CaAl:OLZ 5% and NiAl:OLZ 5% showed

601

better results, but the binary systems in all concentrations, with both LDHs, promoted

602

an increase in the OLZ dissolution rate, which was not observed in any of the physical

603

mixtures. Also, the CaAl:OLZ 5%, NiAl:OLZ 5%, the unloaded CaAl-LDH and NiAl-

604

LDH showed promising results in the antioxidant and toxicity tests, with no in vitro

605

toxicity at all by the unloaded carriers, reinforcing the proposed use of LDHs as

606

functional excipients in the pharmaceutical industry with great expectations for future in

607

vivo tests.

AC C

EP

TE D

596

608 609

FUNDING SOURCES

610 611

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

612 613

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1X-ray diffraction patterns of : (A) CaAl:OLZ systems, unloaded CaAl-LDH, free OLZ and physical mixtures; and (B) NiAl:OLZ systems, unloaded NiAl-LDH, free OLZ and physical mixtures.

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Figure 2: Infrared vibrational spectra of (A) CaAl:OLZ systems, unloaded CaAl-LDH, free OLZ and physical mixtures; and (B) NiAl:OLZ systems, unloaded NiAl-LDH, free OLZ and physical mixtures. Figure 3: TG, DTG and DSC curves of (A) free OLZ, (B) unloaded CaAl-LDH, and (C) unloaded NiAl-LDH under nitrogen atmosphere (50 mL/min) and heating rate of 10° C/min.

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Figure 4: DSC curves of (A) CaAl:OLZ systems, unloaded CaAl-LDH, free OLZ and physical mixtures, and (B) NiAl:OLZ systems, unloaded NiAl-LDH, free OLZ and physical mixtures, obtained under nitrogen atmosphere (50 mL/min) and heating rate of 10° C/min. Figure 5: OLZ dissolution profiles of (A) CaAl:OLZ systems and physical mixtures, and (B) NiAl:OLZ systems and physical mixtures in 900 mL of phosphate buffer solution, pH 6.8, temperature 37° C and 50 rpm.

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Figure 6: Erythrocyte hemolysis inhibition capacity of the CaAl:OLZ 5%, NiAl:OLZ 5% (1000, 2000 and 4000 µg/mL), free OLZ and the unloaded CaAl-LDH and NiAlLDH in concentrations equivalent to those present in the system. The results represent the in vitro inhibition mean ± SD, n = 3, of the experiment in duplicate. Trolox (50, 100 and 200 µg/mL) was used as standard antioxidant. *p ≤ 0.05 vs. negative control (100% hemolysis induced by AAPH) (ANOVA and Student-Neuman-Keuls as post hoc test).

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Figure 7: 24 hours (A) and 48 h (B) toxicity in Artemia salina of CaAl:OLZ 5%, NiAl:OLZ 5% (1000, 2000 and 4000 µg / mL), free OLZ and unloaded LDHs in concentrations equivalent to those present in the systems. The results represent the in vitro mortality rate mean ± SD, n = 3, of the experiment in duplicate. Potassium dichromate (50, 100 and 200 µg/mL) was used as standard toxic agent. *p ≤ 0.05 vs. negative control (PBS buffer) (ANOVA and Student-Neuman-Keuls as post hoc test).

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