Dissolution and physicochemical stability enhancement of artemisinin and mefloquine co-formulation via nano-confinement with mesoporous SBA-15

Accepted Manuscript Title: Dissolution and Physicochemical Stability Enhancement of Artemisinin and Mefloquine Co-Formulation via Nano-Confinement with Mesoporous SBA-15 Authors: Kumaran Letchmanan, Shou-Cang Shen, Wai Kiong Ng, Reginald B.H. Tan PII: DOI: Reference:

S0927-7765(17)30261-8 http://dx.doi.org/doi:10.1016/j.colsurfb.2017.05.003 COLSUB 8532

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

16-2-2017 18-4-2017 1-5-2017

Please cite this article as: Kumaran Letchmanan, Shou-Cang Shen, Wai Kiong Ng, Reginald B.H.Tan, Dissolution and Physicochemical Stability Enhancement of Artemisinin and Mefloquine Co-Formulation via Nano-Confinement with Mesoporous SBA-15, Colloids and Surfaces B: Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.05.003 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.

Graphical Abstract

Highlights    

The dissolution rate of ART and MFQ was enhanced in a single dosage formulation. Characteristic studies clearly indicate the amorphization of the crystalline APIs. ART/MQF/SBA-15 (1:2:3) shows a superior dissolution compared with crystalline APIs. ART/MQF/SBA-15 (1:2:3) possess excellent physicochemical stability for 6 months.

Dissolution and Physicochemical Stability Enhancement of Artemisinin and Mefloquine Co-Formulation via Nano-Confinement with Mesoporous SBA-15

Kumaran Letchmanana,*, Shou-Cang Shena, Wai Kiong Nga, Reginald B.H. Tana,b,** a

Institute of Chemical and Engineering Sciences, A*STAR (Agency for Science, Technology

and Research), 1 Pesek Road, Jurong Island, Singapore 627833, Singapore b

Department of Chemical and Biomolecular Engineering, National University of Singapore,

4 Engineering Drive 4, Singapore 117576, Singapore ABSTRACT The objective of this study is to enhance the dissolution rate, supersaturation and physicochemical stability of combination of two poorly water-soluble anti-malarial drugs, artemisinin (ART) and mefloquine (MFQ), by encapsulating them inside mesoporous silica (SBA-15) via co-spray drying. Characteristic studies such as powder X-ray diffraction (PXRD), transmission electron microscopy (TEM) and scanning electron microscope (SEM) clearly indicate the amorphization of the crystalline drugs. ART/MQF/SBA-15 formulations show a superior dissolution enhancement with a burst release of more than 95% of drugs within 30 min. In addition, the combination formulation exhibits a stable supersaturation enhancement by 2-fold higher than that of the untreated crystalline counterparts. ART/MQF/SBA-15 samples possess excellent physicochemical stability under 2 different moderate storage conditions for 6 months. The amorphization of ART and MFQ via nanoconfinement using mesoporous SBA-15 is a potentially promising approach to enhance the solubility of poorly water-soluble anti-malarial drugs that co-formulated into a single dosage form.

Keywords: Artemisinin-based combination therapy, excipient, supersaturation, dissolution rate, physicochemical stability 

Corresponding author,

E-mail: * [email protected]; Tel: (65) 67963880, **[email protected]; Tel: (65) 6516 6360

1. Introduction Malaria is the most severe parasitic disease and one of the largest infectious killer of human beings [1]. It is a major public health and economic burden in third-world countries and has become a tropical disease of the first priority for the World Health Organization (WHO) [1-4]. Approximately 500 million clinical cases and 1–2 million of deaths are reported annually (85% of these deaths occur in sub-Saharan Africa), accounting for about 4– 5% of total fatalities in the world [5-7]. The traditional drugs employed to fight malaria such as chloroquine and quinine were becoming less effective, due to the rapid development of parasitic resistance [8]. In view of this, ART and its derivatives are the only hope and widely used to treat malaria patients [9]. Unfortunately, cases of increased in vitro tolerance against ART and its derivatives have already been discovered in South America and South East Asia [10, 11]. The emergence of ART resistance in natural parasite populations of Plasmodium falciparum (P. falciparum) may be only a question of time. Therefore, in order to delay the development of resistance and thus prolong the efficacy of ART and its derivatives, artemisinin-combination therapies (ACTs) were introduced and widely implemented. In general, ACTs have shown a remarkable double effect of preventing the emergence and spread of drug resistance due to ART monotherapy [12], and interrupting the transmission of P. falciparum. However, most of the antimalarial drugs (especially ART and its derivatives) exhibit poor oral bioavailability, owing to its poor aqueous solubility which renders their efficacy in ACTs. For example, the clinical use of ART is hampered by its low solubility in both water and blood. ART, which is classified under the Biopharmaceutics Classification System (BCS) class II has a solubility of 48 µg/ml in water at 37°C, with a half-life of 1-2 hours [13-15]. The poor solubility of ART results in a poor and erratic absorption upon oral administration, which induces limited and variable bioavailability (~32%) [16]. In addition, ART may cause

incomplete clearance of malaria parasites due to its short half-life and high first pass metabolism, resulting in recrudescence [13]. In order to address these shortcomings, several techniques have been developed to improve their dissolution rate and solubility [17-22]. However, up to our knowledge limited studies have reported the solubility enhancement of combinations of antimalarial drugs involving ART. Thus, in this study, the solubility enhancement of ART was studied in combination (co-formulation) with another class II antimalarial drug, mefloquine (MFQ). Mefloquine hydrochloride or [(2, 8- bis(trifluoromethyl)quinolin-4-yl]-(2-piperidyl)methanol HCl is an orally administered medication that is a highly effective drug used against multidrug-

resistant strains of Plasmodium falciparum. It is proposed to share a similar mechanism of action with Chloroquine, which is inhibition of Heme polymerase, even though the exact mechanism of action is uncertain [23]. MFQ is slightly soluble in water (1.8 mg/ml) and has high lipophilicity

(logP = 3.9). Plasma protein binding is about 98% and long elimination half-life is 2–4 weeks. It is classified as a Biopharmaceutics Classification System (BCS) class II or IV drug because of its lack of permeability data and low solubility [24]. As MFQ is present as hydrochloride salt, no significant attempts had been made for the enhancement of the solubility. In addition, to our knowledge, no studies have been reported on the solubility enhancement for combinations of two class II drugs in a single formulation using mesoporous carriers. Physical transformation of crystalline drug into a more soluble amorphous form via nano-confinement by using mesoporous carriers is considered as one of the most effective techniques to enhance the solubility of poorly water-soluble drugs [18, 25-27]. The growing interest in this material for drug delivery is mainly due to its important features such as uniform pore size distribution, tunable pore diameter, thick pore walls, large pore volume and specific surface area [25, 26, 28, 29]. In addition, features such as pore size, pore topology

and surface interactions are important parameters that can change the thermodynamics and crystallization kinetics of amorphous drugs to improve their physical stability [30-33]. Therefore, for the first time we have explored the potential use of mesoporous carrier to improve the solubility of the combination of two poorly water-soluble antimalarial drugs in a single dosage formulation. Mesoporous silica (SBA-15) was used as a drug excipient and the amorphization of poorly water-soluble drugs (ART and MFQ) was achieved by encapsulating them within the pore channels of the mesoporous particles using a co-spray drying technique. This work mainly focuses on enhancing the solubility and dissolution rate as well as retaining the storage stability of combination of these 2 drugs. The storage stabilities of formulated of ART/MFQ/SBA-15 samples were investigated under 2 different storage conditions for 6 months.

2. Materials and Methods 2.1.

Materials

Artemisinin (ART) was obtained from Junda Pharmaceuticals, Co. China and Mefloquine hydrochloride (MFQ-HCl) was from Provizer Pharma, India. Tri-block copolymer poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide), pluronics P123 (EO20-PO70EO20, MW: 5800) and tetraethyl orthosilicate (TEOS, 98%) were purchased from SigmaAldrich. All other reagents and solvents used in the study were reagent grade and were used without further purification. Activ-vial® was supplied by CSP technologies, USA.

2.2.

Synthesis of SBA-15

Ordered mesoporous silica, SBA-15, was synthesized via a rapid condensation process as reported by Shen et al. [34].

2.3.

Spray drying

The drug loaded samples were prepared by using BÜCHI B-290 mini spray dryer (BÜCHI Labortechnik AG, Switzerland) that operated at inert loop mode with N2 flow. The inlet temperature was maintained at 81 °C (slightly higher than the boiling point of ethanol, 78 °C) and the resulting outlet temperature at the above operating condition was approximately 4753 °C. In order to formulate ART/MFQ/SBA-15 (1:2:3) sample, 1 g of ART and 2 g of MFQ were dissolved in 200 ml of ethanol (Fisher Scientific Ltd., UK) and 3 g of SBA-15 were dispersed in the solution. The mixture was stirred overnight. The fine liquid suspension of ART, MFQ and SBA-15 was fed to the spray dryer via a peristaltic pump at a feed rate of 6.0 ml/min and sprayed into the chamber from a nozzle with a diameter of 406 μm at a pressure of 0.12-0.15 MPa. All the samples were dried in desiccators with silica gel under reduced pressure for 1 day before the characterizations.

2.4.

Physical mixture

The physical mixture of ART/MFQ at ratio of 1:2 w/w was prepared by thoroughly mixing the crystalline ART and MFQ in a turbula mixer (Turbula® T2F) at 49 rpm for 30 min until a homogeneous mixture was obtained. This physical mixture was characterized immediately after harvesting the sample from the glass vessels at the end of the mixing process. Physical mixture was denoted as P.M.

2.5.

Surface area and pore volume analyzer

Nitrogen adsorption-desorption isotherms were measured by using an Autosorb-6B gas adsorption analyzer (Quantachrome Instruments, Boynton Beach, FL) at the temperature of 196 °C (77 K). Approximately 0.10 g samples were used in each measurement. Prior to adsorption measurements, SBA-15 sample was degassed at 200 °C while drug loaded samples were outgassed at 40 °C under vacuum for 24 h to remove any residues and absorbed water. The specific surface areas of the samples were assessed using the linear region of the Brumauer-Emmett-Teller (BET) plots. The total pore volume was estimated from the amount of N2 adsorbed at a relative pressure of 0.95, while mesoporous size distributions were computed from the adsorption branch of N2 adsorption-desorption isotherms using the conventional Barrett-Joyner-Halenda (BJH) approach.

2.6.

Powder X-ray Diffraction (PXRD)

The PXRD was performed using a D8-ADVANCE (BRUKER, Madison, WI) X-ray diffractometer in steps of 0.028° using monochromatized Cu Kα radiation (λ = 0.1542 nm) as X-ray source and scanned over an angular range from 5 to 50o (2θ). The measurement

conditions were as follows: target, Cu; filter, Ni; voltage, 40 kV; current, 10 mA; scanning speed, 2°/min.

2.7.

Scanning Electron Microscopy (SEM)

The morphology of powder samples was examined by a high resolution scanning electron microscope (SEM, JSM-6700F, JEOL, Tokyo, Japan) operating at 5 keV under lower SEI (secondary electron image) and secondary electron image (LEI) mode. Prior to analysis, samples were mounted on double sided adhesive carbon tapes and coated with gold for 1 min by a sputter coater (Cressington Sputter Coater 208HR, UK).

2.8.

Transmission Electron Microscopy (TEM)

High resolution TEM images were taken by TECNAI F20 (G2) (FEI, Philips Electron Optics, Holland) electron microscope at 200 kV. Before examination, powdered samples were deposited on a copper grid with Formvar carbon film.

2.9.

Contact angle measurement

The contact angle measurements were carried out using the sessile-drop technique with Contact angle Analyzer KSV CAM 100 (Finland). Approximately 50 mg of sample powders were compressed into tablets by a hydraulic press at a pressure of 75 MPa for 1 min. A water droplet was placed on the compact surface using a microsyringe, and the droplet was photographed to determine the contact angles. Three measurements per sample were performed to ensure reproducibility. All the measurements were carried out at room temperature.

2.10.

In vitro drug release studies

The drug release of the formulated samples were measured by using in vitro dissolution tester, USP II, (Agilent 708-DS Dissolution Apparatus) in 900 ml of phosphate buffered saline (PBS) at 37 ± 0.5°C with the paddle speed of 100rpm. Powder samples with an amount equivalent to 50.0 ± 1.0 mg and 200 ± 1.0 mg of ART was used for dissolution test (under sink condition) and supersaturation (non-under sink condition) respectively. Each sample was mixed with 800 mg of cornstarch and pressed to tablet (13mm × 6mm) at a pressure of 75 MPa prior to each test. 3 ml of sample were withdrawn from the vessel at interval of 5, 10, 15, 30, 60 and 120 min and replaced by fresh dissolution medium. The collected samples were filtered over a PTFE membrane filters (pore size: 0.45 μm). The drug concentration was determined by using high performance liquid chromatography (HPLC, Agilent 1100 series). Drug release of the formulated samples was compared to that of their corresponding crystalline drugs. All experiments were performed in triplicates (n = 3) and the results were registered as an average with standard deviation.

2.11.

Chemical and physical stability

Chemical and physical stability tests of co-spray dried samples at controlled temperature and relative humidity (RH) were conducted based on the procedures from International Conference on Harmonization (ICH)-ICH Q1A (R2). The samples were tested for 6 months under 2 different storage conditions: open pan inside desiccators (25 ºC/18% RH) and ActivVial® (25 ºC). The physical stability of all the samples were analyzed using PXRD and the chemical stability were investigated using HPLC (Agilent 1100 series).

2.12.

Method of analysis

The concentration of ART was measured by HPLC (Agilent 1100 series) equipped with Eclipse XDB C18 (150 mm × 4.6 mm (i.d.) × 5 μm) (Eka Chemicals AB, Sweden) column [4, 35]. The mobile phase consisted of 50% of ultrapure water and 50% acetonitrile (HPLC grade). The flow rate was maintained at 1.0 ml/min and the UV detector was operated at a wavelength of 210 nm. Drug content was determined by calculating the peak area at 9.3 min. For MFQ, the concentration of the samples was measured by means of HPLC (Agilent 1100 series) equipped with Inertsil C8-3 (150 mm × 4.6 mm (i.d.) × 5 μm) (GL Sciences Inc., Tokyo, Japan) column [36]. The mobile phase which consisted of MeOH, acetonitrile and 0.05 M KH2PO4 (55:9:35, v/v/v) adjusted to pH 3.9 with 0.5% orthophosphoric acid was filtered through a 0.2 µm nylon filter. The flow rate was maintained at 1.00 ml/min and the UV detector was operated at a wavelength of 284 nm. Drug content was determined by calculating the peak area at 6.8 min. All the measurements were carried out in triplicates (n=3) to ensure replicability.

2.13.

Statistical analysis

Data were processed using Microsoft Excel 2003 software. Each sample was tested in triplicates and the mean ± standard deviation is reported. Two sample comparisons of means were carried out using Student’s t-test analysis and statistical significance was ascertained when p value was less than 0.05.

3. Results and Discussion 3.1.

Structural Characterization and Degree of Drug Loading

The adsorption-desorption isotherms and pore size distributions of SBA-15 before and after co-spray drying are respectively shown in Figure 1(A) and (B), whereas the structural information about SBA-15 is summarized in Table 1. Both SBA-15 and drug-loaded SBA-15 illustrate typical type IV adsorption isotherm containing the H1 hysteresis loop in the range of P/P0 at 0.50–0.77, associated with mesoporous materials [37]. SBA-15 has a uniform pore size distribution with a mean pore size of approximately 8.7 nm and total pore volume of 1.16 cm3/g and BET specific surface area of 809.0 m2/g. The large specific surface area and pore volume, and uniform pore size distributions of SBA-15 are able to encapsulate both the drug particles with drug loading of 47.9 wt% (Table 1). It is shown that more than 94% of drugs in the designed formulation could be loaded onto SBA-15 due to the large porosity of drug carrier and the high efficiency of spray dying technique. The sharp decreases in the total pore volume, BET surface area and pore diameter of SBA-15 (Table 1) implies the incorporation of ART and MFQ into the pores of the carrier via capillary forces. The narrowed hysteresis loop and the reduced amount of N2 adsorption suggest a limited size of pores after the encapsulation of the drug particles. Meanwhile, the tail in the pore size distribution of SBA15 at small pore diameters corresponds to about 12% of micropores [Figure 1 (B)], which vanished upon loading due to either filling or obstruction by the presence of drug particles.

3.2.

Physical Characterization

The physical-state of raw APIs and freshly co-spray dried samples are analyzed with PXRD (Figure 2). Raw ART is crystalline in nature as indicated by the highly intense PXRD peaks mostly between 7.0° and 25.0°, exemplify the typical reflections of the orthorhombic polymorphic form of the drug [38]. Meanwhile, raw MFQ shows PXRD peaks with relatively

reduced intensity as compared with ART. Characteristic peaks at diffraction angles (2θ) between 10.0° to 25.0° indicate the crystalline nature of MFQ, which is in agreement with form D polymorph of the drug [39]. On the other hand, all the co-spray dried samples of ART/SBA-15, MFQ/SBA-15 and ART/MFQ/SBA-15 exhibit halo PXRD patterns without observable peaks attributable to the crystalline ART and MFQ, indicating that no crystalline drugs were detected by X-ray diffraction. The physical state of ART and MFQ are fundamentally changed from the crystalline to amorphous form after incorporated into SBA15. In comparison, all the physically mixed samples showed sharp and specific diffraction peaks attributed to the crystalline MFQ and ART and no changes in their physical state and particle size were observed after the physical mixing (Figure S1 and S2). Pore size at nano-scale [40], drug-carrier interactions [41] and rapid evaporation of the solvent [42] during formulation process appeared to be responsible for the formation of amorphous solid dispersion. SBA-15 with a narrow pore size distribution in the range of a few nanometers is effective in preventing the formation of crystals due to spatial confinement. Additionally, the drug molecules have less time to arrange into a crystal lattice due to the rapid evaporation of organic solvent during co-spray drying, thereby predisposing the molecules into forming a disordered structure instead of an ordered one.

3.3.

Morphology

The crystalline ART appears to be in an orthorhombic form with prism and rod-like particles (particle size of 50–1000 μm); meanwhile, MFQ is in needle-shaped structure (particle size of 1–50 μm), resembling the morphology of the polymorph form D (Figure 3). These two forms are the thermodynamically stable forms of ART and MFQ at 37°C [39]. In the meantime, SBA-15 appears as aggregates of spherical and spheroid shaped particles with particle size of 0.5−1 µm. There are no changes in the morphology and the particles size of

SBA-15 after being co-spray dried with ART or/and MFQ. Drug crystals are hardly noticed on the surface of SBA-15 as most of the drug particles are dispersed and confined within the pore channels of SBA-15 in an amorphous form. Moreover, the presence of SBA-15 particles during the co-spray drying is able to inhibit the particle aggregation and crystal growth of ART and MFQ. The nano-confinement of drug particles inside the pore channels of SBA-15 is validated and confirmed by co-spray dries ART with non-porous silica. Presence of crystalline particles can be detected by PXRD (Figure S5) for the co-spray dried sample of ART/non-porous silica (1:9 w/w). Samples with 1:3 w/w and 1:1 w/w of drug-to-carrier weight ratios show even stronger X-ray diffraction intensity with the same pattern of raw ART. The presence of crystalline particles on the external surface of non-porous silica can be clearly observed by SEM images (Figure S6) for samples with drug-to-carrier weight ratios of 1:3 w/w and 1:1 w/w. These crystalline particles were not observed on the external surfaces of SBA-15 even after co-spray drying at the high ratio of 1:1 w/w. These results indicated that the non-porous silica which lacks in pore channels and total pore volume is unable to accommodate and amorphize even as low as 10 wt% of ART and thus crystalline ART formed on the external surfaces. In comparison, the ability of SBA-15 to present in amorphous form even at high drug loading of 50 wt%, indicating that the ART molecules encapsulated inside the pore channels of SBA-15 after being co-spray dried. Presence of extra particles on the external surface of SBA-15 only can be observed when the drug loading was exceeded the pore volume capacity of SBA-15, as can be seen for ART/MFQ/SBA-15 (2:4:1 w/w/w) (Figure S6).

3.4.

Transmission Electron Micrograph (TEM)

Figure 4 shows the structural features of the SBA-15 examined by TEM. The TEM micrographs reveal the spherical and spheroid shape of SBA-15 micron-particles with a particle size of 0.5–1 µm (Figure 4); in agreement with the shape determined by SEM measurements (Figure 3). At higher magnifications, the ordered cylindrical pore channels of SBA-15 can be observed with an estimated uniform pore size in the range of 8–9 nm (in agreement with BET results) and thick pore walls. Moreover, the pore system of SBA-15 is in a well-ordered hexagonal arrangement and straight lattice fringes were viewed, confirming the existence of a two-dimensional hexagonal structure of p6mm symmetry [43]. The thick pore walls and well-ordered pore channels are able to afford homogeneous drug distribution and restrict drug re-crystallization. In addition, the wide pore size and short pore channels are advantageous for the adsorption and desorption of drug molecules with minimum diffusion resistance, which will be beneficial for a better dissolution and solubility of ART. Figure 4(c)−(e) illustrate the unchanged appearance of SBA-15 pore channels and pore walls after loading with ART and MFQ particles which verifies that the drug encapsulation did not change the appearance and the pore structures of SBA-15.

3.5.

In vitro Drug Release

Figure 5(A) depicts the drug release profiles of co-formulated ART/MFQ/SBA-15 (1:2:3 w/w/w) under sink condition using USP II. The drug release behaviors of the formulated samples are compared with P.M.-ART/MFQ (1:2 w/w). The P.M.-ART/MFQ (1:2 w/w) achieves only marginal dissolution rates: 21.2% of ART and 25.1% of MFQ in the first 15 min. The total drug dissolved is only 43.0% for ART and 59.7% for MFQ in 2 h. This indicates the poor dissolution of the raw drugs due to their crystalline and hydrophobic nature (hydrophobicity of the crystallize drugs is indicated by their relatively large contact angle as

shown in Table S1). Meanwhile, a markedly superior drug release is observed for ART/MFQ/SBA-15 (1:2:3 w/w/w): initial burst release of 94.7% of ART and 92.1% of MFQ is observed in 15 min; and 100% (p < 0.05) of ART and MFQ released in 2 h. Figure 5(B) shows the supersaturation of formulated samples under non-sink condition using USP II. P.M.-ART/MFQ (1:2 w/w) shows poor solubility of ART and MFQ. The supersaturation of crystalline ART to be 48.4 µg/ml, which is similar to the value reported by Ferreira et al. [15]. For crystalline MFQ, the maximum amount of MFQ dissolved is 90.2 µg/ml in 2 h (equilibrium cannot be achieved in 2h). Meanwhile, the solubility of the drugs from ART/MFQ/SBA-15 (1:2:3 w/w/w) is significantly higher than that of those counterpart raw drugs. Co-formulated ART achieved a supersaturation of 124.8 µg/ml (p < 0.05), whereas the MFQ showed a supersaturation of 282.1 µg/ml (p < 0.05), which can be sustained for 120 min. The enhancement of dissolution and apparent solubility of ART and MFQ from ART/MFQ/SBA-15 (1:2:3 w/w/w) are attributed to their amorphous forms with reduced particles size, as suggested by the Noyes-Whitney equation [44] and Ostwald-Freunlich equation [45, 46]. It has been reported that the amorphous forms represents the most energetic solid state with increased molecular motion and reduced binding energy [47, 48]. This culminates in a reduction in the lattice energy of the drug molecules compared with the crystalline forms. In addition, the amorphous drugs incorporated into the pores channels of SBA-15 are smaller than 10 nanometers due to the confined space, which results in higher specific surface areas exposed to the dissolution medium compared with their crystalline counterpart. These differences translate into the fact that no additional energy is required to solvate or hydrate the amorphous drugs, thereby dramatically increasing the apparent solubility and dissolution rate of the drugs.

In addition, factors such as the hydrophilicity of carriers (Table S1) and host-guest interactions facilitate the rapid penetration of dissolution medium influx through the pore channels to break the hydrogen bonds between drug molecules and silanol groups to displace the drug particles. Moreover, the rapid displacement of drug molecules by the dissolution medium attributes to the competitive interaction between water and ART because of the hydrophilicity of the silica pore walls [38]. Meanwhile, the short pore channels of SBA-15 submicron particles accelerate the diffusion of the dissolved drug by minimizing the diffusion distance and the pore restriction, thus resulting in a rapid desorption of a greater amount of drug molecules without any obstructions [26]. The enhanced dissolution rate and apparent solubility of ART and MFQ (both BCS class II drugs) are highly expected to improve their absorption in the gastrointestinal tract and thereby improve their bioavailability.

3.6.

Chemical and Physical Stability Evaluation

The results of chemical stability of ART/MFQ/SBA-15 (1:2:3 w/w/w) are summarized in Table 2. The percentage of ART and MFQ in the combination formulations remains almost unchanged without degradation after stored in desiccators (25 °C/18% RH) and Activ-vial® (25 °C) for 6-months. Approximately 97% of drugs can be preserved, indicating that the chemical stability of combination formulation is not affected by storage under moderate conditions. In the meantime, the co-spray dried samples exhibit excellent physical stability as the samples retained their amorphous forms throughout the storage periods without any traces of crystals [Figure 6(A)]. All the stored samples show the halo PXRD pattern without presence of PXRD peaks assigned to the crystalline ART or MFQ. This indicates that the amorphous drugs entrapped within the pore channels of SBA-15 possess excellent stability during the storage test. Only moderate conditions were chosen

since both the ART and MFQ are not chemically stable at high temperature and high humidity (Table S2 and Table S3). SBA-15 as drug carriers is able to inhibit re-crystallization through the size-constraint effect on nucleation and crystal growth [25, 49], thickness and rigidity of pore walls [30] and the formation of hydrogen bonds through surface silanol groups [50, 51]. These outstanding advantageous of SBA-15 not only play an important role in producing amorphous forms but also stabilize the amorphous forms during long-term storage periods. The durability of mesoporous structures and stability of amorphous APIs in nanoporous channels are synergetic effects to support each other to preserve physical stability during storage. The significance of SBA-15 particles in amorphizing crystalline drugs and stabilizing it for long-term storage can be seen clearly from the physicochemical analysis of co-spray dried ART/MFQ (1:2 w/w). Even though amorphous forms can be obtained by co-spray drying of ART and MFQ (Figure S3), a weak peak at 10.6° could be detected after one-week storage in open pan inside desiccators at 25 °C/18% RH, suggesting the re-appearance of traces of crystalline drugs (Figure S7). This indicates that the amorphous ART/MFQ (1:2 w/w) is physically unstable and has the propensity of re-crystallization during storage, mainly attributed to the formation of ART crystals. The inability of various formulation techniques to amorphize ART without presence of carrier further confirms the significance of SBA-15 particles in stabilizing amorphous solid dispersion (Figure S3 and Figure S4).

3.7.

Dissolution after Storage for 6-months

Figure 6(B) shows the effect of storage on the dissolution rate profiles of ART/MFQ/SBA-15 (1:2:3 w/w/w), as the drug release of the samples was analyzed after 6months of storage at 2 different storage conditions. The dissolution was conducted using USP II apparatus. Both ART and MFQ showed an initial burst release from ART/MFQ/SBA-15

(1:2:3 w/w/w), more than 91% (p < 0.05) of drugs release could be achieved in 2 h of dissolution. The dissolution profile achieved by ART/MFQ/SBA-15 (1:2:3 w/w/w) samples after storage is still superior to P.M.-ART/MFQ (1:2 w/w) and almost similar to those achieved by freshly co-spray dried samples [Figure 5(A)]. The long-term stability of the formulated amorphous ART and MFQ with SBA-15 contribute to an excellent stability with preserved dissolution behavior of dosage forms after 6-months of storage.

4. Conclusion It is technically feasible to generate X-ray amorphous ART and MFQ co-formulation using SBA-15 via co-spray drying. The significance of the SBA-15 is reflected by the fact that the produced amorphous drugs can be stabilized during long-term storage. The remarkably enhanced dissolution kinetics of amorphous ART/MFQ/SBA-15 as compared with the crystalline counterpart is attributed to the amorphization of ART and MFQ due to spatial confinement in the pore structure of SBA-15 and particle size reduction. Moreover, the structural properties and hydrophilicity of SBA-15 could also contribute to the enhanced dissolution and supersaturation. The elucidation of the physicochemical stability of ART and MFQ is vital to prolong the shelf-life of the formulated amorphous samples with preserved drug release profile. It is accordingly recommended that the co-spray dried combination formulations of ART/MFQ/SBA-15 are preferable to be stored at low humidity and temperature for maximal preservation of the physical and chemical stability of the APIs.

Acknowledgement Financial support for this study was provided by project grant R-279-000-329592 from GlaxoSmithKline (GSK) and the Science and Engineering Research Council of A*STAR (Agency for Science, Technology and Research), Singapore. The work was carried out in the Institute of Chemical and Engineering Sciences, A*STAR, Singapore and supported by the National University of Singapore. The authors would like to thank laboratory officers for their technical assistance.

Disclosure The authors report no conflicts of interest in this work.

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List of Figures Figure 1(A) N2 adsorption-desorption isotherms and (B) pore size distributions of SBA-15 before and after co-spray drying with ART and MFQ Figure 2 PXRD patterns of ART and MFQ before and after co-spray drying with SBA-15. (Note: the intensity of ART was reduced 20 times) Figure 3 SEM images of (a) ART, (b) MFQ, (c) SBA-15, (d) MFQ/SBA-15 (1:1 w/w), (e) ART/SBA-15 (1:1 w/w) and (f) ART/MFQ/SBA-15 (1:2:3 w/w/w) Figure 4 TEM images of SBA-15 at scale of (a) 500 nm and (b) 100 nm and (c) ART/SBA-15 (1:1 w/w), (d) MFQ/SBA-15 (1:1 w/w) and (e) ART/MFQ/SBA-15 (1:2:3 w/w/w) Figure 5(A) Dissolution profiles of freshly prepared samples under sink condition and (B) Supersaturation under non-sink condition using USP II: ( ) stands for ART and ( ) stands for MFQ; dotted line: P.M.-ART/MFQ (1:2 w/w) and solid line: ART/MFQ/SBA-15 (1:2:3 w/w/w). n=3, p < 0.05. Figure 6(A) PXRD patterns of ART/MFQ/SBA-15 (1:2:3 w/w/w) after storage for 6-months at different storage conditions and (B) Dissolution profiles of ART/MFQ/SBA-15 (1:2:3 w/w/w) under sink condition using USP II after storage for 6-months: ( ) stands for ART and ( ) stands for MFQ; dotted line: stored in Activ-vial® (25 °C) and solid line: stored in desiccators (25 °C/18% RH). n=3, p < 0.05.

Figure 1(A) N2 adsorption-desorption isotherms and (B) pore size distributions of SBA-15 before and after co-spray drying with ART and MFQ

Figure 2 PXRD patterns of ART and MFQ before and after co-spray drying with SBA-15. (Note: the intensity of ART was reduced 20 times)

Figure 3 SEM images of (a) ART, (b) MFQ, (c) SBA-15, (d) MFQ/SBA-15 (1:1 w/w), (e) ART/SBA-15 (1:1 w/w) and (f) ART/MFQ/SBA-15 (1:2:3 w/w/w)

Figure 4 TEM images of SBA-15 at scale of (a) 500 nm and (b) 100 nm and (c) ART/SBA-15 (1:1 w/w), (d) MFQ/SBA-15 (1:1 w/w) and (e) ART/MFQ/SBA-15 (1:2:3 w/w/w)

Figure 5(A) Dissolution profiles of freshly prepared samples under sink condition and (B) Supersaturation under non-sink condition using USP II: ( ) stands for ART and ( ) stands for MFQ; dotted line: P.M.-ART/MFQ (1:2 w/w) and solid line: ART/MFQ/SBA-15 (1:2:3 w/w/w). n=3, p < 0.05.

Figure 6(A) PXRD patterns of ART/MFQ/SBA-15 (1:2:3 w/w/w) after storage for 6-months at different storage conditions and (B) Dissolution profiles of ART/MFQ/SBA-15 (1:2:3 w/w/w) under sink condition using USP II after storage for 6-months: ( ) stands for ART and ( ) stands for MFQ; dotted line: stored in Activ-vial® (25 °C) and solid line: stored in desiccators (25 °C/18% RH). n=3, p < 0.05.

Table 1 Pore volume and specific surface areas before and after co-spray drying and drug loading of SBA-15 Sample

Surface area (SBET) [m2/g]

Total pore volume (Vpore) [cc/g]

*Drug Loading [wt%]

SBA-15

809.0 ± 26.3

1.16 ± 0.04

−

ART/SBA-15 (1:1 w/w)

112.8 ± 24.5

0.20 ± 0.04

48.8 ± 1.1

MFQ/SBA-15 (1:1 w/w)

188.3 ± 34.9

0.33 ± 0.07

47.1 ± 2.4

ART/MFQ/SBA-15 (1:2:3 w/w/w)

123.6 ± 10.1

0.21 ± 0.10

16.5 ± 0.9 a 31.4 ± 1.8 b

a

refers to ART, b refers to MFQ Data represent mean ± S.D., n=3 *Drug Loading (wt%) = weight of encapsulated drug / total weight sample used×100% Table 2 Amounts of ART and MFQ remaining in ART/MFQ/SBA-15 (1:2:3 w/w/w) samples after storage at 2 different storage conditions for 3 and 6-months. Storage conditions

API

Percentage of drug 3-month

6-month

Open pan inside desiccators@ (25 °C/18% RH)

ART

97.7 ± 2.1

97.1 ± 2.7

MFQ

97.7 ± 1.8

98.2 ± 1.5

Activ-vial®@ (25 °C)

ART

99.8 ± 0.3

99.5 ± 0.2

MFQ

99.7 ± 0.8

99.6 ± 0.6

Data represent mean ± S.D., n=3