Introduction

The therapeutic effectiveness of a drug depends on its bioavailability as well as on the solubility of drug molecules. Solubility is the phenomenon of dissolution of a solute in the solvent to give a homogenous system and is one of the important parameters to achieve the desired concentration of drug in the systemic circulation to produce a pharmacological response. Nearly 40% of the new chemical entities currently being discovered have poor solubility in water. More than one-third of the drugs listed in the U.S. Pharmacopoeia fall into the poorly water-soluble or water-insoluble categories. Low aqueous solubility is the major problem encountered with the formulation development of new chemical entities as well as for the generic development. To achieve high absorption of a drug, it should be present in the form of an aqueous solution at the site of absorption (Lachman, Lieberman, & Kanig, 1991). The insufficient dissolution rate of the drug is the limiting factor in the oral bioavailability of poorly water-soluble compounds. In such cases, dose-escalation would be required until the blood drug concentration reaches the therapeutic drug concentration range. This dose-escalation sometimes causes topical toxicity in the gastrointestinal tract upon oral administration, and such toxicity could lead to a reduction in patient compliance. The formulation design of a drug product with a high dose is generally difficult due to significant higher tablet weight. Increasing drug load might result in poor powder properties and may have different in-process challenges during granulation and compression. In addition to this, the manufacturing cost would also increase since a large amount of active pharmaceutical ingredient (API) might be consumed to develop and manufacture the drug product. The poor solubility of new drug candidates might also affect the chemical properties during the drug discovery stage. During clinical trials, the poor solubility and bioavailability of a drug substance might result in limited therapeutic potential, thereby leading to insufficient clinical outcomes (Kawabata, Wada, Nakatani, Yamada, & Onoue, 2011). Different factors affecting solubility, solubility enhancement techniques, its importance and applications has been reported for poorly water-soluble drugs (Bharti, Attal, Anirudha, Munde, & Birajdar, 2015; Kadam, Shinkar, & Saudagar, 2013; Patil, Shinkar, & Saudagar, 2017; Savjani, Gajjar, & Savjani, 2012; Sikarra, Shukla, Kharia, & Chatterjee, 2012) Use of porous media like mesoporous Sylysia 350 has been investigated as a potential means to increase the solubility of poorly soluble drugs and to stabilize the amorphous drug delivery system. These materials have nano-sized capillaries, Porous and the large surface area which enable the materials to accommodate high drug loading and promote the controlled and fast release. Therefore, mesoporous Sylysia 350 has been used as a carrier in the solid dispersion to form Mesoporous Nanomatrix. Mesoporous Sylysia 350 is also being used as an adsorbent in a conventional solid dispersion, which has many useful aspects. Various methods are available for incorporation of drugs into mesoporous materials like solvent deposition methods, mechanical activation and vapor-phase mediated mass transfer (Chaudhari & Gupte, 2017).

Materials and Methods

Apixaban was obtained as gift samples from Dr. Reddys Laboratories Limited, Hyderabad, India. The Mesoporous material like Sylysia 350, Carbon and Tin oxide were procured from SD Fine Chem Limited, Hyderabad, India and all other chemical used were of analytical grade from Loba Chemie Pvt. Ltd., Mumbai, India.

Mesoporous Nanomatrix

Different trials were performed for selection of different Mesoporous materials, solvents and polymer to prepare Mesoporous Nanomatrix (Jia et al., 2011; Wang et al., 2011; Yin et al., 2018; Zhang et al., 2014; Zhang, Wang, Gao, Li, & Li, 2013; Zhang, Zhi, Li, Gao, & Song, 2013).

Trials for selection of Mesoporous material

Based on a literature survey, different trials for Apixaban and Mesoporous materials in the ratio of 1:1 and 1:3 were performed and evaluated for Apixaban solubility.

Trials for selection of solvent

Different solvents like Dimethyl Sulfoxide (DMSO), Dichloromethane (DCM) and Acetonitrile (solvent class 2 & 3) acceptable to be used in the pharmaceutical as per US FDA Safety guidance (Q3C) for the use of solvents in pharmaceuticals were evaluated. The addition criteria for selection of solvents are based on good solubility for Apixaban and the carrier like HPMC K15M and PVP K30 (Kim, 2013; Liberman, Lachman, & Schwartz, 1989).

Trials for the screening of polymer for preparation of Apixaban–Sylysia 350 /Polymer mesoporous Nanomatrix

The different polymer like PVP K30 and HPMC K15M were screened for the preparation of Apixaban–Sylysia 350 /Polymer mesoporous Nanomatrix by solid dispersion method.

Procedure for preparation of Trials of Mesoporous Nanomatrix

The weighed quantity of Apixaban (10 mg), mesoporous polymer Sylysia 350 (10 mg) and carrier polymer HPMC K15M or PVP K30 (10 mg) was dissolved separately in 1 mL volume of Dichloromethane solvent. Then, allowed to a dispersion of the above Apixaban solution (10 mg/mL) into prepared the 1 mL of Sylysia 350 dichloromethane suspension (10 mg/ml) in a round flask and further sonicated for 30 minutes. Then, again allowed the dispersion of carrier solution (HPMC K15M or PVP K30) into the above Apixaban Sylysia 350 dichloromethane suspension with continues stirring for 24 h. The above-prepared suspension was evaporated under reduced pressure at a temperature of 40°C till to dry. Further, the obtained Nanomatrix was vacuum dried in a hot air oven at room temperature for 48 hr. The dried solid dispersion Nanomatrix was stored in a desiccator for further evaluation (Yin et al., 2018).

Screening of Apixaban–Sylysia 350/polymer Nanomatrix by DOE

Based on the considerable good solubility as compared to the other, the carrier (s) was selected to formulate the Apixaban nanomatrix. However, based on initial learning, the Sylysia 350 and Polymer weight and Carrier/polymer type were selected as a factor for the DOE, and solubility of each DoE run is considered as a response to the DOE. The API concentration i.e., 10 mg and solvent type DCM, was kept constant to all DOE run. The statistical optimization of various process parameters for the experiment was done using Design-Expert^® software.

Evaluation of Apixaban Nanomatrix

Solubility Studies

The plain Apixaban and prepared trials of Apixaban mesoporous Nanomatrix were evaluated for comparative solubility study in distilled water. The Apixaban/Sylysia 350/Polymer mesoporous Nanomatrix was added into 10 mL of aqueous solution in a test tube and was shaken at 37°C in a water bath. After 48-hr equilibrium, the saturated solution was rapidly filtered through a 0.45-μm millipore filter and diluted with distilled water and it was analyzed by UV Spectrophotometer at a specific wavelength (Working document QAS/17.699/Rev2, 2018).

Drug content

Based on obtaining data from the solubility study of Apixaban Nanomatrix, the optimized batch was further evaluated for drug content. The test was estimated by dissolving 10 mg of mesoporous Nanomatrix in 10 mL of DCM. The samples were sonicated for 15 min and filtered through Whatman filter paper 0.45µm and analyzed by using UV Spectrophotometer and absorbance were taken.

Particle size measurement and Polydispersity Index

Based on obtaining data from the solubility study of Apixaban Nanomatrix, the optimized batch was further evaluated for mean particle size distribution and the polydispersity index by particle size analyzer.

Differential Scanning Calorimetry (DSC)

The optimized batch was evaluated for DSC (Mettler Toledo) study to get the compatibility reactions.

In vitro drug release study

The in-vitro drug release was performed in USP apparatus Type II (Electrolab Dissolution tester USP TDT-08L) using the paddle method at 75 rpm of rotation speed. The phosphate buffer pH 6.8 (900mL) was used as a dissolution medium and media temperature maintained at 37 ± 0.5ºC during the study. An accurately weighed amount of the Apixaban plain drug and Nanomatrix (all equivalent to 10 mg of Apixaban) were transferred into separate vessels of the dissolution apparatus. 10mL aliquot was removed at predetermined time intervals i.e., 10, 20, 30, 40, 50, 60 minutes from dissolution medium and replace with same buffer solution maintained at 37 ± 0.5ºC for maintaining sink condition and the samples were filtered through a 0.45-μm millipore filter and analyzed by using UV Spectrophotometer at specific wavelength (Liberman et al., 1989).

Formulation Immediate Release Tablet

Based on the above-mentioned evaluation parameters, the optimize Apixaban Nanomatrix was further selected to formulate the immediate-release tablets. The tablets were prepared by direct compression method using the different composition of diluents, binders and lubricants (Liberman et al., 1989).

Procedure for preparation of Immediate Release Tablet

The required quantity of Apixaban plain drug and optimized Nanomatrix were weighed separately. The other excipients (Lactose Monohyderate, Microcrystalline cellulose and Sodium starch glycolate), as mentioned in the formula composition, were also weighed and shifted together through #20 sieve. The sifted blend was mixed together for 10 min in a polybag. The required quantity of Magnesium Stearate, as mentioned in the formula composition, was weight and shifted through #40 sieve. The sifted Magnesium Stearate was mix together with the blend of Step 2 in a polybag for 1 min. The Lubricated blend of the prepared granule mixture was compressed manually on a compression machine fitted with 6 mm diameter round shape Punch and 6 mm Die were selected and tablet were formulated by direct compression method.

Evaluation of compressed Tablet

In vitro drug release of an immediate-release tablet

The drug release from the Immediate release tablet of Apixaban was performed by the In-vitro dissolution test in the USP apparatus Type II (Electrolab Dissolution tester USP TDT-08L) using paddle at 75 rpm rotation speed in 900 ml of phosphate buffer pH 6.8 as a dissolution medium by maintaining temperature 37±0.5ºC. 10mL aliquots were removed at predetermined time intervals i.e., 10, 20, 30, 40, 50, 60 minutes from dissolution medium and replace with the same buffer solution maintained at 37 ± 0.5ºC for maintaining sink condition. The samples were filtered through a 0.45-μm millipore filter and analyzed for the drug release using a UV Spectropho

tometer at a specific wavelength of 278 nm (Liberman et al., 1989).

Assay of Tablet

Weighed accurately 20 tablets and an average weight of twenty tablets was calculated. These tablets were crushed and powdered using mortar and pestle. Powder equivalent to 50mg Apixaban was accurately weighed and transferred to a 50 mL of volumetric flask. Sample was sonicated for 30min and diluted up to mark with diluent [water: acetonitrile (60:40 v/v)]. The solution was filtered through a 0.45-μm millipore filter. This filtered solution was further diluted to 10 µg/mL with diluent and analyzed for the drug release using a UV Spectrophotometer at the wavelength of 278 nm. This procedure was performed in triplicate.

Results and Discussion

Mesoporous Nanomatrix

Trials for selection of mesoporous material

Based on a literature survey, the different mesoporous materials, as mentioned in Table 1 in the ratio of 1:1 and 1:3, were evaluated for solubility of Apixaban and Mesoporous material.

The screening outcome of the above trials resulted that the batch APX2 contains the ratio of 1:3 of Apixaban and Sylysia 350 was showing the maximum solubility as compared to the other mesoporous materials like Carbon and Tin Oxide. Hence, Sylysia 350 mesoporous material was finalized for further trials.

Trials for selection of solvent

The solubility of Apixaban in different solvents were evaluated and discussed in Table 2.

Different solvents like DMSO, DCM and Acetonitrile (solvent class 2 & 3) acceptable to be used in the pharmaceutical as per US FDA Safety guidance (Q3C) for the use of solvents in pharmaceuticals were evaluated. These selected solvents are showing good solubility for Apixaban. The DCM is showing good solubility and lower boiling points (400 0C) as compared to the other evaluated solvents like DMSO (1890 0C) & Acetonitrile (820 0C). Hence, DCM was selected for further trials.

Trials for a screening of polymer for preparation of Apixaban–Sylysia 350/Polymer Nanomatrix

The different polymers like PVP K30 and HPMCK15M were screened for preparation of Apixaban–Sylysia 350/Polymer mesoporous Nanomatrix by solid dispersion method. The comparative solubility results of these polymers with mesoporous Nanomatrix against the Apixaban API is presented in Table 3.

The HPMC K15M carrier is showing considerable good solubility as compared to the other evaluated carrier PVPK30. Hence, the HPMC K15M carrier was selected for further trials.

DOE Screening Apixaban–Sylysia 350/polymer Nanomatrix by Solubility

The comparative solubility results for the selected I-Optimal screening DOE are presented in Table 5; Table 4 and Table 6 respectively. The ANOVA for the selected DoE model is presented in Table 7 and the normal plot is shown in Figure 1 and Contour surface plot for PVP K30 and HPMC K 15M are presented in Figure 2 and Figure 3 respectively.

Actual Equation-PVP

Solubility (microgram/mL) = 3.03889 + 1.30007 * weight of Sylysia 350 + 0.783127 * weight of carrier + -0.0116353 * weight of Sylysia 350^2 + -0.00808981 * weight of carrier^2

Actual Equation-HPMC

Solubility (microgram/mL) = 20.9758 + 0.959774 * weight of Sylysia 350 + 0.783127 * weight of carrier + -0.0116353 * weight of Sylysia 350^2 + -0.00808981 * weight of carrier^2

Coded Equation

47.5053 + 8.22232 * A + 5.67955 * B + 4.71477 * C + -2.5522 * AC + -2.61794 * A^2 + -1.82021 * B^2

From the above DoE trial, the maximum solubility was found 58.19±0.38µg/mL in run no.7 (batch no. APX 15) with a combination of Apixaban (10 mg), Sylysia 350 (37 mg) and HPMC K15M (38 mg). However, the other evaluated polymer run no.16 (batch no. APX 24) with a combination of Apixaban (10 mg), Sylysia 350 (40 mg) and PVP K30 (40 mg) showing solubility 56.27±0.63µg/mL. Hence, HPMC K15M polymer was selected for further formulation trials.

Evaluation of Optimized Apixaban Nanomatrix

Drug content, Particle size measurement and Polydispersity index

The DoE trial run no.7 (batch no. APX 15) with combination of Apixaban 10 mg, Sylysia 350, 37 mg and HPMC K15M, 38 mg mesoporous Nanomatrix is showing highest solubility and was further evaluated for Drug content, particle size estimation and Polydispersity index and is presented in Table 8 and Figure 4, respectively. The observed mean Particle size of batch no. APX 15 (DoE run no.7) Nanomatrix was around 298±2 nm and has a uniform controlled distribution.

DSC of Apixaban mesoporous Nanomatrix

The optimized trial batch APX 15 and API was evaluated for DSC and is presented in Figure 5 and Figure 6, respectively. The batch APX 15 shows sharp endothermic peak onset from to be in 231.56 ºC. This corresponds to the melting point of Apixaban which shows the crystalline nature of Apixaban Nanomatrix.

In vitro drug release

The comparative in vitro dissolution of Apixaban plain 10 mg drug and optimized Apixaban Mesoporous Nanomatrix (APX 15) 10 mg blend in pH 6.8 phosphate buffer is shown in Table 9 and Figure 7. From the dissolution study, it was clear that the batch APX 15 (Mesoporous Nanomatrix) shows a greater rate of drug release as compared to the APX (plain drug).

Formulation of Immediate Release Tablet

Based on the above-mentioned evaluation parameters, the optimized Apixaban Nanomatrix batch no. APX 15 and plain drug batch no. API (was further selected to formulate the immediate-release tablets as per the formula composition is shown in Table 10. The tablets were prepared by direct compression method using the different composition of diluents, binders and lubricants and were evaluated for in vitro drug release and an assay of the formulation.

Evaluation of compressed Tablet

In vitro drug release Study of an immediate-release tablet

The evaluated comparative dissolution profile of test batches in pH 6.8 phosphate buffer is presented in Table 11 and Figure 8.

From the dissolution study, it is evident that the immediate-release tablet manufactured with the Mesoporous Nanomatrix (batch no. APX 15) shows gradual and complete drug release within 1h. However, the tablet manufactured with a plain drug does not showed complete drug release for evaluated till 1 hr.

Assay of Formulation

The evaluated Assay for test formulations is presented in Table 12. On the basis of the assay result, it was concluded that the tablets contain the potency within the specification of 90-110 %.

Conclusions

From the present study, we can concluded that the optimized Apixaban mesoporous Nanomatrix is a suitable potential option for solubility enhancement, increased in-vitro drug release and can be used for effective delivery of BCS class IV drugs.