*Article* **Study of Formulation and Process Variables for Optimization of Piroxicam Nanosuspension Using 32 Factorial Design to Improve Solubility and In Vitro Bioavailability**

**Yahya Alhamhoom <sup>1</sup> , Sandip M. Honmane 2,\*, Umme Hani 1,\* , Riyaz Ali M. Osmani <sup>3</sup> , Geetha Kandasamy <sup>4</sup> , Rajalakshimi Vasudevan 5, Sharanya Paramshetti <sup>3</sup> , Ravindra R. Dudhal 2, Namrata K. Kengar <sup>2</sup> and Manoj S. Charde <sup>6</sup>**

	- <sup>3</sup> Department of Pharmaceutics, JSS College of Pharmacy, JSS Academy of Higher Education and Research (JSS AHER), Mysuru 570015, Karnataka, India
	- <sup>4</sup> Department of Clinical Pharmacy, College of Pharmacy, King Khalid University, Abha 62529, Saudi Arabia
	- <sup>5</sup> Department of Pharmacology, College of Pharmacy, King Khalid University, Abha 62529, Saudi Arabia
	- <sup>6</sup> Department of Pharmaceutical Chemistry, Government College of Pharmacy, Karad, Shivaji University, Kolhapur 415124, Maharashtra, India
	- **\*** Correspondence: sandiphonmane@gmail.com (S.M.H.); uahmed@kku.edu.sa (U.H.); Tel.: +91-8600392878 (S.M.H.); +965-98712387 (U.H.)

**Abstract:** Piroxicam is a Biopharmaceutical Classification System (BCS) Class II drug having poor aqueous solubility and a short half-life. The rationale behind the present research was to develop a Piroxicam nanosuspension to enhance the solubility and thereby the in vitro bioavailability of the drug. Piroxicam nanosuspension (PRX NS) was prepared by an anti-solvent precipitation technique and optimized using a full-factorial design. Herein, the nanosuspension was prepared using polymer polyvinylpyrrolidone (PVP) K30® and Poloxamer 188® as a stabilizer to improve the solubility and in vitro bioavailability of the drug. Nine formulations were prepared based on 3<sup>2</sup> full-factorial experimental designs to study the effect of the formulation variables such as concentration of poloxamer 188 (%) (X1) and stirring speed (rpm) (X2) as a process variable on the response of particle size (nm) and solubility (μg/mL). The prepared NS was characterized by phase solubility, Fourier-transform infrared (FT-IR), differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), transmission electron microscopy (TEM), particle size, zeta potential, entrapment efficiency, and percent drug release. DSC and XRPD analysis of freeze-dried NS formulation showed conversion of PRX into a less crystalline form. NS formulations showed a reduction in the size from 443 nm to 228 nm with −22.5 to −30.5 mV zeta potential and % drug entrapment of 89.76 ± 0.76. TEM analysis confirmed the size reduction at the nano level. The solubility was increased from 44 μg/mL to 87 μg/mL by altering the independent variables. The solubility of PRX NS in water was augmented by 14- to 15-fold (87.28 μg/mL) than pure PRX (6.6 μg/mL). The optimized formulation (NS9) at drug-to-stabilizer concentration exhibited a greater drug release of approximately 96.07% after 120 min as compared to the other NS formulations and pure PRX (36.78%). Thus, all these results revealed that the prepared NS formulations have improved the solubility and in vitro dissolution compared to the pure drug. Furthermore, an increase in the drug release was observed from the NS than that of the pure PRX. All these outcomes signified that the prepared PRX NS showed an increase in solubility and in vitro dissolution behavior; which subsequently would aid in attainment of enhanced bioavailability.

**Keywords:** polymers; nanosuspension; solubility; bioavailability; anti-solvent precipitation; in vitro drug dissolution

**Citation:** Alhamhoom, Y.; Honmane, S.M.; Hani, U.; Osmani, R.A.M.; Kandasamy, G.; Vasudevan, R.; Paramshetti, S.; R. Dudhal, R.; K. Kengar, N.; Charde, M.S. Study of Formulation and Process Variables for Optimization of Piroxicam Nanosuspension Using 32 Factorial Design to Improve Solubility and In Vitro Bioavailability. *Polymers* **2023**, *15*, 483. https://doi.org/10.3390/ polym15030483

Academic Editor: Edina Rusen

Received: 14 December 2022 Revised: 8 January 2023 Accepted: 16 January 2023 Published: 17 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

There has been a growing need for alternative drug delivery systems and techniques owing to the increasing number of newly investigated drugs, increased sensitivity to clinical findings, and escalating healthcare costs. The current drug delivery system is rapidly evolving and has increased productivity. There has been significant advancement and research in delivery systems for the optimization of therapy and its cost-effectiveness. Newer pharmaceutical and biopharmaceutical product categories are accelerating the development of drug delivery technology. Often, conventional methods are unable to deliver these novel entities effectively. Thus, advanced delivery systems have therefore become increasingly important in today's world. Most of the recently developed drug molecules have poor solubility, which can lead to significant formulation issues and poor bioavailability.

Solubility is one of the key parameters to achieve the desired concentration of drug in the systemic circulation necessary for achieving the required pharmacological response. Following oral administration, poorly water-soluble drugs require high dosages to attain therapeutic plasma concentration. Water insolubility is one of the central elements which restrict the usage of many potential drug moieties and other active compounds. Because of this, the bioavailability of the drug is less and it fails to reach the site of action [1]. Solubility is closely associated with bioavailability. It greatly increases the bioavailability of the dosage form. Only highly soluble drug molecules can cross the cell membrane and show their desired therapeutic effect by reaching the site of action [2,3]. In vitro dissolution of the drug is related to its in vivo bioavailability [2]. The primary strategy in this study domain is enhancing the solubility of BCS class II drugs. Solubility of the drugs can be improved by a variety of techniques, such as particle size reduction, micro-emulsion, micellar solubilization, solvent deposition, Super Critical Fluid (SCF) process, solid dispersion, nanosuspension (NS), cryogenic techniques, inclusion complex formation-based techniques, hydrotropy, co-crystallization, complexation, liquid-solid system, etc. [2,4].

The majority of the aforementioned solubility enhancement methods, including NS, may be utilized to make drugs more soluble. In developing an ideal formulation, several factors, such as stability at various temperatures, solubility, compatibility of the solvent and excipients, and photostability, are essential. Thus, the present study aimed in developing NS to resolve issues relating to low solubility and poor bioavailability [5]. A drug that is weakly water soluble and free of any matrix material can be utilized to develop NS [6]. NS improves medication safety and effectiveness by resolving the challenges of low solubility and bioavailability as well as by changing the pharmacokinetics of the drug [7]. NSs are colloidal dispersions having surfactant-stabilized drug particles that are nanoscale in size [4,8]. Pure drug particles are dispersed in the aqueous medium to create a biphasic system known as an NS. The suspended particle has a diameter of less than 1 μm. The increase in surface area and saturation solubility of the drug particles results from the reduction in drug particle size, which accelerates the dissolution. The increased vapor pressure of the particles leads to increased saturation solubility and solution velocity of the nanoparticles. Because of these properties, NS is the best technique for enhancing the water solubility and dissolution rate of drugs [9,10]. The particle size distribution, surface charge, crystalline state, dissolution rate, and saturation solubility are the main characteristics of oral NSs. A zeta potential of at least ±30 mV is needed for an electrostatically stabilized NS to be physically stable. A general guideline line of ±20 mV will be sufficient in the case of a combined steric and electrostatic stabilization. For pharmaceuticals that exist in several polymorphs, the crystallinity of the NS is crucial [11].

The drug Piroxicam(PRX) [4-Hydroxy-2-methyl-N-(2-pyridinyl)-2H-1, 2-benzothiazine-3-carboxamide 1,1-dioxide] belongs to the class of anti-inflammatory drugs. PRX demonstrates prolonged and delayed oral absorption [12]. It is a highly protein-bound medication that is slowly removed from the body, increasing the half-life to up to 36 to 86 h. It has various side effects such as diarrhea, constipation, headache, dizziness, and ringing in the ears. Although it has a variety of side effects, PRX has a stronger pharmacological efficacy because it is a potent anti-inflammatory drug [12]. In the US, PRX is approved

for the treatment of rheumatoid arthritis and osteoarthritis [13]. PRX is a BCS class II medication that has high permeability and low solubility [2]. It exhibits a slow and gradual absorption when taken orally and is proven to be ulcerogenic. Therefore, the need of the hour is to develop novel formulations that would accelerate its absorption in the GI tract and might give quick relief from rheumatoid arthritis and osteoarthritis with a reduction in its dose and dose-dependent side effects. The NS development approach has viable potential for enhancing solubility of poorly soluble drugs and is also cost effective, simple and robust. PRX in the form of NS is a practically executable and promising way to mitigate this problem. Hence, the objective of the current study was to investigate the effects of a formulation variable (polymer concentration) and process parameters (stirring speed and time) on the NS formulation of PRX with the intent of attainment of improved solubility and in vitro bioavailability. In the present study, NS has been chosen as an approach to enhance the solubility, dissolution release and in vitro bioavailability of PRX. Differential scanning calorimetry (DSC), X-ray powder diffraction (XRPD), and transmission electron microscopy (TEM) analysis were performed to determine the solid-state properties of the drug in physical mixtures and NSs in comparison with the free PRX.

#### **2. Materials and Methods**

#### *2.1. Materials*

PRX was procured from Zydus Cadila Healthcare Ltd., Goa, India. The following chemicals were obtained commercially: PVP K30®, Poloxamer 188®, Methanol, Hydrochloric Acid and Dichloromethane (Loba Chemie, Mumbai, India). All the other chemicals, solvents and reagents used were of analytical grade and were stored and used as per the supplier's instructions.

#### *2.2. Methods*

#### 2.2.1. Pre-Formulation Study

Pre-formulation studies were carried out to determine the characteristics of the drug and excipients, particularly on the physicochemical, physicomechanical, and biopharmaceutical aspects [14].

#### 2.2.2. Organoleptic Evaluation

The drug sample was evaluated for organoleptic properties. The organoleptic evaluation was conducted by observing the appearance, color, and odor of the drug sample.

#### 2.2.3. Melting Point

Using a micro-controlled based melting point apparatus (SMP10/1, Stuart, UK), the melting point of the drug was determined. The drug sample was inserted into a capillary tube with one end closed. The capillary tube was inserted into a silicone oil bath, which was heated with the help of an electrical heating coil in a controlled manner. The temperature at which the drug sample started melting was noted as the melting point temperature. The average of triplicate readings was noted and compared with the literature value.

#### 2.2.4. Ultraviolet-Visible (UV-Visible) Spectrophotometry

The absorbance maxima (λmax) of PRX were determined in various solvents such as methanol, methanolic HCl, phosphate buffer solution (PBS) pH 6.8, and PBS pH 7.4 in the range of 200–400 nm by using a UV-Visible double beam spectrophotometer (Shimadzu 1800, Tokyo, Japan) [14].

#### 2.2.5. Determination of Calibration Curve

The calibration curve of PRX has been investigated in different solvents such as methanol, methanolic HCl, PBS pH 6.8, and PBS pH 7.4 at specific wavelengths [14].

#### 2.2.6. Solubility of Drug

Drug solubility was assessed in a variety of solvents, including distilled water, PBS pH 7.4, and methanol. In order to create saturated solutions, the excess drug was added to the vehicles, which were then shaken continuously for 48 h at a temperature of 25 ± 0.5 ◦C. The solutions were filtered, diluted and analyzed using UV spectrophotometry (Shimadzu 1800, Japan) [14].

#### 2.2.7. Fourier-Transform Infrared (FT-IR) Spectroscopy

Drug-excipient compatibility was confirmed by FT-IR spectroscopy (Bruker Alpha II). The spectra were obtained using the KBr pellet method within the range of 4000 cm−<sup>1</sup> to 400 cm−1. Briefly, the pellets were prepared with KBr in a ratio of 1:100 and force was applied for several minutes to obtain uniform thin pellets. These pellets were placed in between two plates in a sample holder and scanned. The absorbance was plotted against their corresponding wavenumber [12].

#### 2.2.8. Preparation of NS

**Preparation of drug solution:** the required amount of the drug was dissolved in 4 mL dichloromethane to obtain a clear solution [15].

**Preparation of polymer solution:** the required amounts of Poloxamer 188 and PVP K30 were dissolved in water. Using a mechanical stirrer (Remi RQT 124 AD, Mumbai, India), the polymer solution was homogenized at 1000–1200 rpm. Then, the drug solution was added dropwise into the polymer solution using a syringe and stirred continuously followed by sonication for 20 min. From the preliminary study, based on complete mixing and the optimal particle size, stirring time was optimized at 15 min [15,16].

#### *2.3. Optimization of Formulation*

A randomized, 32 full factorial design with two factors and three levels was employed to systematically study the nanosuspension formulation (Table 1). A total of nine experimental trials were performed at all possible combinations. The concentration of stabilizer and stirring speed were identified as the independent variables based on the experiments conducted during the optimization, which were altered at three different levels, i.e., low, medium, and high. Solubility (μg/mL) and particle size (nm) were considered as dependent variables (responses) The response variables used were solubility and particle size (nm). The development and evaluation of the statistical experimental design were accomplished by utilizing the Design-Expert 8.0 software (Stat-Ease Inc., Minneapolis, USA). The effect of two independent variables, stabilizer concentration (X1) and stirring speed (X2), on the response (Y) was studied.


**Table 1.** 3<sup>2</sup> full factorial design with composition and independent variables.

−1: low level, 0: medium level, and +1: high level of independent variables.

**Formulation optimization using the desirability function:** using Design-Expert 8.0 software (Stat-Ease Inc., USA), various response surface methodology (RSM) computations were carried out for the current optimization research. All of the response variables were developed in a linear model with quadratic terms. Additionally, using the Design-Expert software output files, linear plots and 3D graphs were developed. Analysis of variance (ANOVA) was used to determine the importance of these characteristics on the variables.

The optimization process employed the desirability function once the mathematical model had been fitted. The results were combined to find a product with the desired properties during formulation optimization. The desirability function predicts the ideal values for the independent variables by combining all the results into one variable. The least desirable value for the replies is represented by a desirability value of 0, while the most desirable value is represented by a desirability value of 1.

#### 2.3.1. Freeze-Drying of Optimized Formulation

The optimized batch (NS9) was lyophilized using mannitol as a cryoprotectant for 24 h under controlled conditions to obtain dry powder using a laboratory-scale lyophilizer (Alpha 1–2 L Dplus, Martin Christ Gefriertrocknungsanlagen GmbH, Germany). The product was stored in an airtight container until further characterization [17].

#### 2.3.2. Characterization of NS

**Determination of phase solubility of NS:** for the determination of the phase solubility of PRX, different concentrations of Poloxamer 188 were prepared. Briefly, an excess amount of the drug (1 g) was added to each of the 250 mL flasks containing 25 mL of stabilizer poloxamer 188 having three distinct concentrations (0.1%, 0.3%, and 0.5%) and at different speeds. The flasks were properly sealed and agitated for 48 h at 37 ◦C at 100 rpm in an orbital shaker cum incubator (Orbit™ 1000 multipurpose digital shaker). For the establishment of the equilibrium, they were kept in the incubator for a further 24 h. Five (5) mL of the supernatant was filtered and appropriately diluted, and the amount of drug in the filtrate was evaluated photometrically using a UV-Visible spectrophotometer (Shimadzu 1800, Tokyo, Japan) at 354 nm [18].

**Particle size, polydispersity index and zeta potential analysis:** by using the dynamic light scattering (DLS) technique, the mean particle size, polydispersity index and zeta potential were determined using a particle size analyzer (Horiba Scientific SZ-100). Freshly prepared NS was diluted 100 times with distilled water and analyzed [17,19].

**Determination of Entrapment Efficiency (EE):** entrapment efficiency (EE) of the optimized formulation was determined by quantitatively estimating the amount of drug loaded into the NS. The NS formulation was ultracentrifuged (Optima KE-90-IVD, Beckman Coulter, Pasadena, CA, USA) for 20 min; the resulting supernatant was diluted sufficiently with methanol for subsequent UV-spectrophotometric analysis at 334 nm [20].

$$\% \text{EE} = \frac{\text{Total amount of drug } - \text{ Free drug}}{\text{Total amount of drug}} \times 100\tag{1}$$

**Transmission Electron Microscopy (TEM):** the size and shape of the optimized NS were evaluated by means of TEM (H-7500, Hitachi, Tokyo, Japan).

**Differential Scanning Calorimetry (DSC) analysis:** a differential scanning calorimeter (Mettler-Toledo DSC 821e, Columbus, OH, USA), was used to carry out the thermal analysis for confirming the compatibility between the drug and the excipient. The thermal behavior of the drug and the optimized formulation was investigated via DSC analysis. Briefly, the samples were weighed accurately and sealed hermetically in aluminum pans and crimped and were heated from 25 to 250 ◦C at a heating rate of 10 ◦C/min. Throughout the measurement, nitrogen gas was purged over the sample cell with a flow rate of 50 mL/min [21].

**X-ray Powder Diffraction (XRPD):** XRPD is a crucial method used to determine the crystalline or amorphous nature of the sample. Using a powder X-ray diffractometer (AXS

D8 Advances, Bruker Ltd., Germany), diffractograms of the pure drug and NS formulation were obtained with tube anode Cr over the interval of 10–70◦/2θ using copper as an X-ray target, with 1.54 Å wavelength [21,22].

**In vitro dissolution study:** a USP Type II dissolution testing paddle apparatus was used for determining the in vitro dissolution. An amount of sample which is equivalent to 10 mg of PRX was added to the glass jar of apparatus containing 900 mL of PBS pH 6.8 maintained at a temperature of 37 ◦C [18,23]. Then, for 2 h, the paddle was rotated at 75 rpm. Three (3) mL of sample was taken out at predetermined intervals, filtered, and properly diluted. The concentration of the drug dissolved in the medium was then determined with the help of a UV spectrophotometer (Shimadzu 1800, Tokyo, Japan). To maintain a constant volume of a medium during the dissolution, 3 mL of fresh medium was always replaced in the glass jar.

### **3. Result and Discussion**
