Materials
TEM obtained as a gift sample from Connexions Life sciences, Bangalore. HPMC E15 and PVP K-25 were procured from SD Fine-Chem Limited. All the reagents used for analytical development in the study were of analytical grade and were obtained from Sigma Aldrich, Bangalore.
Methods
HPLC method development of TEM
Agilent HPLC (contained UV–VISIBLE detector Model) with the EZChrom Elite software was used for analyzing in vitro dissolution samples and for data collection and integration. A reverse-phase Ecosil-C18 column (250 × 4.6 mm, 5 μm) was used for the TEM chromatographic separation at 25 °C. The flow rate was selected as one of the factors in the experimental design, and it was set at 0.80 to 1.0 ml/min, and detection was done at a wavelength of 291 nm. Twenty microliter was the injection volume [7].
Validation as per ICH guidelines was performed for the developed method to assess various parameters like the specificity in which placebo sample solutions were prepared in the same manner as in sample preparation and were injected into the chromatographic system without TEM at various concentrations, and the chromatogram data was recorded.
Linearity was determined by preparing seven standard TEM solutions with concentrations ranging from 50 to 150% of the target standard concentration were prepared and injected into the chromatographic system, and the coefficient of determination was calculated. The precision of the developed method was confirmed by repeatability and intermediate precision. Intraday and interday analyses were performed by repeating the method for three times in the same day and on three successive days. The absorbance was determined and percentage RSD was calculated; six sample solutions (TEM) were prepared at lower (50%) and higher (150%) levels and three intermediate sample solutions (100%) to assess accuracy.
Robustness was determined by using two mobile phases, one containing 90% acetonitrile and the other containing 110% acetonitrile, and the system suitability parameters were assessed by standard solution injection; flow rate robustness was assessed by increasing the flow rate from 0.8 to 1.0 ml/min. The effect of pH on robustness was assessed by injecting the solutions consisting of the mobile phase of varying pH from 4.4 to 4.7 into the chromatographic system.
Standard and blank solution preparation
The standard solution was prepared by dissolving the drug TEM 40 mg into a 25-ml volumetric flask containing phosphate buffer of 15 ml and acetonitrile of 10 ml (60:40 v/v), sonicated for 5 min, and then filtered through 0.45-μm membrane filter. The concentration of the solution was found to be 1.6 mg/ml. The blank solution was prepared as similar to standard but without the pure drug in the ratio of 60:40% v/v mixture of phosphate buffer and acetonitrile, the same solution used as mobile phase [8].
Sample preparation
The collected dissolution sample was transferred to a 25-ml volumetric flask. The sample was then dissolved and diluted with pH 4.4 phosphate buffer: acetonitrile (60:40 v/v) and subjected to sonication for 10 min, filtered through a 0.45-μm membrane filter [8].
Solubility studies
The solubility studies were performed by placing the pure drug in excess quantity and optimized nanosuspension in two different flasks which consist of 10 ml of distilled water. The samples present in the sealed flasks were placed on the orbital shaker for agitation (Cintex Industrial Corporation, Mumbai) at room temperature for 48 h. Then, the centrifugation of samples was done to separate the undissolved content. The analysis was carried out by the above developed HPLC technique for the quantification of TEM present in the supernatant layer [7].
Drug-excipient compatibility studies
The compatibility between the drug and the excipients (HPMC E15, sodium hydroxide, sodium bicarbonate) was studied via FT-IR, using Bruker alpha E ATR spectrophotometer (KBr pelletization method) [7].
Formulation development of TEM nanosuspensions
Bottom-up anti-solvent precipitation method
Accurately weighed 100 mg of pure TEM was dissolved in 2 ml of solvent (chloroform) at room temperature. The above solution was poured into a beaker having a fixed volume (100 ml) of anti-solvent (water) containing 100 mg of polymer (HPMC E15) at room temperature with a stirring speed of 1500 rpm using mechanical Remi stirrer. The above solution was stirred continuously for 2 h to evaporate the solvent. The formed nanosuspension was transferred into a container and stored at 25 °C until further use. The same procedure was adopted using PVP K-25 as a stabilizer, instead of HPMC E15 [9].
Emulsification solvent evaporation method
To 5 ml of chloroform, 100 mg of TEM was added and dissolved at room temperature. The resulting solution was added into a 50 ml aqueous solution of 100 mg HPMC E15 at the same temperature under high-speed homogenization (8000 rpm) for 5 min. After homogenization, the preparation was subjected to ultrasonication for 15 min, followed by mechanical stirring (10 min) for the evaporation of the organic solvent. The nanosuspension was obtained after centrifugation for 10 min at 4000 rpm. The same procedure was adopted using PVP K-25 as a stabilizer instead of HPMC E15 [10].
Acid-base neutralization method
TEM (100 mg) was weighed and dissolved in 5 ml mixture of hydrochloric acid solution (3 N) and chloroform (1:5 v/v). HPMC E15 was dispersed in 100 ml of sodium hydroxide (0.0375 N, 1% w/v) solution. The acidic drug solution was added dropwise to the sodium hydroxide solution under moderate stirring (1500 rpm) for 1 h to produce the nanosuspension [11].
The selected drug is highly lipophilic. The solubility of the drug in aqueous solutions is strongly pH-dependent, with maximum solubility observed at high and low pH. In the pH range of 3–9, it is poorly soluble. According to the Henderson-Hasselbalch equation, an alteration of 1 unit in pH value will cause 10 times to change in solubility of weak electrolyte drugs [12]. It is possible to accomplish an acid-base neutralization method. The formulations produced nanosized particles by opting the acid-base neutralization method.
Experimental design
Based on the literature survey and trials, the acid-base neutralization method was further selected for the formulation of TEM nanosuspensions. From the preliminary experimental trials, it was observed that with a change in speed (1500–2000 rpm) there was no change in particle size. However, change in the stirring time resulted in reduced particle size. It was also observed that stabilizer concentration (HPMC E15) and speed of stirring [13] were major factors that affected the formation of nanosized particles. Particle size and in vitro drug release were selected as responses. Hence, the above parameters were selected as factors and experimented with low and high levels by opting 22 factorial design with midpoint using the Design-Expert software (version 11).
Particle size and Zeta potential
The particle size and Zeta potential of nanosuspensions were measured using HORIBA SCIENTIFIC SZ-100 ZETA SIZER at 25 °C. The samples were diluted (1:500) prior to analysis with distilled water and were measured in triplicates [7].
Drug content
Nanosuspension equivalent to 40 mg of drug was transferred into a 25-ml volumetric flask consisting of chloroform: methanol (1:5 v/v), and the flask was shaken for 20 min and was filtered. The filtrate was diluted using 0.1 N HCl: methanol (1:9 v/v), and the drug content was estimated by the above developed HPLC method [7].
In vitro drug release studies
The dissolution study of nanosuspension has been performed by using USP type–II, paddle apparatus. The accurately measured volumes of nanosuspension (5 ml) were transferred into the dissolution apparatus (Electrolab TDT-08 L, India) consisting of 900 ml of 0.1 N HCl which served as medium and operated at a speed of 50 rpm at 37 ± 0.5 °C. Five milliliters of the sample was withdrawn periodically and replaced with an equal volume of fresh medium up to 12 min. Collected samples were then filtered using the 0.1-μm PTFE syringe filter [7] (Whatman Inc., Clifton., USA).
Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM, Thermo Fisher Scientific India Private Ltd., Mumbai, India) studies were conducted to study the morphology of suspended nanoparticles. The sampling cell contained a thin film of a nanosuspension solution. At × 300 magnification, images were taken with Zeiss tabletop scanning electron microscopy [7].
Stability
As per ICH Q1A (R2) guidelines, the stability studies were performed for the optimized formulations. The formulations were placed in bottles and stored at 40° ± 2 °C/75 ± 5% RH and evaluated for changes in particle size at the end of 30, 60, and 90 days [14].
In vitro drug diffusion studies
Franz diffusion apparatus was used to perform diffusion studies. A fresh goat mucosa was collected from a local slaughter shop, within an hour of excision and was taken into the lab in normal saline and used as a biological membrane. TEM equivalent to 100 mg nanosuspension was placed on the biological membrane fixed to the diffusion apparatus; 0.1 N HCl was used as a diffusion medium maintained at 37 ± 0.5 °C and rotated at 50 rpm. At predetermined time intervals, 3 ml of samples was withdrawn and replaced with the fresh medium and analyzed the drug concentration by the above developed HPLC method. The study was conducted for both plain suspension and TEM nanosuspension [15].
Ex vivo permeability studies
A fresh goat mucosa was collected from local slaughter shop (Anantapuramu, Andhra Pradesh, India), within an hour of excision and was taken into the lab in normal saline. The collected mucosa was cleaned properly and with continuous aeration; at 37 °C, the tissue was placed in an organ bath. The organ tube (receptor compartment) filled with 30 ml of 0.1 N HCl. At predetermined time intervals, 3 ml of samples was withdrawn and replaced with fresh medium and analyzed the drug concentration by the developed HPLC technique. The study was conducted for both plain suspension and TEM nanosuspension [15].
The organ tube (beaker with 30 ml of 0.1 N HCl) used as a receptor compartment. A part of the tissue in the form of a sac filled with TEM nanosuspension and placed in an organ tube with continuous aeration to study the permeation of drug (ex vivo permeability studies mimics the in vivo behavior by aeration to assess the permeation of drug). In vitro diffusion and ex vivo permeability studies were the methods to study the amount of drug that diffuses or permeates at a given time which serves as alternate to in vivo studies and provides information to conduct in vivo studies.