Development, Characterization, and Pharmacodynamic Evaluation of Hydrochlorothiazide Loaded Self-Nanoemulsifying Drug Delivery Systems

The objective of the current work was to develop optimized self-nanoemulsifying drug delivery systems (SNEDDS) and evaluate their in vitro and in vivo performance. The research comprised various studies which includes solubility studies in various vehicles, pseudoternary phase diagram construction, and preparation and characterization of SNEDDS along with in vitro dissolution and in vivo pharmacodynamic profiling. Based on dissolution profile, a remarkable increase in rate of dissolution was observed in comparison with plain drug and marketed formulation. Optimized SNEDDS formulation was composed of Capmul MCM (19.17% w/w), Tween 80 (57.5% w/w), Transcutol P (12.7% w/w), and HCT (4.17% w/w). In vivo pharmacodynamic evaluation in Wistar rats showed considerable increase in pharmacological effect of HCT by SNEDDS formulation as compared with plain HCT.

Lipid based formulation represents a distinctive and relatively novel solution to delivery of poorly soluble compounds. A lipid dosage form usually consists of one or more drugs dissolved in a blend of lipophilic excipients such as triglycerides, partial glycerides, surfactants, or cosurfactants [11]. Among the lipid based systems, the self-nanoemulsifying drug delivery system (SNEDDS) is a potential technology to improve the rate and extent of absorption of poorly watersoluble drugs [12]. SNEDDS are isotropic mixtures of drug, lipids, and surfactants, usually with one or more hydrophilic cosolvents or cosurfactants [13]. Hydrophobic drugs can be dissolved in these systems, enabling them to be administered as a unit dosage form for peroral administration. When such a system is released in the lumen of the gastrointestinal tract, it disperses to form a fine oil in water emulsion (micro/nano) with mild agitations provided by gastric mobility. This leads to in situ solubilization of drug that can subsequently be absorbed by lymphatic pathways, by passing the hepatic firstpass effect [14].
Hydrochlorothiazide (HCT) is a potent diuretic drug that is practically insoluble in water and has a solubility of only 250 g/mL in 0.1 N HCl at 25 ∘ C [15,16]. HCT has low toxicity and is widely used in combination with cardiovascular drugs for the treatment of hypertension [17]. Poor water solubility causes possible deviation in the oral bioavailability and, therefore, there is great interest in the development of new drug delivery systems that could enhance the solubility and permeability of HCT and, as a consequence, its bioavailability. According to Biopharmaceutical Classification System 2 The Scientific World Journal (BCS), HCT is classified as class IV drug having low solubility and low permeability [18,19]. SNEDDS can be utilized to enhance drug solubilization in GIT and it has also an impact on permeability [20][21][22]. Rapid disintegrating tablet strategy has been experimented for HCT using various carriers [23]. However, at present, no HCT marketed products arising from this approach are available, probably because of the unsatisfactory performance of the studied systems. The main objectives of the present study were to develop an optimized selfemulsifying drug delivery system for HCT and assess its pharmacodynamic effect in terms of diuretic efficacy.

Preparation of SNEDDS Formulations.
From the solubility study and ternary phase diagram studies, SNEDDS components were selected for drug incorporation and a series of SNEDDS were prepared ( Table 2) with varying ratio of oil to Smix. The series contained Capmul MCM (oil) and Tween 80/Transcutol P (Smix). HCT (4.17% w/w) was loaded into each mixture. The HCT-SNEDDS was prepared by dissolving drug into Smix in glass vials and accurately weighed oil was added. Components were mixed and heated (45-50 ∘ C) to form a homogenous mixture and stored at room temperature till further use.    Table 4.

Emulsification Time and Precipitation Assessment.
The emulsification time of SNEDDS formulation was assessed on USP II dissolution apparatus (Dolphin, India) ( Table 4). Each formulation (600 mg) was added dropwise to 500 mL of distilled water maintained at 37 ± 0.5 ∘ C. Gentle agitation was provided by a standard stainless steel dissolution paddle rotating at 50 RPM. These studies were carried out in USP type II dissolution test apparatus (Dolphin, India) at 100 RPM in 900 mL of phosphate buffer (pH 6.8). The temperature was maintained at 37 ± 0.5 ∘ C. 600 mg of each SNEDDS formulation (F1-F6) was filled in hard gelatin capsule (size 0) and used for dissolution studies; results were compared with plain HCT and marketed tablet of HCT (AQUAZIDE). 2 mL aliquots were withdrawn at 5, 10, 20, 30, 45, and 60 min intervals and filtered using 0.22 nylon membranes (Pall Life Sciences, India). The withdrawn samples were diluted suitably and analyzed for the drug content UV spectrophotometrically at 270 nm against phosphate buffer (pH 6.8). An equal volume of the dissolution medium was replaced in the vessel after each withdrawal to maintain the sink condition. Each test was performed in triplicate ( = 3), and calculated mean values of cumulative drug release were used while plotting the release curves ( Figure 3).  [25,26]. One end of pretreated cellulose dialysis tubing (7 cm in length) was tied with thread and 0.3 mL of SNEDDS formulation (equivalent to 10 mg HCT) was placed in it along with 0.7 mL of dialyzing medium (phosphate buffer pH 6.8). The other end of tubing was also tied with thread and was allowed to rotate freely in the dissolution vessel of a USP type II dissolution test apparatus that contained 900 mL dialyzing medium (phosphate buffer pH 6.8) maintained at 37 ± 0.5 ∘ C and stirred at 100 RPM. Aliquots were collected periodically and replaced with fresh dissolution medium and analyzed spectrophotometrically at 270 nm for HCT content.
2.5. 10. In Vivo Studies. In vivo study was approved and performed in accordance with the guideline of the animal ethics committee. The rats were housed individually in metabolic cages, controlled conditions of temperature (25 ∘ C), and a 12:12 h light/dark cycle. The study was conducted in four groups consisting of three male Wistar rats weighing 250-280 g. Animals were grouped as follows.
Group I: three rats for plain HCT drug suspension in 0.25% carboxymethyl cellulose (HCT).
Group IV: three rats for blank SNEDDS formulation (placebo).
Fifteen hours prior to each experiment food and water were withdrawn. Suspension of HCT (10 mg/kg) and optimized SNEDDS formulation F2 (equivalent to 10 mg of HCT) was administered to animals by gavage performing doses. The four groups of rats were allocated to one of four different treatments as summarized in Table 5. The groups were inverted after providing washout period of 72 hours to each group [27,28].
Cumulative urine output was recorded at 2, 4, 6, and 8 hours after oral administration of compounds. The urine volume was measured and a urine sample was taken for further analysis. Urinary sodium was determined in a flame photometer (F129, Systronics, India). Results were presented

Results and Discussion
3.1. Solubility Studies. Solubility of drug substance is a key criterion for selection of components for developing a SNEDDS formulation. The self-emulsifying formulations consisting of oil, surfactant, cosurfactant, and drug should be a clear and monophasic liquid at ambient temperature. Solubility studies were performed to identify suitable oils, surfactants, and cosurfactants that possess good solubilizing capacity for HCT ( Table 1). As HCT was found to have maximum solubility in Capmul MCM, Tween 80, Transcutol P, and PEG 400, further studies were conducted using various combinations of these oils and surfactants to identify the self-emulsifying area. Two sets of Smix and oil in different ratios were used to construct ternary phase diagrams. They were (1) Tween 80 and PEG 400 as Smix and Capmul MCM as oil phase and (2) Tween 80 and Transcutol P as Smix and Capmul MCM as oil phase. For both sets the selected ratios of Smix were 1 : 1, 2 : 1, 3 : 1, and 4 : 1.

Pseudoternary Phase Diagram Studies.
Self-nanoemulsifying systems form fine oil-water emulsions with gentle agitation, upon their introduction into aqueous media. Surfactant gets preferentially adsorbed at the interface, reducing the interfacial energy as well as providing a mechanical barrier to coalescence. Figures 1(a) tightly packed [29]. From the phase diagram studies it can be observed that as the Smix ratio increases the emulsion area decreases. Therefore the cosurfactant plays vital role in the emulsion formation for both of the Smix combinations. The phase study revealed that the emulsion region was more with Tween 80-Transcutol P (Smix) in comparison with Tween 80-PEG 400 combination. Hence the Tween 80-Transcutol P (Smix) was selected for drug loading and further studies.

Dilution Test.
The objective of dilution study was to study the degree of emulsification and recrystallization of the drug, if any. Dilution may better mimic conditions in the stomach following oral administration of SNEDDS preconcentrate. Accurate mixture of emulsifier is necessary to form stable nanoemulsion, for the development of SNEDDS formulation, when one part of each SNEDDS formulation was diluted with 10 parts of distilled water, 0.1 HCl, and phosphate buffer (pH 6.8). It was observed that the formulations F1 to F4 were found to be most stable because they do not show any precipitation or phase separation on storage in various dilution media (Table 3). Table 4, which was in the limit (95-102%). agitation of GIT due to peristaltic activity. It has been reported that self-emulsification mechanism involves the erosion of a fine cloud of small droplets from the monolayer around emulsion droplets, rather than progressive reduction in droplet size [30]. The ease of emulsification was suggested to be related to the ease of water penetration into the colloidal or gel phases formed on the surface of the droplet [31,32]. It was observed that an increase in the proportion of Tween 80 from 33.54 to 57.5% w/w in the composition resulted in increased self-emulsification time from 6 to 18 seconds (Table 4). This might be because of high viscosity imparted by Tween 80 which increases the free surface energy of system thereby increasing the emulsification time with increase in content of surfactant. Below 44% concentration of surfactant, there was turbid and unstable dispersion (Table 4). This may be due to excess penetration of water into the bulk oil causing massive interfacial disruption and ejection of droplets into the bulk aqueous phase [26]. ( max 560 nm). The percentage transmittance of the six selected optimized formulations was determined. As the value closer to 100% is formulation which is isotropic in nature, optimized formulations of F2-F3 from Smix ratio of 3 : 1 gave maximum percentage transmittance (Table 4). Nanoemulsion formed by SNEDDS in GIT meets with patient acceptabilty but isotropic nature of formulations or percentage transmittance closer to 100% gives an indication of globule size in nanometer range. The droplet size of the emulsion is a crucial factor in selfemulsification performance, because it determines the rate and extent of drug release as well as absorption [33]. Thus, the The Scientific World Journal formulation has the capacity to undergo enhanced absorption and thus the ability to have increased oral bioavailability.

Viscosity Determination.
Viscosity of SNEDDS without dilution was found to be in between 302 and 364 cP, which was suitable for filling in hard gelatin capsule without risk of leaking problem. As SNEDDS was diluted 10 and 100 times with water, viscosity of the system was decreased, which indicates that oral administration of SNEDDS formulation will be diluted with the stomach fluid and viscosity will be decreased and therefore absorption from the stomach will be fast (Table 4).

Droplet Size Analysis.
The droplet size of the emulsion is an essential factor in self-emulsification performance because it determines the rate and extent of drug release as well as drug absorption. Also, it has been reported that the smaller particle size of the emulsion droplets may lead to more rapid absorption and improve the bioavailability [34].
It is well known that in nanoemulsion systems the addition of surfactants stabilizes and condenses the interfacial film, while the addition of cosurfactant causes the film to expand; thus, the relative proportion of surfactant to cosurfactant has varied effects on the droplet size. From Table 4, it can be seen that formulation F2 has the smallest droplet size of 42.84 nm.
3.4.6. Zeta Potential Determination. Emulsion droplet polarity is also a very essential factor in characterizing emulsification efficiency [35]. The significance of zeta potential is that its value can be related to the stability of colloidal dispersions. Zeta potential indicates the degree of repulsion between adjacent, similarly charged particles in dispersion.
For molecules and particles that are small enough, a high zeta potential will present stability. When the potential is low, attraction exceeds repulsion and the dispersion will break and flocculate. So, colloids with high zeta potential (negative or positive) are electrically stabilized. Negative values of zeta potential of the optimized formulations indicated that the formulations were negatively charged. Formulation F2 was found to be the most stable formulation (Table 4) (Figure 4).

In Vitro Dissolution Studies. SNEDDS formulation F2
showed significantly higher drug release as compared to plain HCT and marketed HCT tablet (AQUAZIDE) (Figure 3). F2 showed more than 90% of drug release in 10 minutes while plain HCT and marketed tablet showed 44 and 49%, respectively. Spontaneous formation of nanoemulsion of SNEDDS formulation F2 could be the reason for the faster rate of drug release into the aqueous medium. Dramatic increase in the rate of release of HCT from SNEDDS compared to plain HCT and marketed formulation can be attributed to its quick dispersibility and ability to keep drug in solubilized state. Thus, this greater availability of dissolved HCT from the SNEDDS formulation could lead to higher absorption and higher oral bioavailability.

In Vitro Diffusion Studies.
Conventional dissolution testing can only provide a measure of dispersibility of SNEDDS in the dissolution medium of SNEDDS. Alternatively, in vitro performance of SNEDDS can be evaluated by drug diffusion studies using the dialysis technique. It is very popular and well documented in many literatures [25,26]. SNEDDS formulations F1, F2, and F3 were selected for diffusion studies, as these formulations show smaller droplet size among other formulations. Though SNEDDS F6 has smaller droplet size than F3, the former was found to be unstable and turbid on precipitation and clarity test. The release of HCT from these dosage forms was evaluated in phosphate buffer pH 6.8; the release percentage of F2 was significantly higher than that of F1 and F3 ( Figure 5). It suggests that HCT dissolved perfectly in SNEDDS form and could be released due to the small droplet size, which permits a faster rate of drug release into aqueous phase. The release rate of HCT from SNEDDS F2 (mean droplet size: 42.84 nm) was faster than SNEDDS F1 and F3 (mean droplet size: 98.48 and 151 nm, resp.). In this study, diffusion profiles of all three formulations (F1, F2, and F3) did not show any differences during initial 1 h; however, at the end of 12 h, formulation F2 showed 97.5% diffusion while F1 and F3 showed 85.5% and 81% diffusion, respectively ( Figure 5).
Results clearly indicate the effect of mean droplet size on drug diffusion across dialyzing membrane. Hence decreasing the particle size of nanoemulsion could increase the release rate of drug. Therefore, F2 was selected as optimized formulation for in vivo studies.

3.4.9.
In Vivo Studies. This study was performed to evaluate the pharmacodynamic potential of an optimized formulation (F2) against plain HCT. Cumulative volumes of excreted urine after oral administration compounds are shown in Figure 6(a). Statistically significant diuretic effect of SNEDDS was observed after 4 hours of administration in comparison with HCT, control, and placebo. This effect was maintained up to 24 hours. The diuretic effect of SNEDDS was significantly different in comparison with HCT, control, and placebo between 4 and 24 hours. Sodium and chloride ions quantification is one of the best methods to determine diuretic effect of drugs [36,37]. Values of concentration of sodium in excreted urine are shown in Figure 6(b). SNEDDS group showed significant increase in the amounts of electrolyte in comparison with control, placebo, and HCT groups after 8 hours of administration; however this effect was more but not significant as compared to HCT group up to 6 hours.
Diuretic activity data suggest that SNEDDS formulation increased the pharmacological effect of drug. The higher diuretic activity of the SNEDDS is due to complete dissolution of HCT in SNEDDS, which could have increased absorption. Solubility is a crucial characteristic for increasing the bioavailability of drugs according to the BCS [38]. Moreover, SNEDDS play an important role in improvement of permeability too. Higher permeability may be attributed to Capmul MCM, Tween 80, and Transcutol P as these components have the ability to enhance the permeability [20][21][22]39]. However the present work did not deal with the permeation experiments using cell models and this aspect will be developed in future studies.

Stability Studies.
Optimized SNEDDS formulation (F2) filled into hard gelatin capsules as the final dosage form.