MATERIALS AND METHODS

Study area: The formulation development and characterization were carried out in industrial lab, whereas the sexual behavior study was conducted in pharmaceutical preparation lab of pharmaceutics department, Prince Sattam bin Abdulaziz University, AlKharj, Saudi Arabia. This research project was conducted from November 2019-June 2020.

Chemical collection: Sildenafil (SL) and Glycyrrhizin, (GZN) was obtained as gift samples from Jazeera Pharmaceuticals (JPI), Riyadh. Soluplus^® (SLP) was obtained from BASF (Ludwigshafen, Germany) with no commercial value. Acetone (ACN) and Acetic Acid (AA) were purchased from Sigma Aldrich, Germany. All the other chemicals used were of analytical grades. Freshly prepared ultra-purified water was used throughout the work.

Development of Spray-dried amorphous solid dispersion of sildenafil: Spray drying technique was used to prepare Spray-dried amorphous solid dispersion; common solvents of different polarity were used to make the dispersion of drug and polymers combination. Model drug sildenafil and soluplus were dissolved in acetone and whereas glycyrrhizin was solubilized in acetic acid (1%). The concentration of drug carrier was fixed with 1:1 for GZN and SLP. The sildenafil (SL) to drug carriers (GZN and SLP ratios were 1:0.5, 1:1 and 1:1.5 (w/w). Solute to solvent ratio was selected as 1.5, 2 and 2.5% w/v for batches SN1, SN2 and SN3, respectively (Table 1). The drug-carriers solution was ultra-sonicated for about 2 hrs. Once solids get saturated with the solvents, the solution was placed on the magnetic stirrer (100 rpm). The solution was then allowed to pass through the pump into the spray drier through the nozzle feed rate of 30% and aspirator rate of 100%. The inlet temperature was set in such a way that; the outlet temperature should be equivalent to boiling point of the solvent(s) used. For the present study inlet temperature adjusted to get the outlet temperature of 60°C.

Droplet within a millisecond converted to solid. Nitrogen gas will be used along with the temperature to evaporate the solvents. The dry solid will be collected by cyclone mechanism in the collector and preserved in a desiccator for the characterization²⁸.

Characterization
Yield, particle size, polydispersity and zeta-potential: The product after spray drying was weighed and correlated with the weight of the precursors. The percentage yield was calculated using equation-1

(1)

Prepared SDASD of SL was characterized for particle size, polydispersity index and zeta-potential by Dynamic Light Scattering (DLS) theory using a Malvern particle size analyzer at 25^oC. All the samples were measured in triplicate with 20 cycles in each measurement.

Table 1:	Yield, particle size, polydispersity and zeta-potential of SDASD
SN1, 2 and 3: Formulation code given by the author, SL: Carrier Ratio : Drug-carrier ratio, PDI: Polydispersity index, DEE : Drug entrapment efficiency

The sample under investigation diluted with (1:200) milli-Q water (Milli-Q^® Direct 8 water purification system) followed by ultrasonication for 3 min. The suspended particles in the solvent will collide with each other and solvent molecules, considered being in Brownian motion. Following equation involved in the Quasi-Elastic Light Scattering technique using Non-Invasive Back Scatter technology in which the sample under investigation exposed with a laser beam in multidirectional to measure particle size²⁹.

Autocorrelation function³⁰ was carried out by:

(2)

Particle size measurement in movement calculated by Stokes-Einstein equation³¹.

(3)

The instrument also gives the polydispersity index reading in the same run.

Zeta potential over a particle of (diameter >25 μm), measured by Phase Analysis Light Scattering in the same instrument (Malvern Instruments Ltd, made in the USA) using Helmholtz and Smoluchowski equation that correlates streaming potential and current passed.

For planar solids zeta potential investigations, surface charges calculated by the equation.

(4)

For irregularly shaped samples solids zeta potential calculated by the equation³².

(5)

Where:

Quantification of the sildenafil calibration plot: Quantification of the sildenafil was done by the UV- spectroscopy technique (Jasco V-630 Made in Japan), maximum absorption was observed and reported at λ-max 294 nm. A stock solution was prepared by dissolving 10 mg SL drug in 5 mL Dimethyl sulfoxide (DMSO) followed by dilution with methanol to give the concentration of 1000 μg mL^–1. Different aliquots were prepared from 0-200 μg mL^–1, absorbance was noted against the blank (methanol). The calibration graph was plotted by concentration vs absorbance.

Drug encapsulation efficiency: Encapsulation efficiency (EE) was determined by measuring the amount of drug in the filtrate after the encapsulation process using a UV- spectrophotometer at λ-max 294 nm (Jasco V-630 Made in Japan). Following equation is used in the calculation of encapsulation efficiency.

(6)

X-ray diffraction: XRD diffractograms of pure drug SL, GZN, SLP and optimized SDASD of SN2 batch were obtained using the X-ray diffractometer of Cu target/filter monochromator (Ultima IV Multipurpose X-ray Diffraction System, Rigaku, Tokyo, Japan). The diffractometer was run at voltage/current of 40 kV/40 mA with continuous scan mode and a scan speed of 0.500 deg./min at scan-axis; 2 theta/theta.

Scanning electron microscopy (SEM): The selected SDASD SN2 was subjected to the SEM after suspending the sample in water, vortexed suspension then smeared on the glass slide followed by drying. The dried sample was mounted on carbon tape, and sputter- adhered on SEM stubs using gold sputter under reduced pressure evaporator (Zeiss EVO LS10; Cambridge, UK). The sample in the micro stage was then observed and spotted with the magnification to analyze and study the microstructure of the prepared SDASD of sildenafil³³.

In vitro Drug Release study: All three batches SN1, SN2, SN3 along with pure drug SL and marketed formulation were subjected to the in vitro drug release study using the USP-Paddle–II method (Erweka DT 600, Heusenstamm, Germany). The sample under investigation was submerged in a 900 mL 0.1 N HCl dissolution medium. The paddle was rotated at 50 rpm, dissolution medium was maintained at 37±0.5°C, and at a predetermined time interval 5 mL samples were withdrawn and replaced with 5 mL fresh dissolution media. The aliquots were passed through 0.4 μm syringe filters and suitably diluted. The drug concentration was measured by a UV- spectrophotometer at λ-max 294 nm (Jasco V-630 Made in Japan). The mean of at least three determinations was considered to calculate the drug release. The drug released was cumulated and presented against the respective time interval³⁴.

In vivo studies in male rats: Sexual behavior analysis: The experiment was conducted in a calm laboratory under faint red light in transparent cages. Male albino rats (300-350 g weight aged around 5-6 months and female albino rats of weighing 150-180 g, aged 3-4 months were procured from the animal house of the Pharmacology Department, Prince Sattam Bin Abdulaziz University-Alkharj, Saudi Arabia. Three groups of sexually experienced male rats that were showing reactive sexual activity (n = 5) were selected for the experiment and kept singly in separate cages. Group I served as normal control and was given the vehicle; 1% sodium carboxymethylcellulose. Group II (Reference): received sildenafil at a dose of 5 mg kg^–1. The SN2 test Group-III was also administered with 5 mg kg^–1 of the optimized formulation SN2. The vehicle and other drugs were administered orally as single doses through an orogastric tube. SDASD batches of SN2 were considered as test (GROUP III) whereas, 1% sodium carboxymethylcellulose and sildenafil at a dose of 5 mg kg^–1 served as control (GROUP I) and reference (GROUP II), respectively.

Sexual behavior analysis: After 30 min, female rats were introduced into the male cages with one female to one male ratio and the sexual behavior of the male animals was immediately begin and continued for the end of the first mating series. The following parameters were measured as mentioned by Fouche et al.³⁵.

Depending on the previously mentioned parameters, the followings can be computed:

(7)

(8)

Statistical Analysis: The significance of the results values was determined by one-way analysis of variance (ANOVA) with post –hoc t-test, p-value<0.05 was considered as significant.

RESULTS AND DISCUSSION

Spray dried amorphous solid dispersions were prepared using different drug to drug carriers ratio as shown in Table 1. Drug: carriers ratio was varied in all three batches whereas the drug amount was kept constant. The proportion of solvents and process parameters were kept constant in all the batches. The results of change in the drug-carrier ratio on yield, drug entrapment efficiency, average particle size, polydispersity index and zeta potential are shown in Table 1. SDASDS were formed by rapid evaporation of solvent by spray drying technique. According to Table 1 and based on the equation-1, SN1, SN2 and SN3 showed 85.6, 90.3 and 98.8% of product yield, increase in the drug-carrier ratio increases the production yield³⁶. The variation in the yield and loss of the product may be related to the adherence or deposit of powder to the cyclone walls and piping of the spray chamber and the outlet filter of the receiver chamber. To avoid oxidation of the product during the drying process inert gas such as nitrogen and carbon dioxide alone or in combination could be used³⁷. These gases could alter the yield (%) and solid-state behavior of the product, for instance, carbon dioxide may act as a plasticizer. SN2 and SN3 batches could be considered as successful with optimized parameters and cost-effective processes³⁸, as the yield from these batches was found to be ≥90 %.

Fig. 1:	Calibration plot of Sildenafil citrate

Fig. 2:	XRD spectrums of SL, GZN, SLP and optimized SDASD SN2

The particle size of prepared solid dispersion was found to be ranged between; 0.710-1.45 μm. All prepared SDASD were more or less monodispersed based on the PDI value which was ≤0.3³⁹. The results of particle size, PDI, and zeta potential are shown in (Table 1). The difference in the particle sizes could be due to variation in the viscosity of the spray solution and solid loading, moreover atomizer size and conditions applied also plays a crucial role in end product solid sizes. The SN2 formulation showed particle size 0.710 μm and PDI of 0.239, considered as optimized. Small collapsed hollow spheres with reduced particle size, increased surface area could improve the dissolution rate and bioavailability⁴⁰. Zeta potential indicates charges over the particles; all batches showed a negative charge. SN2 batch showed -33.7 mV zeta potential, any dispersion with an electrokinetic intensity of ≥30mV considered as stabilized formulation⁴¹. The calibration graph was plotted (Fig. 1) good correlation coefficient (R²) 0.9966 was obtained that obeyed the Beer-Lambert's law. The results of the calibration curve were sensitive, accurate, precise, and reproducible. The results of SN1, SN2 and SN3 showed 41.62, 40.90 and 53.89% drug encapsulation respectively, from these results it could be revealed that an increase in the drug: polymer ratio enhances the drug encapsulation⁴². The x-ray diffraction spectrum of SL, GZN, SLP and optimized SN2 SDASD peaks at 2θ showed in Fig. 2. Intense peaks of SL revealed the crystallinity nature of the drug that affects the solubility and bioavailability⁴³. Besides, GZN and SLP showed broad peaks.

Fig. 3:	SEM microphotographs of SN2 solid dispersion obtained using a spray drying technique

Fig. 4:	Cumulative (%) drug release of SDASD and sildenafil (SL)

However, XRD spectra of Sildenafil in SN2 SDASD powder showed the absence of characteristic intense peaks of SL, which mean the transformation of SL from crystalline to an amorphous state (Fig. 2). The amorphous form of drug entity regarded as higher solubilized form with improved bioavailability⁴⁴. The SEM photomicrographs of optimized SN2 SDASD obtained by exposure of sample under study with the electron beam followed by higher magnifications. The image revealed spray-dried sildenafil product was roughly spherical with a shrivelled surface, small dents on the outer surface could be due to rapid solvent evaporation under reduced pressure of the spray drying process⁴⁵. Irregular surfaced particles have an increased effective surface area, that could increase the solubility, dissolution, and bioavailability of model drug used (Fig. 3).

The drug release from the prepared SDASD of sildenafil was studied for 3 h; the results were plotted in (Fig. 4).

Table 2:	Effect of sildenafil SDASD on male rats
ML : Mount latency, MF : Mount frequency, IL: Intromission latency and IF : Intromission frequency, EL : Ejaculation latency and SL: Sildenafil

Fig. 5:	Effect of SL and SN2 complex on the copulatory efficiency and intercopulatory efficiency of male rats

Drug release was found to be 55.88, 75.34 and 49.21% for SN1, SN2 and SN3 solid dispersions respectively, whereas for the pure drug SL it was only 32.93%, this indicate increased in drug release with the carrier combination and at the same time further increase in drug carrier ratio reduces the drug release³⁴. As per the Fig. 4, it was evident that there is a rapid and continuous drug release from the SDASDs compared to pure drug. Optimized SN2 showed the highest drug release amongst all prepared formulation and in comparison to pure drug. The drug release was ordered as SN2>SN1>SN3>SL.

The MF and the ML reflect sexual interest or libido, whereas the IF and IL are useful indices of the sexual excitement and the efficiency of erection. Therefore, the reduced duration of ML and IL as well as the increased values of MF and IF recorded in SN2-treated rats suggests enhanced sexual interest, libido, and erection in comparison with both C and D. Besides, SN2 prolonged ejaculation latency of male animals as compared to rats of C and D groups (Table 2). The significant increase in the duration of ejaculation latency confirms that SN2 has the potential to improve the copulatory performance of male rats.

The percentages of copulatory and the intercopulatory efficiencies were the highest in the male rats Group III administered with SN2 compared to control and the reference groups (Fig. 5). The present results revealed that the highest aphrodisiac activity in male rats was exhibited by SN2.

Spray-dried amorphous soiled dispersions (SDASD) is a novel approach and are applied for many poorly soluble drugs to increase the solubility in order to achieve improved bioavailability and biological activity.

CONCLUSION

The aforementioned results revealed that Spray-dried amorphous solid dispersion developed by using Glycyrrhizin and soluplus has an optimum yield, particle size, and drug entrapment. Drug release results showed relatively higher release compared with pure drug. Enhanced drug release data was supported by the better sexual behavior of optimized formulation compared to pure drug. Therefore, SDASD of sildenafil could be a potential remedial complex for the treatment of erectile dysfunction.

SIGNIFICANCE STATEMENT

This study discovers the increased solubility and enhanced sexual activity of prepared sildenafil SDASD that can be beneficial for the treatment of erectile dysfunction and may restore perished sexual behavior and increases relationship bonding.

This study will help the researcher to uncover the critical areas of spray-dried amorphous solid dispersions by the use of hydrophilic polymers to increase the solubility of BCS – class II drugs that many researchers were not able to explore. Thus a new theory on this glycyrrhizin and PDE-5 inhibitor and possibly other combinations of polymers may be arrived at.

ACKNOWLEDGMENTS

This Research Project was fully supported by Deanship of Scientific Research at Prince Sattam Bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia with grant number 2020/03/16705.