Effect of Amphiphilic Graft Co-Polymer Carrier on Solubility and Dissolution Enhancement of Ambrisentan

Aim: Ambrisentan is a endothelin type A selective receptor antagonist used in the management of pulmonary arterial hypertension. Ambrisentan is BCS Class II drug haves very poor solubility in water and shows incomplete absorption after oral administration. The present work was aimed to study the effect of amphiphilic graft co-polymer carrier on enhancement of solubility and dissolution rate of poorly water soluble drug ambrisentan. To improve the aqueous solubility of ambrisentan solid dispersion was formulated by using novel carrier amphiphilic graft co-polymer (Soluplus® ). Materials and Methods: Solid dispersion was prepared by kneading technique by utilizing various ratios of carrier. Obtained solid dispersions ware evaluated for solubility, percentage yield, drug content and in vitro dissolution study. Powder characterization was performed by infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and X-ray diffraction (XRD). Results: FTIR spectroscopy shows no interaction between drug and polymer. DSC study showed that endothermic peak of drug was completely disappeared in Solid dispersion suggesting Original Research Article Radke and Jain; JPRI, 33(50A): 329-338, 2021; Article no.JPRI.77077 330 complete miscibility of drug in Soluplus®. XRD study suggest the conversion of crystalline ambrisentan in to amorphous form. All solid dispersions prepared with Soluplus® as a carrier showed increase in solubility. Solubility of ambrisentan was found to be increased 7.17 fold in optimized SD formulation ASD5. In vitro dissolution study showed the faster drug release from SD formulation compare to its pure form. All solid dispersion formulation’s release more than 50% of drug in first 10 min. Conclusion: This study conclude that the preparation of amphiphilic graft co-polymer based solid dispersion prepared by kneading technique is found to be useful in enhancement the solubility and dissolution rate of ambrisentan.


INTRODUCTION
Administartion of medication through oral route is more usual because of its simplicity and ease of ingestion. Swallowing a dose form by oral route is a comfortable and familiar method of taking medication from the patient's standpoint. Apart from its popularity for drug administration, there are number of variables that can limit drug absorption from the gastrointestinal tract. The most common problem among those is poor water solubility of the drug. Drug with low aqueous solubility will show dissolution rate limited absorption and ultimately gives incomplete bioavailability [1]. Aqueous solubility and intestinal permeability is the important parameter to achieved the desired bioavailability of drugs through oral route. In order to achieved intended plasma drug concentration and optimum therapeutic effect, the aqueous solubility of drug is important consideration [2]. Drugs with low solubility is responsible for high dose administration and increased frequency of administration of dosage form. Hence enhancement of solubility and dissolution rate of low aqueous soluble drugs is an challenging task in the drug development and in bioavailability improvement [3]. According to Biopharmaceutical classification system, BCS Class II are class of drugs that have low aqueous solubility and high membrane permeability. Such drugs shows poor or limited absorption from GI tract after oral administration [4]. Hence such drug always demand the solubility enhancement so as to improve its absorption rate.
There are many methods are discussed in literature in the past regarding improvement of solubility and dissolution properties of poor water soluble drug molecule. Solid dispersion is one of the oldest and effective technology successfully utilized by many researcher to increase the drug solubility during formulation and development. Solid dispersion possess several advantages like reduction in particle size, improve wettability of drug, higher particle porosity and amorphous formation of materials [5]. There are various methods are available for the preparation of solid dispersion such as solvent evaporation method, fusion method, hot melt extrusion method and kneading method. Kneading method of SD preparation provide additional advantages of particle size reduction during processing and industrially feasible technique [6].
Polyvinyl caprolactam-polyvinyl acetatepolyethylene glycol graft co-polymer (Soluplus®) is novel polymer designed by BASF corporation widely useful in increasing solubilizing properties of poorly soluble drugs. Soluplus® is available commercially as white free flowing granular powder [7]. Soluplus® has been widely researched carrier in solubility enhancement using different technique and model drugs like hot melt extrusion, spray drying, high shear dispersions, electrospinning/ electrospraying, microwave radiation and solvent evaporation technique [8].
Ambrisentan is (2S)-2-[(4, 6-dimethylpyrimidin-2yl) oxy]-3-methoxy-3, 3-diphenylpropanoic available as white to off-white crystalline powder. Ambrisentan is a BCS class II drug and is practically insoluble in water. Ambrisentan is categorized as endothelin type A (ETA)-selective receptor antagonist used in the management of pulmonary arterial hypertension. Because of its very poor solubility in water, the rate limiting step in the absorption of drug is its solubility in GI fluids. As the solubility of drug increased in water, it eventually fasten the absorption GI membrane which results in improvement of bioavailability of drugs. In this research, it was aimed to study the effect of amphiphilic graft copolymer carrier (Soluplus® ) on improvement of aqueous solubility and dissolution rate of ambrisentan by utilizing solid dispersion technique [9,10].

Materials
Ambrisentan was obtained as a gift sample from Cadila Pharmaceuticals, Mumbai India. Soluplus® was gifted by BASF Corporation India as a free sample. All other chemical and reagents used were of analytical grade.

Saturation Solubility Study of Drug
Saturation solubility study of pure drug ambrisentan was estimated in distilled water, acetate buffer pH 1.2, phosphate buffer pH 6.8 and phosphate buffer pH 7.4. Extra amount of drug was added to 10 ml study fluid in a glass vial. Samples were then shaken at constant speed on rotary shaker at 25°C±2°C for 48 hr. The resultant saturated solutions were then filtered and analyzed spectrophotometrically after suitable dilution [11].

Phase Solubility Study
To analyzed the possible solubilizing effect of carrier Soluplus® on solubility of drug, the phase solubility study was carried out. Excess amount of ambrisentan (approx. 100 mg) were added to 10 ml glass vial containing 0.25%, 0.50%, 0.75%, 1% and 2% aqueous solution of carriers. The solution was then shaken for 48 hr on rotary shaker at a controlled temperature of 25 0 C±2 0 C. The obtained solutions were then filtered through whatman filter paper no 1 and analyzed by UVspectrophotometer to determine the concentration of the dissolved drug [12,13].

Gibbs-Free Energy Transfer
Gibbs free energy transfer was most commonly utilized method to judge the possible favorable or unfavorable effect of carrier on solubilisation of drug in aqueous medium. The Gibbs free energy of transfer (ΔG∘tr) values was calculated using the Gibbs-Helmholtz equation. Negative Gibbsfree energy values obtained after calculation indicates improvement in drug dissolution. The Δ ∘tr values of drug were calculated using the following equation: Where S 0 /S is the ratio of the molar solubility of drug before and after treatment with polymer.
is the value of gas constant 8.31 JK−1 mol−1 and is temperature in degree kelvin [14].

Preparation of Ambrisentan -Soluplus® Physical Mixture
A physical mixture of ambrisentan with Soluplus ® was prepared by mixing of drug and carrier using mortar and pestle in different ratio (1:1, 1:2, 1:3, 1:4, 1:5). This mixture passed through sieve no 60 and store in desiccators till further use. The composition was shown in Table 1.

Preparation of Solid Dispersion
Solid dispersion of Ambrisentan with Soluplus ® in different weight ratio (1:1, 1:2, 1:3, 1:4, 1:5) ware prepared by kneading method. Mixture of drug and carrier was placed in a mortar and was kneaded thoroughly with water and methanol (1:1) for 20 min. The kneaded mixtures were then dried in oven at 40°C. After drying the mass was then pulverized and screened through 60mesh and stored in desiccator for further study [15,16]. The composition was shown in Table 1.

Determination of Solubility of Solid Dispersion and Physical Mixture
The solubility study of physical mixture and solid dispersion was determined in distilled water and phosphate buffer pH 6.8 in shake flask method. Excess quantities of sample were added in 25 ml of distilled water and phosphate buffer in conical flask and shaken for 24 hours at room temperature on rotary flask shaker. After shaking the samples containing undissolved solid suspended in the test medium were centrifuged at 10,000 rpm for 5 min, the clear supernatants obtained were filtered using whatman filter paper. Further sample ware suitably diluted and analyzed by spectrophotometer at 263.5 nm [17].

Determination of Percent Yield of Solid Dispersion
The percent yield of ambrisentan solid dispersions was determined by using the following formula:

Determination of Drug Content
Ambrisentan solid dispersion equivalent to 10 mg of drug was accurately weighed and dissolved in methanol (100 ml). The solution was filtered after vigorous shaken. The drug content was analyzed at 263.5 nm against blank by UV spectrometer after appropriate dilution [18].

Fourier Transform Infra-Red Spectroscopy (FTIR)
Compatibility studies of ambrisentan and with carrier Soluplus® was performed using FTIR spectroscopy (Shimadzu FTIR-8700). Spectrum of pure drug and solid dispersion was recorded over the frequency range of 400 to 2000 cm-1 at 4cm resolution [19].

Differential Scanning Calorimetry (DSC)
The thermal analysis was carried out using Shimadzu Thermal analyzer DT 40 (Japan). The samples were placed in sealed aluminum pans and heated at a rate of 10°C per min in the temperature range of 20-300°C under a nitrogen flow rate of 40 ml/min. SDC thermogram was recorded for pure ambrisentan, Soluplus ® and solid dispersion formulation [20].

Powder X-Ray Diffraction (XRD)
X-ray powder diffraction patterns of drug, carrier and solid dispersion was recorded on an X-ray powder diffraction system (Rigaku, Mini Flex 600) The scanning was done over range of 5º to 60º. The position and intensities of diffraction peaks were considered for the comparison of crystallinity [21].

In vitro Dissolution Study
In vitro dissolution study of prepared solid dispersions were determined using USP dissolution test apparatus II (Paddle type) (Esico International, Mumbai). Accurately weighted SD preparation equivalent to 10 mg of ambrisentan ware added to 900 ml of phosphate buffer pH 6.8 as dissolution medium, maintained at 37±0.5°C and stirred at 50 rpm. 5 ml samples were withdrawn at specific time and same volume was replaced with fresh media in order to maintain the sink condition. Collected samples were analyzed at 263.5 nm using UV-visible spectrophotometer against the blank after suitable dilution. Similar procedure was conducted for dissolution study of plain ambrisentan for comparision with SD formulations. The release profile data was analyzed for cumulative percent dissolved at different time intervals [22].

Saturation Solubility Study of Drug
The solubility of ambrisentan in distilled water, acetate buffer pH 1.2, phosphate buffer pH 6.8 and phosphate buffer pH 7.4 is shown in Fig. 1. The solubility of ambrisentan in distilled water was found to be 7.351 μg/ml, suggesting its poor water solubility. Solubility of ambrisentan increases as the pH of aqueous media increases. The solubility of ambrisentan in buffer pH 1.2, 6.8 and 7.4 was found to be 5.16 μg/ml, 28.50 μg/ml and 35.41 μg/ml respectively.

Phase Solubility Study
Phase solubility study of ambrisentan was studied using increasing concentration of hydrophilic polymer. A linear increase of solubility of ambrisentan was seen with an increasing concentration of Soluplus® carriers in water. This increased solubility of drug may be due solubilizing effect of carriers. At 2% w/v concentration of Soluplus® , the aqueous solubility of ambrisentan was increased by 6.2 fold. showed good affinity between drug and polymer. The phase-solubility diagram constructed for Soluplus® in distilled water was linear giving A L type solubility curve. The apparent stability constant (Kc) calculated from the linear plot of the phase solubility diagram, were found to be 143.98 M -1 indicate stronger interactions between the drug and carrier [23]. The solubility of ambrisentan at 0.25, 0.5, 0.75, 1 and 2% aqueous solution of carrier was shown in Fig. 2.

Gibbs-Free Energy Transfer
Gibbs free energy transfer was studied for ambrisentan in aqueous solutions Soluplus® in order to determine the possible favorable or unfavorable effect of carrier on solubility of drug. The Δ ∘ tr of drug at different concentration of Soluplus® are shown in 2. Negative values of Gibbs-free energy indicate improved dissolution [24]. In our study, increased in negative Δ ∘ tr value with increasing concentration of polymer was seen at all concentration levels of polymer, which suggests that treatment of drug and polymer was favorable for improving the solubility of drug [25].

Solubility Study of Solid Dispersion and Physical Mixture
The solubility of ambrisentan PMs and SDs was determined in distilled water and phosphate buffer pH 6.8. Ambrisentan is poorly soluble in water, having a maximum solubility of 7.351 μg/ml in distilled water. In the present study, a solid dispersion was prepared to increase the solubility and dissolution rate of ambrisentan. All physical mixture of ambrisentan showed improvement in solubility of drug as compare to pure drug. It was found that as the concentration of hydrophilic carrier increases, the solubility also increases. Solubility study of solid dispersion showed multi fold increase in solubility of drug in both the solvent. SD formulation (ASD5) showed maximum increase in solubility of drug in both solvent. Solubility was increased 7.17 fold in distilled water when compare with pure drug and hence considered as optimized formulation. This increased solubility of drug was might be because of conversion of drug in amorphous form or by increased wettability of drug by Soluplus® [26] The solubility of drug from SD formulation was higher in phosphate buffer solution than distilled water. The solubility data for all the PMs and SDs formulations are presented in Table 3.

Percentage Practical Yield and Drug Content
The percentage practical yields calculated for all SDs formulation was shown in Table 4.
Percentage yield for all SDs formulation was found optimum showing effectiveness of kneading technique. Drug content for solid dispersion formulations ASD1 to ASD5 was found to be in the range of 97.99% to 98.70.%. The percentage drug content for all SDs formulation was found within pharmacopoeial limit which indicate uniform distribution of drug in solid dispersion.

Fourier Transform Infra-Red Spectroscopy
IR spectra of pure ambrisentan, Soluplus® and its SD with Soluplus® are presented in Fig. 3. Sharp characteristic peaks of pure ambrisentan ware also appears in the spectra of optimized SD formulation. The peaks of drug are almost unchanged in the optimized solid dispersion which indicates that the symmetry of drug molecule is not affected significantly [27].

Differential Scanning Calorimetry
Thermogram of pure drug showed a single sharp endothermic peak at 194.62 0 C, corresponding to its melting point indicating the crystalline nature of drug, whereas Soluplus® does not showed any endothermic or exothermic peak representing its amorphous nature. Optimized SD formulation (ASD5) showed complete absence of endothermic peak of drug, which suggests that the drug is miscible in Soluplus® completely. This further proved the crystalline transformation of drug in to amorphous form [28]. Similar observation was reported by Ha et al. [29]. DSC thermogram of pure ambrisentan, Soluplus® and solid dispersion ASD5 are shown in Fig. 4.  Values are mean ± SD, n=3

In vitro Dissolution Study
In vitro dissolution study was carried out for all solid dispersion formulations and the pure ambrisentan. The study was conducted using phosphate buffers pH 6.8 as the dissolution media. The pure ambrisentan has shown very low drug release of 40.25% at the end of 60 min, indicating poor solubility. Dissolution study showed that, all SD formulation gives faster drug release as compare with pure form and it was found that as concentration of polymer increased the drug dissolution also increased. SD formulations ASD1 to ASD5 showed the drug release of 90.44%, 91.69%, 93.62%, 96.26% and 98.87% respectively at the end of 60 min. All SD formulations showed more than 50% drug release in 10 min showing its significant improvement in drug dissolution. Optimized SD formulation (ASD5) containing drug and Soluplus® in 1:5 ratio showed highest drug release. The drug release profile of all SD formulation and pure ambrisentan are shown in Fig. 6.

CONCLUSION
In this investigation, an attempt was made to improve the aqueous solubility and dissolution rate of drug using novel amphiphilic graft copolymer (Soluplus® ) carrier. Results of prepared SD formulation suggest that solubility of ambrisentan was improved manyfold. DSC and XRD study demonstrate the that crystallinity of drug was reduce and amorphous state of drug was formed. From this study, it was concluded that the solid dispersion prepared using Soluplus® is an effective way of enhancement of solubility, dissolution and bioavailability of poorly water soluble drugs.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONCENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.

ACKNOWLDGMENT
Authors are thankful to Cadila Pharmaceuticals for providing gift sample of Ambrisentan to conduct this research. Also authors are thankful to BASF, India for supplying Soluplus for completion of this research.