Combination of Phospholipid Complex and Matrix Dispersion Techniques for Improving Oral Bioavailability and Hypolipidemic Activity of Simvastatin

INTRODUCTION

Cardiovascular diseases (CVDs) are a group of non-communicable ailments which involves heart and blood vessels. To date, CVDs are ranked one to cause deaths globally on an annual basis. The CVDs account for an estimated 17.8 million deaths by the end of 2017 worldwide which forms approximately 31% of total deaths globally (1, 2). Hyperlipidemia is the keystone of CVDs responsible for global burden of cardiovascular disease. This complex metabolic disorder refers to abnormal elevation in lipids levels in systemic circulation (3,4,5).

Simvastatin (SIM), synthetically obtained from a fermentation product of Aspergillus terreus, is categorized as a statin class of hypolipidemic agents and indicated for dyslipidemia, hypercholesterolemia, and coronary heart disease as a drug of choice (6,7,8). SIM exerts its hypolipidemic action through inhibition of HMG CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase, a pivotal enzyme for catalytic reduction of HMG CoA to mevalonate, a crucial intermediate in the process of hepatic cholesterol biosynthesis (9,10,11). Besides, it is beneficial for the management of cancers and osteoporotic fractures (12).

SIM is considered as a BCS (Biopharmaceutics Classification Systems) class II candidate and displays low solubility, higher GI permeability, and low dissolution. Furthermore, it undergoes high first pass metabolism mediated by CYP3A in liver and intestine. These factors collectively result into slow and erratic oral absorption which consequently leads to suboptimal plasma concentration of SIM. Thus, only 5% of orally administered dose of SIM is bioavailable for systemic effect (8, 13,14,15). Therefore, these factors pose a major challenge in oral dosage development to achieve therapeutic plasma concentration.

The formulation scientists have reported diverse strategies to mitigate these impediments to successful delivery of SIM, such as dendrimer (14), nanosuspension (6), nanostructured lipid carriers (8), self-microemulsifying drug delivery systems (11), polymeric micelles (16), and solid lipid nanoparticles (17). Among the aforementioned approaches, lipid-based delivery systems have gained immense attention on account of their versatility and potential to circumvent solubility and permeability concerns. In addition, lipid-based delivery systems tend to absorb through lymphatic route in the GI tract (9).

Over the last two decades, phospholipid complexation has been researched critically to modulate biopharmaceutical performance and systemic exposure of BCS class II, III, and IV drugs via oral route (18,19,20). Being a biocompatible, amphiphilic, and endogenous compound (part of cell membrane), phospholipids are at the forefront of drug delivery for safe and effective administration of poorly bioavailable drug candidates (21). Phospholipid complexes are derived from molecular interactions between drug and phospholipid when reacted in stoichiometric ratios at suitable conditions (22, 23). However, phospholipid complex was found to be sticky and poorly dispersible which resulted into an unsatisfactory dissolution profile. Thus, there is void for further modulation of complex in order to attain optimum bioavailability. The literature references demonstrated significant improvement in drug performance with dual formulation strategies such as drug-phospholipid complex–loaded micelles (24), nanolipospheres (25), matrix film (26), and matrix dispersions (27, 28).

In the present work, the dual strategy is adopted by loading SIM-PLC in Soluplus® matrix dispersion to overcome shortcomings of SIM-PLC without impairing molecular interactions between SIM and phospholipid. Thus, Soluplus®, an amphiphilic polymer, was selected as a carrier for fabrication of matrix dispersion due to its vast merits Soluplus® is bifunctional polymer acts as a matrix as well as active solubilizer through micellization, thereby widely utilized in drug products aimed at increase in solubility and oral bioavailability. Moreover, several reports indicated P-gp efflux inhibition potential of Soluplus® (29,30,31,32). In the initial set of experiments, complexation efficiency of SIM with phospholipid was determined to confirm the complexed amount of SIM in SIM-PLC and SIM-PLC-loaded matrix dispersion (SIM-PLC-MD). FTIR (Fourier transform infrared spectroscopy) was utilized to ensure complexation between SIM and phospholipid. The physical state of SIM was monitored by employing an analytical tool such as PXRD (powder X-ray diffractometry). The morphological examination was carried out using SEM (scanning electron microscopy) and TEM (transmission electron microscopy) techniques. Then after, in vitro experiments like solubility, dissolution, and n-Octanol–water partition coefficient studies were undertaken and profiles of SIM, SIM-PLC, and SIM-PLC-MD were compared. The oral pharmacokinetic study was conducted in SD rats. Finally, developed formulations with promising in vitro and pharmacokinetic outcome were further subjected to pharmacodynamic study (hypolipidemic study) to corroborate in vivo efficacy by means of their potential to decline cholesterol and triglycerides levels.

MATERIALS AND METHODS

Materials

SIM was generously given as a gift sample by Cipla Pvt. Ltd. (Mumbai, India). Cilnidipine was purchased from Kimia Biosciences Pvt. Ltd. The various soybean phospholipids such as Lipoid S 75 (Phosphatidylcholine 70%), Phospholipon® 90 G (Phosphatidylcholine ≥ 94%), and Phospholipon® 90 H (hydrogenated phosphatidylcholine ≥ 90%) were provided as gift sample by Lipoid (Ludwigshafen, Germany). Tyloxapol was collected from Sigma-Aldrich Chemicals Pvt. Ltd. (Mumbai, India). The solvents (acetonitrile and methanol) were of HPLC grade and collected from Merck Specialties Pvt. Ltd. (Mumbai, India).

In Vitro Drug Analysis

The in vitro analysis of the samples was performed using HPLC (Waters 717plus autosampler) equipped with Waters 2487 dual ʎ absorbance UV detector set at ʎmax of 240 nm. The C18 (5 μm, 250 mm × 4.6 mm) column (Agilent Technologies, Inc., USA) was selected for chromatographic separations using acetonitrile and 0.1% glacial acetic acid (85:15 v/v) as mobile phase pumped isocratically. After sample injection of 10 μL, the analysis was carried out at elution rate of 1 mL/min for 11 min as run time.

Then after, validation of developed analytical method in terms of accuracy, precision, LOD, and LOQ was performed. The method was meeting specified requirements as analytical detection method according to ICH guidelines Q2 (R1) and was found to be linear over a wide range of concentration (1–50 μg/mL) with correlation coefficient of r2 ~ 0.999.

Fabrication of Simvastatin-Phospholipid Complex (SIM-PLC)

Preparation Method

SIM-PLC was generated by employing a simple solvent evaporation technique. Herein, the required amounts of SIM and phospholipid according to molar ratios were precisely weighed in a round-bottom flask and dissolved in dichloromethane to get clear solution. The reaction mixture was then carefully refluxed for 2 h at 60°C preceded by solvent evaporation using a Rota evaporator operated at reduced pressure to get final solidified complex. For overnight, the solidified complex was placed in a vacuum oven to remove solvent traces. The resultant product was stored at room temperature in a desiccator until further studies.

Investigation of Phospholipid Type and Appropriate Molar Ratio of SIM and Phospholipid

In this study, different phospholipids were screened to select the suitable phospholipid for the complexation. Afterwards, different molar ratios of SIM and screened phospholipid were investigated to obtain the optimized formulation. In brief, different phospholipids (Lipoid S 75, Phospholipon® 90 G, and Phospholipon® 90 H) having different phosphatidylcholine content were screened for higher complexation efficiency and drug content. The selected phospholipid was further utilized in different molar ratio. Besides, various ratios of SIM and phospholipid such as 1:0.5, 1:1, 1:2, and 1:3 were studied to check their effect on SIM complexation.

Fabrication of Simvastatin-Phospholipid Complex–Loaded Matrix Dispersion (SIM-PLC-MD)

SIM-PLC-MD was generated using a solvent evaporation method with minor modification referred to as the discontinuous solvent evaporation method. Herein, Soluplus® was utilized as the third component to prepare matrix dispersion in order to improve flow properties along with dissolution profile of SIM-PLC. The matrix dispersion was prepared by taking Soluplus® in different ratios with SIM-PLC including SIM:phospholipid:Soluplus® ratio 1:1:1 and 1:1:2. Briefly, SIM and phospholipid were weighed accurately and dissolved in dichloromethane at room temperature. The reaction mixture was then carefully refluxed for 2 h at 60°C. Subsequently, solvent was evaporated at 40°C under vacuum using a Rota evaporator. After the solvent volume was reduced to half, Soluplus® in different molar ratios was added to the reaction mixture and continued the evaporation until complete removal of the solvent. The resultant product was vacuum-dried overnight and stored in a desiccator at room temperature. The molar ratio was finalized on the basis of unaltered complexation efficiency of SIM-PLC after addition of Soluplus®.

Determination of the Complexation Efficiency of SIM-PLC and SIM-PLC-MD

The complexation efficiency was determined to investigate the complexation between SIM and phospholipid in SIM-PLC and SIM-PLC-MD. The difference in solubility among SIM, SIM-PLC, and SIM-PLC-MD was the basic criterion for selecting cyclohexane as a solvent. In brief, SIM-PLC and SIM-PLC-MD were accurately weighed and dispersed in 2 mL of cyclohexane separately. In cyclohexane, uncomplexed SIM practically remained insoluble whereas complexed SIM and phospholipid were completely soluble. The content of SIM in complexed form was separated and quantified by injecting aliquots into HPLC system equipped with auto sampler unit and UV detector. The detection parameters were kept the same as described in the “In Vitro Drug Analysis” section. Afterwards, complexation efficiency of SIM was calculated by applying the following equation:

$$ \mathrm{Complexation}\ \mathrm{efficiency}=\mathrm{M}1/\mathrm{M}2\times 100 $$

where M1 represents complexed amount of SIM in SIM-PLC and SIM-PLC-MD and M2 represents total amount of SIM in SIM-PLC and SIM-PLC-MD.

In vitro Evaluation of SIM-PLC and SIM-PLC-MD

Physicochemical Characterization

Fourier Transform Infrared Spectroscopy

The interactions in SIM-PLC and SIM-PLC-MD were characterized using FTIR spectrophotometer (FT-IR Spectrometer, Perkin Elmer, Inc., Waltham, MA, USA). The samples were mixed with diluent KBr, pelletized, and analyzed by setting wavenumber in the range of 4000 to 400 cm−1.

Powder X-ray Diffractometry

The samples were subjected to a powder X-ray diffraction spectrometer (Bruker D8 Advance, Germany) to check solid state of SIM, phospholipid, physical mixture of SIM and phospholipid, SIM-PLC, and SIM-PLC-MD. The instrument was equipped with a radiation source (Cu Kα 1) and regulated at fixed voltage (40 kV) and current (40 mA). The diffraction patterns were collected by scanning samples in the 2θ angle range of 5 to 40° and at scanning rate of 0.01° 2θ/min.

Scanning Electron Microscopy

The morphological examination of SIM, phospholipid, SIM-PLC, and SIM-PLC-MD was carried out using a scanning electron microscope (S-3400, Hitachi Ltd., Japan) regulated at 10 kV as acceleration voltage. The gold-palladium was utilized as sputter coating source for samples, which were further observed and captured at various magnifications.

Transmission Electron Microscopy

The visual examination of colloidal dispersions of SIM-PLC and SIM-PLC-MD prepared in purified water was performed using a transmission electron microscope (FEI Tecani G2F20, The Netherlands). The carbon-coated copper grid was taken and carefully placed with a drop of dispersion followed by staining of the sample using phosphotungstic acid. The overflowing solution was drained off and the grid was dried prior to the examination under the transmission electron microscope.

n-Octanol–Water Partition Coefficient Studies

The effect of phospholipid complexation on log P of SIM was determined using an agitation method. Firstly, SIM, SIM-PLC, and SIM-PLC-MD (equivalent to 20 mg of SIM) were accurately weighed and then separately added to vials having n-octanol phase (5 mL) in each. These vials were placed in water shaker bath maintained at 37 ± 0.5°C and agitated at 100 rpm for 24 h. Afterwards, double-distilled water was poured to each vial and agitated for an additional 24 h at constant conditions. On completion, vials were kept undisturbed for layer formation of each phase. Then after, each phase was carefully separated and solid residues were removed by centrifugation for 15 min at 10,000 rpm. The resultant solutions were filtered (0.45 mm PVDF syringe filter, Millipore Millex-HV) and injected to HPLC system for quantification of SIM. The following equation was used for log P values calculation:

log P = log10 (P)

P indicates n-octanol/water partition coefficient.

Apparent Aqueous Solubility Study

The solubility of SIM after formulated as SIM-PLC and SIM-PLC-MD was determined and compared with plain SIM and PM. The aqueous buffers including 0.1 N HCl having pH 1.2 and phosphate buffer having pH 6.8 were prepared as per the United States Pharmacopoeia (USP XXXII). Initially, glass vials containing aqueous buffers (5 mL) were separately added with an excess of SIM, PM, SIM-PLC, and SIM-PLC-MD. These sealed glass vials were then incubated for 72 h at 100 rpm in water shaker bath (EQUITRON, Medica Instrument Mfg. Co., Mumbai, India) controlled at 37 ± 0.5°C. Afterwards, samples were centrifuged (10,000 rpm, 10 min) to separate solid residue and clear supernatant was further filtered using a 0.45-μm PVDF syringe filter. The resultant solution was quantitatively analyzed for SIM amount using the previously described method (“In Vitro Drug Analysis” section).

In Vitro Dissolution Study

The automated dissolution test apparatus of USP 37 type II (ElectrolabTDT-08L, Mumbai, India) was utilized to investigate the release pattern of SIM, PM, SIM-PLC, and SIM-PLC-MD in various release media including 0.1 N HCl having pH 1.2 and phosphate buffer having pH 6.8. Herein, the dissolution apparatus was regulated at 37 ± 0.5°C and 50 rpm. Then, samples (equivalent to 10 mg of SIM) were precisely weighed and carefully added to dissolution flasks having 900 mL buffers previously equilibrated at the abovementioned conditions. The sampling was performed at designated time intervals and the same volume was replaced with blank buffers in order to maintain sink conditions throughout the study. After centrifugation (10,000 rpm for 10 min) and filtration, the samples were injected into HPLC system in order to quantify SIM release.

In Vivo Evaluation of SIM-PLC and SIM-PLC-MD

Animals

Firstly, Institutional Animal Ethics Committee (IAEC/17/01), NIPER SAS Nagar approved the animal protocol (IAEC/17/69) for handling of animals and commencement of the study. Then after, healthy female SD rats were issued from Central Animal Facility (CAF) and housed at 25 ± 2°C/50–60% relative humidity in natural light/dark atmosphere before study. The animals were given a standard pellet diet along with water ad libitum.

Oral Pharmacokinetic Study

Pharmacokinetic Experimentation

Eighteen SD rats (180–200 g) were arbitrarily divided into 6 groups wherein each group has 3 animals. For overnight, all animals were fasted with water access prior to the conduct of study. A single oral dosing (40 mg/kg body weight) of SIM suspension (0.25% w/v sodium carboxymethyl cellulose) was done in groups I and II. On other hand, groups III and IV were orally administered with a single dose of SIM-PLC (dose equivalent to 40 mg/kg body weight of SIM). Lastly, SIM-PLC-MD was orally given at the same dose to groups V and VI.

At fixed time intervals, blood samples were withdrawn from the tail vein in micro centrifuge tubes previously sprinkled with 4% w/v of EDTA solution (50 μL) to avoid coagulation. The collected samples were immediately centrifuged (6000 rpm, 10 min) at 4°C and supernatant was reserved at −40°C until HPLC analysis.

Plasma Samples Processing and Quantification of SIM

The stored plasma samples were thawed at room temperature and processed further for SIM extraction and determination using a reversed phase HPLC method. Herein, the mixture of plasma sample (100 μL) and internal standard (clindipine, 50 μL) was vortexed (30 s) and added with protein precipitating solution that consists of methanol and acetonitrile (1:1 ratio). The resultant mixture was further vortexed for additional 3 min, followed by centrifugation (6000 rpm, 10 min). The supernatant was injected to HPLC system after filtration through a 0.25-μ membrane filter. The acetonitrile and 0.02% of glacial acetic acid (pH 3.4) were used as mobile phase (70:30 ratio) and pumped in isocratic mode (1 mL/min). The linear (r2 0.998) calibration curve (concentration range 100–50,000 ng/mL) was obtained and the method was validated as per ICH guidelines.

Data Analysis

The pharmacokinetic profiles of SIM, SIM-PLC, and SIM-PLC-MD were generated using Kinetica software version 5.0 (Thermo Fisher Scientific Inc., USA) and compared.

Pharmacodynamic Experimentation

This study was undertaken to evaluate hypolipidemic potential of SIM, SIM-PLC, and SIM-PLC-MD and comparison of their profiles. Twelve SD rats (180–200 g) were arbitrarily distributed into 4 groups with 3 animals in each. Then after, SIM suspension (0.25% w/v sodium carboxymethyl cellulose), SIM-PLC, and SIM-PLC-MD were administered as a single oral dose (equivalent to 40 mg/kg body weight of SIM) to groups II, III, and IV respectively whereas group I was designated as the control group. The constant dose of SIM, SIM-PLC, and SIM-PLC-MD was repeated for four subsequent days. After 1 h of last dosing, Triton WR1339 (Tyloxapol) solution in saline was injected (0.1 mL) via intraperitoneal route to all control (I) and test animals. At the time interval of 24 h, blood samples were collected from the tail vein. The biochemical analysis of samples was performed for cholesterol and triglycerides levels.

Statistical Analysis

In the current work, quantitative results are presented as mean ± standard deviation. The MicrocalTM Origin® software version 6.0 (Microcal software, Inc., Northampton, USA) was used to apply one-way analysis of variance (ANOVA). Significant difference was regarded as p < 0.05.

RESULT AND DISCUSSION

Investigation of Phospholipid Type and Appropriate Molar Ratio of SIM and Phospholipid

This preliminary study involved screening of appropriate phospholipid to generate SIM-PLC with higher complexation efficiency. Table I presents the increment in complexation efficiency in the following pattern: Phospholipon® 90 H < Lipoid S 75 < Phospholipon® 90 G. This outcome is attributed to the structure of these phospholipids. Phospholipon® 90 H is a saturated phospholipid whereas Lipoid S 75 and Phospholipon® 90 G are unsaturated phospholipids. Unsaturated fatty acids generate a gap between tightly packed tails owing to double bond which makes them highly permeable to aid interactions with drug molecules in comparison to saturated phospholipids (33, 34). In addition, Phospholipon® 90 G has higher phosphatidylcholine content in comparison to other phospholipids which can be correlated with favorable interactions between SIM and phospholipid and maximum complexation efficiency (35). Thus, Phospholipon® 90 G was further subjected to an optimization experimentation to find out suitable molar ratio of SIM and Phospholipon® 90 G.

Table I Screening of Various Phospholipids for Generation of SIM-PLC
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The mixing ratio of drug and phospholipid is an imperative criterion to obtain higher complexation efficiency. Therefore, complex was prepared in different ratios viz. 1:0.5, 1:1, 1:2, and 1:3. The result of optimization study is depicted in Table II. The least ratio (1:0.5) of SIM and phospholipid shown less complexation efficiency accredited to limited amount of phospholipid to interact with SIM that leads to less interactions vis-à-vis less complexation efficiency (20). Then after, further increase in ratio results in incline in complexation efficiency. The ratio 1:1 has shown maximum complexation efficiency of 70.48 ± 1.50%, which markedly decreased to ~ 60% on further increase in ratio up to 1:3. Additionally, complex was more viscous when formulated in ratios more than 1:1. Hence, SIM-PLC in an equimolar ratio of SIM and Phospholipon® 90 G with maximum complexation efficiency was further opted for formation of SIM-PLC-MD. Thus, a conclusion was made that a single bond combination of SIM and phospholipid was involved in the complexation process (36).

Table II Optimization of SIM to Phospholipid Ratio
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Fabrication of Simvastatin-Phospholipid Complex–Loaded Matrix Dispersion (SIM-PLC-MD)

In spite of good complexation efficiency, SIM-PLC was found to be sticky and inconvenient to disperse. Hence, SIM-PLC was further loaded into Soluplus® matrix dispersion to overcome the aforesaid drawbacks without compromising complexation state between SIM and Phospholipon® 90 G. Table III shows complexation efficiencies of matrix dispersions when generated in different ratios (SIM:phospholipid:Soluplus® ratio 1:1:1 and 1:1:2). The complexation efficiency of SIM-PLC-MD in ratio 1:1:1 was not significantly affected when fabricated as matrix dispersion, whereas complexation efficiency was significantly reduced to 63.59 ± 1.35% when ratio was escalated to 1:1:2 which may be due to partial destroyed complex state between SIM and phospholipid by Soluplus®. Hence, SIM-PLC-MD in ratio 1:1:1 was taken forward for in vitro and in vivo evaluation.

Table III Optimization of SIM:Phospholipid:Soluplus® Ratio for Preparation of SIM-PLC-MD
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In vitro Evaluation of SIM-PLC and SIM-PLC-MD

Physicochemical Characterization

Fourier Transform Infrared Spectroscopy

Figure 1 illustrates the FTIR overlay of SIM, phospholipid, SIM-PLC, Soluplus®, and SIM-PLC-MD. The characteristic peaks of SIM were displayed at 3551.27 cm−1 (O─H stretching vibration), 2970.05 cm−1 and 2872.28 cm−1 (methyl and methylene, C─H asymmetric and symmetric stretching vibrations), 1698.11 cm−1 (C=O stretching vibration, ester and lactone carbonyl functional group), 1267.63 cm−1 and 1226.25 cm−1 (lactone C─O─C bending vibrations), and 869.86 cm−1 (trisubstituted olefinic C─H wagging vibrations). Additional peaks at 1468.67 cm−1, 1390.79, and 1369.47 (methyl and methylene, C─H bending vibrations) were also obtained in SIM spectra. The phospholipid exhibited characteristic peaks at 3382.65 cm−1 (O─H stretching vibration), 2924.08 and 2854.16 cm−1 (aliphatic C─H stretching vibration), 1726.34 cm−1 (C=O stretching vibration), and 1465.99 cm−1 (fatty acids chains of phospholipid) along with polar head group peaks at 1227.81 cm−1 (P=O stretching vibration) and 1086.98 cm−1 ((P─O─C stretching vibration). In the case of SIM-PLC, the SIM peak at 3551.27 cm−1 disappeared probably indicating its participation in hydrogen bonding. The SIM peaks at 1698.11 cm−1 and 1468.67 cm−1 were also missing in SIM-PLC, which may be attributed to removal, weakening, or shielding by phospholipid molecule. The phospholipid peaks at 1465.92 cm−1, 2923.78, and 2853.88 cm−1 were found intact in SIM-PLC spectra suggesting non-involvement in complexation. Whereas, phospholipid characteristic peaks at 1726.34 cm−1, 1227.81 cm−1, and 1086.98 cm−1 were shifted to 1735.96 cm−1, 1244.38 cm−1, and 1089.86 cm−1 respectively indicating successful complexation and involvement of polar head groups in interactions. These findings suggesting complexation involved van der Waals forces and other hydrophobic interactions. The Soluplus® spectra presented peaks at 3454.28 cm−1 (O─H stretching vibration), 3052.18 cm−1 (N─H stretching vibration of primary amine), 2858.78 cm−1 and 2928.54 cm−1 (dual signal of aromatic C─H stretching vibration), 1736.00 cm−1 and 1634.95 (dual signal of C=O stretching vibration), 1477.09 cm−1 (C─O─C stretching vibration), and 1442.69 cm−1 and 1243.76 cm−1 (CH2 stretching vibration). In the case of SIM-PLC-MD, the characteristic peaks of SIM at 3551.27 cm−1, 1698.11 cm−1, and 1468.67 cm−1 were still missing indicating presence of SIM in complex form with phospholipid (as found in SIM-PLC). The Soluplus® characteristic peak at 3454.28 cm−1 was broaden and shifted to 3409.45 cm−1 indicating intermolecular hydrogen bonding. The phospholipid peak at 3382.65 cm−1 disappeared suggesting involvement in interaction. But the phospholipid peak at 3382.65 cm−1 was found intact in SIM-PLC, so it was not involved in complexation. Additionally, the peak at 1737.67 cm−1 that was broadened may be due to the overlap of C=O stretching vibration of SIM-PLC (due to interaction of SIM and phospholipid) and C=O stretching vibration of Soluplus®. Moreover, the characteristic peaks of Soluplus® at 2856.22 cm−1, 2927.25 cm−1, 2325.83 cm−1, 1634.97 cm−1, 1476.38 cm−1, 1444.52 cm−1, 1372.92 cm−1, and 1243.72 cm−1 were still found intact in SIM-PLC-MD indicating non-involvement in interactions. The results suggest no interference of carrier material (Soluplus®) at the ratio of 1:1:1 (SIM:phospholipid:Soluplus®) to the complexation of SIM and phospholipid.

Fig. 1
figure 1

FTIR overlay of a SIM, b phospholipid, c SIM-PLC, d Soluplus®, and e SIM-PLC-MD

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Powder X-ray Diffractometry

The PXRD graph of SIM, phospholipid, physical mixture of SIM and phospholipid, SIM-PLC, and SIM-PLC-MD is demonstrated in Fig. 2. The multiple prominent diffraction peaks presented in Fig. 2(a) indicated high degree of crystallinity of SIM. Whereas, Fig. 2(b) represents amorphous nature of the phospholipid. The physical mixture spectra that presented a distinguishable SIM pattern with decreased intensity might be ascribable to the following possible aspects: (a) less amount of SIM in prepared physical mixture, (b) shielding and interference by phospholipid, (c) formation of partial aggregates in in situ conditions (26). On the contrary, characteristic peaks of SIM are missing in SIM-PLC revealing amorphization of SIM after complexation with phospholipid. In parallel, Fig. 2(e) illustrates a hollow pattern revealing amorphous nature of SIM-PL-MD.

Fig. 2
figure 2

PXRD graph of a SIM, b phospholipid, c physical mixture SIM and phospholipid, d SIM-PLC, and e SIM-PLC-MD

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Scanning Electron Microscopy

Figure 3 illustrates the SEM images of SIM, phospholipid, SIM-PLC, and SIM-PLC-MD. SIM appeared as non-uniform and irregular-shaped crystals. Whereas, phospholipid that existed as an irregular mass indicated its amorphous nature. Figure 3(c) exhibits that SIM-PLC particles are irregular block shaped and original crystallinity of SIM disappeared after complexation with phospholipid. In addition to the disappearance of SIM crystals, SIM-PLC-MD existed as smaller and irregular particles without aggregation in comparison to SIM-PLC. These findings suggest that SIM-PLC is well dispersed on carrier material Soluplus® (37). Thus, it was concluded that amorphous nature and enhanced surface area might contribute to improved solubility and dissolution profiles.

Fig. 3
figure 3

Scanning electron micrographs of a SIM, b phospholipid, c SIM-PLC, and d SIM-PLC-MD

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Transmission Electron Microscopy

This experiment was undertaken to investigate structural behavior of SIM-PLC and SIM-PLC-MD in water because these formulations would be dispersed in gastrointestinal fluids after oral administration. Upon dispersion of SIM-PLC in aqueous medium, phospholipid molecules complexed with SIM organized in order and formed nano size, well-defined, dark spherical vesicles having visible striations. This is attributed to regular self-assembling of complexed phospholipid molecules into vesicles at aqueous conditions, which resembled with multilamellar vesicles/liposomes despite of their distinguished internal structure (36).

On other hand, SIM-PLC-MD after dispersion into aqueous medium, arranged into regular micellar-shaped vesicles composed of dark inner core enveloped by light outer layer probably accredited to the interactions of complexed phospholipid with amphiphilic polymer Soluplus® (Fig. 4).

Fig. 4
figure 4

TEM photographs of a SIM-PLC and b SIM-PLC-MD in aqueous medium. Inset shows enlarged view

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n-Octanol–Water Partition Coefficient Studies

log P represents the distribution of drug in two unlike phases such as water and n-octanol. It is a key factor for permeability determination of drugs. As per data, the aqueous solubility of SIM-PLC-MD (390.12 ± 13.59 μg/mL) was markedly elevated compared with SIM-PLC (235.23 ± 8.50 μg/mL) (p < 0.001) and SIM (166.29 ± 8.80 μg/mL) (p < 0.001). In contrast, n-octanol solubility of SIM-PLC-MD (19,609.88 ± 13.59 μg/mL) (p < 0.05) and SIM-PLC (19,764.76 ± 8.50 μg/mL) (p < 0.05) were lower than plain SIM (19,833.71 ± 8.80 μg/mL). Collectively, log P values were notably declined in the following pattern: SIM ˃ SIM-PLC ˃SIM-PLC-MD as illustrated in Fig. 5. These findings are attributed to amorphization of complexed SIM and solubilization capacity of phospholipids that leads to decreased lipophilicity and increased partitioning into the aqueous phase (28). In addition to phospholipid complex, the hydrophilic structure of Soluplus® in SIM-LC-MD results into superior profile than SIM-PLC (38).

Fig. 5
figure 5

The calculated log P values of SIM, SIM-PLC, and SIM-PLC-MD

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Apparent Aqueous Solubility Study

This study was undertaken to evaluate the solubility potential of plain SIM, PM (physical mixture of SIM, phospholipid, and Soluplus®), SIM-PLC, and SIM-PLC-MD in 0.1 N HCl buffer (pH 1.2) and phosphate buffer (pH 6.8) at specified conditions. Figure 6 depicts that SIM possesses a pH-dependent solubility profile which was in accordance with literature reports (16). In comparison to SIM, SIM-PLC displayed enhanced solubility at pH 1.2 and pH 6.8 conditions by 2.66-fold (non-significant at p < 0.05) and 45.30-fold (p < 0.001) respectively. There are two primary factors that governed improved solubility of SIM-PLC. Firstly, amorphization of SIM occurs after complexation with phospholipid as revealed by PXRD technique. Secondly, amphiphilic phospholipid is having wetting and solubilizing potential. Altogether, these factors lead to remarkable solubility enhancement of SIM-PLC (28). Similarly, SIM-PLC-MD exhibited a dramatic increment in solubility in comparison to SIM, SIM-PLC, and PM. The enhancement in solubilization is by approximately 94-fold (p < 0.001) and 431-fold (p < 0.001) in pH 1.2 and 6.8 respectively owing to micellization, reduced viscosity, and increased dispersibility of SIM-PLC by amphiphilic Soluplus® due to its good solubilization potential (39, 40). On other hand, PM shown enhanced solubility in comparison to SIM and SIM-PLC accredited to wetting and solubilization potential of amphiphilic phospholipid and Soluplus®.

Fig. 6
figure 6

The solubility profiles of SIM, SIM-PLC, PM, and SIM-PLC-MD at different pH conditions

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In Vitro Dissolution Study

The dissolution experiment was conducted to investigate the release pattern of SIM, PM, SIM-PLC, and SIM-PLC-MD. Figure 7 depicts a dissolution pattern in 0.1 N HCl having pH 1.2 and phosphate buffer having pH 6.8 at specified conditions. By the end of the dissolution study, SIM displayed the slowest dissolution rate of 7.92% and 16.64% in pH 1.2 and 6.8 respectively. The dissolution pattern of PM demonstrated a trivial increase in release compared with SIM in both buffers. Likewise, SIM-PLC release was insignificantly higher than SIM owing to sticky nature of complex that leads to compromised dispersibility and poses a hindrance to drug release (27, 41). The slight increase in dissolution of SIM-PLC is ascribed to amorphous nature of SIM in complex and solubilization potential of amphiphilic phospholipid. These results are in consonance with proposed hypothesis and SEM analysis. On the contrary, the dissolution profile of SIM-PLC-MD revealed the considerable enhancement in release by ~2.53-fold (p < 0.05) and ~1.5-fold (p < 0.05) in pH 1.2 and 6.8 respectively. This outcome can be explained by three predominant factors. Firstly, SIM is present in easily dissolving amorphous state. Secondly, Soluplus® imparts SIM-PLC-MD with favorable dispersibility, wettability, and reduced viscosity, thereby increasing contact with dissolution medium. Additionally, Soluplus® aids in solubilization by means of micellization with phospholipid complex as elaborated in TEM investigation. Overall, these possible factors played a key role in accelerated dissolution rate of SIM-PLC-MD than SIM and SIM-PLC (28, 38).

Fig. 7
figure 7

Comparative dissolution pattern of SIM, PM, SIM-PLC, and SIM-PLC-MD at a pH 1.2 and b pH 6.8

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In Vivo Evaluation of SIM-PLC and SIM-PLC-MD

Oral Pharmacokinetic Study

Figure 8 illustrates the mean plasma concentration-time profiles of SIM, SIM-LC, and SIM-PLC-MD after a single oral dose. The Kinetica software was used to analyze pharmacokinetic data, which is compiled in Table IV. The bioavailability results suggest varied rate and extend of SIM release from SIM suspension, SIM-PLC, and SIM-PLC-MD, which were in line with in vitro data. The SIM-PLC displayed higher AUC0-6 h of 4.495 μg h/mL in comparison to SIM (2.447 μg h/mL), which was further significantly enhanced to 7.83 μg h/mL in the case of SIM-PLC-loaded matrix dispersion (SIM-PLC-MD). Parallelly, the peak plasma concentration (Cmax) of SIM-PLC-MD was obtained as 0.3850 μg/mL, which was considerably higher than the Cmax of SIM (0.3235) and SIM-PLC (0.3491). Although, SIM-PLC exhibited improved pharmacokinetic profile than plain SIM, but only 1.83-fold enhancement in oral bioavailability is ascribed to sticky nature and compromised dissolution as per in vitro experiment. SIM-PLC-MD shown remarkable improvement in oral bioavailability by 3.19-fold and 1.74-fold when compared with SIM and SIM-PLC respectively.

Fig. 8
figure 8

Mean plasma drug concentration–time curve of SIM following oral administration of SIM, SIM-PLC, and SIM-PLC-MD in SD rats. Data are mean ± SEM (n = 3)

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Table IV Pharmacokinetic Parameters of SIM, SIM-PLC, and SIM-PLC-MD Following Oral Administration in Sprague Dawley Rats
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According to the results, SIM-PLC-loaded matrix dispersion having Soluplus® as carrier (SIM-PLC-MD) attained optimum systemic concentration after the oral administration. The several contributing aspects including amorphous state of SIM, wetting potential of phospholipids, higher dispersibility, and formation of micellar structures collectively augmented solubility, dissolution, and permeability profile, which eventually contributed to enhanced oral bioavailability of SIM-PLC-MD (39). Furthermore, the enhanced elimination half-lives of SIM-PLC (11.1 h) and SIM-PLC-MD (15.3 h) in comparison to SIM (5.46 h) might be favorable to cut down dose and dosing frequency. Similarly, increased mean residence time of SIM-PLC (16.91 h) and SIM-PLC-MD (22.26 h) compared with plain SIM (7.88 h) suggested extended release pattern (8, 14).

Pharmacodynamic Experimentation

After showing promising outcome in bioavailability studies, prepared formulations were further evaluated for their effectiveness to reduce blood triglycerides and cholesterol levels. The SD rats were intraperitoneally administered with Tyloxapol in order to develop hyperlipidemic animal model. Tyloxapol inhibits lipoprotein lipase enzymes in periphery that regulates the removal of lipid particles in body. Therefore, introduction of Tyloxapol results into transitory enhancement in lipid levels (42). The results of hypolipidemic study are interpreted and compiled in Fig. 9. The results presented that SIM-PLC-MD treatment significantly decreased total cholesterol level (86.5 mg/dL) in comparison to control (111 mg/dL) and SIM (105 mg/dL) and SIM-PLC (95.79 mg/dL). Furthermore, the remarkable reduction in triglycerides level by SIM-PLC-MD treatment (58.5 mg/dL) was observed in contrast to control (98 mg/dL), SIM (84.5 mg/dL), and SIM-PLC (73 mg/dL).

Fig. 9
figure 9

Effect of different treatments on plasma triglycerides and cholesterol levels in SD rats. Data are mean ± SEM

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The improved efficacy of SIM-PLC-MD on triglycerides and cholesterol levels in contrast to SIM and SIM-PLC is in agreement with pharmacokinetic findings. The improved systemic availability of SIM as a result of augmented solubility and dissolution effectively declined triglycerides and cholesterol levels. In conclusion, improved therapeutic efficacy of treatment may help to reduce dose, dosing frequency, and hence potential toxicities of SIM.

CONCLUSION

The present work was envisaged to investigate and compare the potential of SIM-PLC and SIM-PLC-MD to circumvent oral bioavailability obstacles and promote in vivo efficacy of SIM. Initially, SIM-PLC and SIM-PLC-MD were successfully formulated and complexation efficiency values revealed unaffected complexation between SIM and phospholipid after loading into matrix dispersion. The data obtained from analytical tools including FTIR and PXRD suggested amorphization of SIM and successful complexation between SIM and phospholipid involving van der Waals forces and other hydrophobic interactions. Additionally, SEM demonstrated morphological characteristics, whereas TEM experiments indicated conversion of SIM-PLC and SIM-PLC-MD into nano size colloidal structures upon dispersion in aqueous medium. Furthermore, the considerable increment in hydrophilicity, thereby solubility of SIM-PLC and SIM-PLC-MD, was observed. The limited dissolution rate of SIM-PLC was considerably accelerated by loading it into Soluplus®-based matrix dispersion (SIM-PLC-MD). Collectively, these factors lead to improved oral bioavailability of SIM-PLC-MD and SIM-PLC by 3.19-fold and 1.83-fold respectively. Finally, SIM-PLC and SIM-PLC-MD with improved oral bioavailability significantly reduced cholesterol and triglycerides levels, wherein SIM-PLC-MD exhibited superior hypolipidemic activity. In a nutshell, a dual/combination delivery system can be a promising strategy to augment oral bioavailability and efficacy of poorly water soluble drugs like SIM. These findings can be further beneficial to cut down dose, dosing frequency, and dose-dependent side effects.