Impact on HDL and LDL of Hyperlipidemic Rat Models: Designed Solid Self-Nanoemulsifying Drug Delivery Systems with Atorvastatin and Ezetimibe combination


Ahmed R. Gardouh1,2*, Ahmed M. Nasef3, Yasser Mostafa4, Shadeed Gad1

1Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy,

Suez Canal University, Ismailia, 41522, Egypt.

2Department of Pharmaceutical Sciences, Faculty of Pharmacy, Jadara University, 21110 Irbid, Jordan.

3Production Department, Medical Union Pharmaceutical (MUP) Co., Ismailia, 41617, Egypt.

4Department of Pharmacology and Toxicology, Faculty of Pharmacy,

Suez Canal University, Ismailia, 41522, Egypt.

*Corresponding Author E-mail:



The main purpose of this study was to develop and evaluate solid self-nanoemulsifying drug delivery systems (S-SNEDDs) of Atorvastatin/ Ezetimibe combination to combine the advantages of liquid SNEDDs with those of solid dosage forms and investigate the effect of solidification on both lipid lowering efficiency and the ability to enhance oral bioavailability of included poorly water soluble drugs. Spray dried solid powder was prepared using Aerosil 200 based on its high adsorption capacity and the ratio of liquid SNEDDs: Aerosil was (4:1) due to the smaller droplet size produced after reconstitution compared to other ratios. Surface morphology characteristics and drug-excipients interactions were evaluated via Scanning Electron Microscopy (SEM) and Fourier Transformed Infrared Spectroscopy (FTIR). Crystallinity nature affect drug dissolution so, it was determined by Differential Scanning Calorimetry (DSC) and Powder X-ray Diffraction (PXRD). Pharmacokinetic study investigated the ability of (S-SNEDDS) to improve oral bioavailability of included drugs while, pharmacodynamic study evaluate its efficiency to control serum cholesterol levels compared to pure drugs suspension and liquid SNEDDs. Solid spray dried powder showed very good flowability (3.41±0.23g/s) and rapid dispersion in water with maintaining the self-emulsifying efficiency of liquid formula. Physicochemical evaluation of powder showed spherical separated particles with no significant drug-excipients interactions and drugs are molecularly dispersed or in amorphous state that improve dissolution rate as proven by in-vitro release studies. Pharmacokinetic and pharmacodynamic studies proved that the solidification process had no remarkable effect on the efficiency of liquid formula to enhance oral bioavailability of incorporated drugs and control serum cholesterol level compared to pure drugs suspension. S-SNEDDS was proved as efficient candidate to improve oral bioavailability of Atorvastatin / Ezetimibe combination and control cholesterol serum levels.


KEYWORDS: Poorly water soluble, Atorvastatin, Ezetimibe, Solid self nanoemulsifying, oral bioavailability.




Cholesterol which is a waxy substance made by animal liver or obtained from diet plays a vital role in human heart health and it is very important for human body. Cholesterol can be classified into High-density lipoprotein (HDL-C) which is the good cholesterol and low-density lipoprotein (LDL- C) which is the bad type of cholesterol1. Atorvastatin (ATV) is one of most important3-hydroxy-3-methyl-glutaryl-CoA(HMG Co-A) reductase inhibitors2 which are the basis of lipid-lowering therapy to decrease cardiovascular risk through reducing the hepatic production of LDL- C and hence, the high doses of atorvastatin can reducing LDL- C serum levels by up to 50%1,3. Ezetimibe (EZT) was developed to lower serum levels of low-density lipoprotein cholesterol (LDL- C)4 through selectively hindering the transportation of both biliary and dietary cholesterol across the intestinal wall5,6


The low oral bioavailability of pure atorvastatin and ezetimibe (14% and 35% respectively) is due to that the drugs come under the Biopharmaceutics Classification System (BCS) class II7,8, and its high  presystemic clearance, and/or extensive first-pass metabolism9. When compared with statin monotherapy, Ezetimibe has a synergistic effect on the reduction of triglycerides and low density lipoprotein (LDL-C) cholesterol and increasing high density lipoprotein (HDL-C) cholesterol10. However, the incorporation of both drugs in single drug delivery system is very difficult because of the different physicochemical properties.


One of the most efficient strategies to improve the oral bioavailability is altering the physical nature of poorly water-soluble lipophilic compounds through its incorporation into lipid vehicle such as oil and self-nanoemulsifying drug delivery systems (SNEDDs)11,13. Conventionally, self-emulsifying medicines are liquid formulations filled into capsules or dispensed as oral solutions14 which results in many disadvantages including lack of stability, irreversible precipitation of excipients/drugs, and leakage of volatile ingredients through the shell of gelatin capsule15. The solidification of liquid SNEDDs (L-SNEDDs) through various techniques like: spray drying  is a promising approach to surpass the limitations of L- SNEDDs (such as drug precipitation)16 and combine the benefits of SNEDDs (enhancement of both solubility and bioavailability) with the advantages of solid dosage forms (ease of handling and portability, higher stability and reproducibility, compact dosage form and better patient compliance)17.


The objective of this study was to prepare and characterize the spray dried solid SNEDDs (S-SNEDDs) using previously optimized L-SNEDDs. We also evaluated both pharmacokinetics and pharmacodynamics characteristics of prepared S-SNEDDs in rats. 




Atorvastatin calcium, Olive oil, propylene glycol, Isopropyl myristate, Polyethylene glycol 400, Castor oil, Aerosil 200®, Micro Crystalline Cellulose PH102 , Sodium carboxy methylcellulose, Poloxamer 407® and hydroxyl propyl - β – cyclodextrine gifts from MUP Co. (Egypt). Ezetimibe was a kind gift from LIPTIS Pharmaceuticals Co. (Egypt). Labrasol ALF®, Capryol 90®, Labrafac lipophile WL 1349®,Plurololeique CC 497®, Peceol®, Maisine CC®, Transcutol HP® and Gelucire 44/14® were kind gifts from Gattefossé (France). Kolliphor grades (HS 15®, RH 40®, ELP® and EL®) were gifts from BASF (Germany). Oleic acid was purchased from Merck Co.(Germany). Span 80®, Tween grades (80®, 60® and20 ®) were purchased from Kolb Ltd. Co. (Switzerland). All the other used chemicals and solvents were of HPLC grade and used without further purification.



Preparation of liquid self-nanoemulsifying drug delivery systems (L-SNEDDs):

According to our study18, the liquid self-nanoemulsifying system containing Atorvastatin/ Ezetimibe combination was prepared using Capryol 90 (10%) as oily phase, Tween 80/ Kolliphor RH40 (1:1) (42.71%) as surfactant mixture and Transcutol HP (47.29%) as co-surfactant. The concentration of each component was optimized by Design Expert software ( of Stat‑Ease, Inc. Minneapolis, USA. based on desirability value.


Preparation of solid (S-SNEDDs) using spray drying technique:

Solid carrier was selected based on oil adsorption capacity (OAC) where; the higher (OAC) value, the better flow and compaction can be achieved. Both hydrophobic like Micro Crystalline Cellulose (MCC) PH102, Aerosil-200 (A-200) and hydrophilic carriers like sodium carboxy methylcellulose (Na-CMC) and hydroxyl propyl - β - cyclodextrine (HP β-CD) were used. OAC was determined using gravimetric method where, the amount of porous carrier needed to convert the unit dose of the oily liquid formula to free flowing powder was determined19. Briefly, 250mg of Aerosil 200 was suspended in 100 mL of solvent (water/ethanol). After sonication for 3 minutes in the case of ethanol and for 3 minutes at 50˚C in the case water, 1gm of the optimized liquid formula was added with stirring constantly until a uniform suspension was formed. Using a Buchi mini spray dryer (Buchi, B-190, Switzerland), the resulting suspension was spray dried under the following conditions: inlet temperature of 60˚C and 140˚C, outlet temperature of 45˚C and 66 ˚C for ethanol and water respectively, atomization air pressure of 6.5 kg/cm and the suspension feeding rate was adjusted to 4 ml/min. The spray dried powders were then collected from the collecting chamber and cyclones20. The emulsion droplet size was measured, and the powders were kept at room temperature in a desiccator containing dry silica crystals20.



To determine the optimal concentration of used solid porous carrier, different amounts of carrier (125, 250, 333, and 500mg) were tested and the emulsion droplet size for each concentration was measured.


Flow rate and angle of response (Ө):

The angle of repose can be defined as the constant three dimensional angle measured relatively to the horizontal base, calculated using a cone-like pile of powder formed when the powder is passed through a funnel-like container21. The angle of repose was determined by funnel method using the following formula22,23:



Where, h is height of the pile, θ is angle of repose and r is average radius of the powder cone.


Bulk density and tapped density24:

The bulk density (BD) of a powder is the ratio of powder total mass to its bulk volume including the volume of the interparticular voids where, the tapped density (TD) of a powder can be defined as the powder total mass to its tapped volume. The tapped volume was determined by tapping the powder to constant volume. Both the bulk and tapped density can be calculated using the following equations25,26:


Compressibility index (Carr's index)

Carr’s compressibility index is used to evaluate the powder flowability and measures the tendency of a powder to consolidate. Carr’s Index was calculated using the following equation27,28:




Hausner ratio:

It is the ratio of the powder tapped density to its aerated bulk density.


With a Hausner ratio lower than 1.25 indicates a free-flowing powder22. It can be calculated according to the following equation29:



Reconstitution properties of S-SNEDDs

The effect of dilution on S-SNEDDs was investigated to mimic the stomach condition following the administration. In brief, 100mg of S-SNEDDs was added with gentle stirring to 100ml of double distilled water in a glass beaker and the temperature was kept at 37ºC. The emulsification ability of S-SNEDDs was judged “good” when a clear/transparent emulsion is formed rapidly in less than one minute25.


Fourier Transformed Infrared Spectroscopy (FTIR):

It is generally used to determine the presence of any drug-excipient interaction. About 2mg of sample (Aerosil 200, atorvastatin, ezetimibe, physical mixture and optimized S-SNEDDs) was triturated with 10mg of dry potassium bromide (KBr) and then pressed into the pellet by a pneumatic press. Infra-red spectrums of the prepared discs were obtained using FTIR spectrophotometer (Shimadzu 8400, Japan). The spectrums were taken over the range of 400–4000cm-1with resolution of 4cm-1. 30. The obtained spectrums were compared with that of the pure drugs for any interactions.


Differential Scanning Calorimetry (DSC):

It is a thermo analytical technique used to determine the nature of crystallinity by measuring the melting point temperatures and its associated enthalpies31. Thermograms of atorvastatin, ezetimibe, Aerosil 200, physical mixture and optimized S-SNEDDs formulation were obtained using differential scanning calorimeter (Setaram, DSC-131 evo, France). Approximately 3mg of each sample was placed in standard aluminum pan and crimped on lids. To obtain the endothermic peaks, in the presence of nitrogen flow at rate of (30ml/min), the samples temperature was raised from 0 to 200°C gradually at heating rate of 10°C/min32.


Powder X-ray diffraction:

Both drug dissolution rate and its bioavailability may be affected by polymorphic alterations of drug and drug-excipients interaction33. So, PXRD studies were used to determine solid state and examine the crystallographic structure of pure drugs and optimized S-SNEDDs. X-ray diffraction patterns of pure drugs (atorvastatin and ezetimibe), physical mixture of drugs with Aerosil 200 and optimized S-SNEDDs were recorded on PANalytical Empyrean X-ray diffractometer (Malvern, United Kingdom), a voltage of 40 kV and a 25-mA current. The scanning rate was kept at 1° min−1 over 2θ range of  10–40°.


Surface Electron Microscope (SEM):

It is used to study the surface morphology and check if the crystalline structures characteristic of drug are not observed in S-SNEDDs micrographs proposing that the drug exists in a completely dissolved state in the S- SNEDDs34. Scanning electron micrographs for Aerosil 200, atorvastatin, ezetimibe and prepared S-SNEDDs formulae were taken using SEM (JEOL, JSM 50A, Japan) at 15 kV accelerating voltage.



In-vitro release studies:

In vitro drug release from both liquid and solid SNEDDs was compared using the USP paddle (apparatus II) method. SNEDDs formulae equivalent to single dose (10 mg atorvastatin and 10mg ezetimibe) were encapsulated in hard gelatin capsules (HGCs) of size (00). The capsules were kept in sinking condition throughout the dissolution study in (Pharmasource PDT 806, Wilmington, USA) dissolution apparatus.


The dissolution medium for both drugs was acetate buffer (pH 4.0) with volume of 900ml and the temperature was kept at 37ºC±0.5 and the speed of paddles was of 75rpm. Samples of 3ml were withdrawn at the preset intervals of 5, 10, 20, 30, 40 and 60 minutes and filtered through 0.45µm filter35. The volume withdrawn each time was immediately replaced with fresh medium17,36. The filtered samples were diluted with methanol then drug concentration was obtained using a validated HPLC-UV method using Waters Alliance 2695 system (USA)and Hypersil ODS C18 column (5µm, 4.6mm×250mm; USA) for chromatic separation. The mobile phase was a mixture of (0.1M ammonium acetate buffer: acetonitrile = 2:3) of pH 6.035. All measurements were done in triplicate37.


In-vivo studies:

In-vivo evaluation studies investigated both pharmacokinetics and pharmacodynamics of solid SNEDDs compared to atorvastatin/ezetimibe suspension after oral administration in laboratory animals38.


Pharmacodynamic activity in rats:

Healthy albino rats were kept for a week before the beginning of the under controlled environmental conditions. The institution research ethics committee at Faculty of pharmacy, Suez Canal University approved the protocol and animal care procedures [201710PHD1] The study followed NIH guidelines for care and use of laboratory animals (8th edition). Well trained professional staff was responsible for animal feeding, delivery of drugs throughout the study, handling of feces frequently and paying attention to minimize the animal suffer. The blood samples were analyzed to determine the baseline of both total cholesterol (TC) and high-density lipoproteins (HDL). A rapid onset of hyperlipidemia can be developed after a single intraperitoneal dose of the non-ionic surfactant (Poloxamer 407) so, the antihyperlipidemic efficacy of pure atorvastatin/ezetimibe suspension, L-SNEDDs and S-SNEDDs formulations were evaluated by poloxamer-induced hyperlipidemic rat model39. Fifteen albino rats (300±50g) were randomly classified into five groups as follows: normal group (group I) receiving normal saline, hyperlipidemic control group (group II) receiving water orally treated group (group III) with drugs suspension, orally treated group (group IV) with optimized L–SNEDDs and orally treated group (group V) with optimized S–SNEDDs. Each dose for the treated groups was of (−1 atorvastatin and 25−1 ezetimibe). Both the % of initial TC level and % of initial non-HDL-C level were calculated at each time point using the following equations40:



Pharmacokinetic study:

The efficacy of solid SNEDDs to improve the oral bioavailability of both atorvastatin and ezetimibe was investigated through the pharmacokinetic study. Rats were randomly divided in three groups each of three animals (n=3); group I orally treated with pure atorvastatin/ezetimibe in suspension form, group II orally treated with optimized L- SNEDDs where, group III received optimized S- SNEDDs . Without anesthesia and by restraining rats by hand, the animals received single oral dose (equivalent to dose of 25−1 atorvastatin and−1 ezetimibe) through intubation using an 18-gauge feeding needle41. The samples were freshly prepared and the overnight fasted rats before the beginning of the study were fed four hours after the oral treatment. At preset time intervals (1, 2, 4, 8, 12 and 24 hrs.) the blood samples (0.75ml) were collected in heparinized capillary tube from the etherized rats through the caudal vein. The plasma samples were centrifuged for 10 minutes at 3000rpm using (Hettich zentrifugen, Mikro 22 R, Germany) and then freezed at–20ºC for further analysis with the previously mentioned valid HPLC method42. The plasma concentration–time profile data were analyzed with pharmacokinetic software (PK function for Microsoft Excel, Pharsight Corporation, CA, USA) to calculate the pharmacokinetic parameters. The maximum concentration (Cmax), area under the plasma concentration-time curve from zero to infinity (AUC 0-∞), elimination half-life (t½)and time required to reach maximum concentration (Tmax), were calculated for both atorvastatin and ezetimibe. The relative bioavailability (Fr%) of S-SNEDDs after oral administration was calculated according to the following equation43:




Statistical analysis:

All the experiments were carried out in triplicates and the shown data were expressed as mean ± standard deviation (SD) and analyzed by one-way analysis of variance (ANOVA) using (JMP 15.1.0, NC., USA). Differences between means were considered statistically significant for 0.05 >p ≥ 0.01, and highly significant for p< 0.01. The differences were considered as statistically non-significant for p values > 0.05.



Selection of solid carrier and solvent:

Oil adsorption capacity of used carriers can be arranged as follow: Aerosil 200 (250mg) > MCC PH 102 (375mg) > HP β-CD (480mg) > Na-CMC (575mg). Aerosil 200 was selected as solid carrier as it showed the highest oil adsorption capacity while, it was minimum with Na-CMC. A marked difference in emulsion droplet size was observed when using ethanol as a solvent instead of water. As shown in Table (1) the droplet size was found to be 185±0.47and 452±1.63nm for water and ethanol, respectively. According to these observations water was used as solvent during spray drying process.


Effect of loading on globule size:

The optimal concentration of solid carrier (Aerosil 200) was determined by droplet size of S-SNEDDs. As shown in Table (1), the amount of Aerosil 200 significantly affects dispersion particle size. Formula prepared using Aerosil 200 amount of 125mg did not form S-SNEDDs and this may be due to the shortage of solid carrier. Above the amount of carrier required to form SNEDDs (250mg), it was observed that particle size increased with increasing amount of carrier. According to these findings, amount of carrier of 250mg was chosen to prepare optimized S-SNEDDs (ratio of L-SNEDDs: Carrier was 4:1).


Table 1: Effect of solvent type and solid carrier amount on particle size of S-SNEDDs


Droplet size (nm)


185 ± 0.47


452 ± 1.63

Carrier amount

Droplet size (nm)

125 mg


250 mg

185 ± 0.47

333 mg

213± 0.82

500 mg

276 ± 0.47


Micrometric properties:

The micrometric properties of S-SNEDDs prepared were shown in Table (2).


Table 2: Micrometric properties of spray dried S-SNEDDs




Flow rate

3.41±0.23 g/s

Very free flowing44

Angle of response

28.32±0.19 degree

Bulk density

0.570 ± 0.01 g/cm3


Tapped density

0.65 ± 0.01 g/cm3


Carr’s index

13.2 ± 1.26%

Good flowability(45)

Hausner ratio

1.15 ± 0.02


Reconstitution of S-SNEDDs:

The reconstitution property of optimized S-SNEDDs was evaluated visually since; upon dilution the formula showed rapid dispersion within one minute without any agglomerations or signs of phase separation. So, it can be said that S-SNEDDs maintained the self-emulsifying efficiency of L-SNEDDs.


Fourier Transformed Infrared Spectroscopy (FTIR):

The FTIR spectra of Aerosil 200, Atorvastatin, Ezetimibe, physical mixture, and the formula are demonstrated in (Fig.1). FTIR spectrum of atorvastatin and ezetimibe were similar to those reported by (Panghal, D., et al.,2013)46 and (Kunam, V., et al.,2019) 47 respectively. The spectrum of both physical mixture and spray dried S-SNEDDs showed that the characteristic peaks are in acceptable ranges of pure atorvastatin and ezetimibe indicating that there is no chemical interactions between the carrier and two drugs48.










Fig.1: FTIR spectra of (a) Aerosil 200, (b)Atorvastatin, (c) Ezetimibe, (d) Physical mixture and (e)S-SNEDDs


Differential Scanning Calorimetry (DSC):

The physical status of two drugs in the prepared S-SNEDDs were investigated since it would have a significant influence on both in-vitro and in-vivo release characteristics. The thermograms of Aerosil 200, pure atorvastatin, pure ezetimibe, physical mixture and S-SNEDDs were shown in (Fig.2). The thermogram of pure atorvastatin showed sharp melting endotherm at 165.669˚C (153.176 - 172.65˚C) while, pure ezetimibe showed characteristic sharp melting endothermic peak at 165.042˚C (163.013 – 168.908˚C) indicating the presence of drugs in crystalline form. Aerosil 200 thermogram showed small peak at 183.754˚C and the physical mixture thermogram showed the characteristic endothermic peaks of atorvastatin and ezetimibe. The thermogram of S-SNEDDs (Fig. 2.e) showed the disappearance of characteristic peaks of either atorvastatin or ezetimibe indicating that the drugs are present in amorphous form.


Powder X-ray diffraction (PXRD):

The pattern of Aerosil 200 (Fig. 2.f.1) shows no characteristic peaks while, PXRD pattern of both atorvastatin (Fig. 2.f.2) and ezetimibe (Fig. 2.f.3) showed a large number of sharp peaks indicating the presence of unprocessed drugs in pure crystalline form since, the intensity of the XRD peak is based on the crystal size49. PXRD pattern of atorvastatin showed sharp characteristic peaks at 2θ diffraction angles at 9.0464º, 9.4414º, 10.1523º, 10.4577º, 11.7333º, 12.0651º and 16.9329º which match with the values reported by (Shete, G., et al.,2010)50. PXRD pattern of ezetimibe showed sharp distinct peaks at 2θ diffraction angles at 12.0651º, 13.8751º, 15.7549º, 18.6242º, 18.8797º, 19.3523º, 20.6209º, 20.8738º, 21.7690º, 22.9028º, 23.3927º, 24.5105º and 26.2967º which match with the values reported by (Dixit, R.P., et al.,2008)51 and these sharp diffraction peaks could be detected in the physical mixture with Aerosil 200.PXRD pattern of spray dried S-SNEDDs (Fig.2.f.5) showed only one peak at 19.9684º and the disappearance of the distinct sharp crystalline peaks of two drugs confirming the existence of the drugs in molecularly dissolved state. The results of both DSC and PXRD confirmed that the drugs may be either distributed in amorphous state or molecularly dispersed in the S-SNEDDs formulation49.

Fig.2: DSC pattern of (a) Aerosil 200, (b) Atorvastatin, (c) Ezetimibe, (d) Physical mixture, (e) S-SNEDDs and PXRD of (1) Aerosil 200, (2) Atorvastatin,(1) Ezetimibe, (4) Physical mixture and (5) S-SNEDDs

Scanning Electron Microscopy (SEM):

The surface morphology of Solid carrier (Aerosil 200), pure drugs (atorvastatin and ezetimibe) and S-SNEDDs were investigated using scanning electron microscope and micrographs are shown in (Fig.3). Aerosil 200 (Fig.3.a) appeared as agglomerates of amorphous particles. Pure atorvastatin (Fig. 3.b) appeared as crystalline powder with irregular or triangular plate shape crystals. Pure ezetimibe (Fig.3.c) appeared as smooth surfaced isolated crystals. S-SNEDD (Fig.3.d) appeared as well separated un-agglomerated spherical particles showing neither distinct crystals of atorvastatin nor ezetimibe on its surfaces.


Fig. 3: SEM micrographs of (a) Aerosil 200, (b) atorvastatin, (c) ezetimibe, (d) S-SNEDDs





Fig. 4: In-vitro release pattern for (a) Atorvastatin and (b) Ezetimibe

Table 3: Pharmacokinetic parameters of pure atorvastatin /ezetimibe suspension and atorvastatin /ezetimibe S-SNEDDs after single oral dose

Pharmacokinetic parameter

Pure Atorvastatin suspension in CMC

Atorvastatin in


Atorvastatin in


Pure Ezetimibe     suspension in CMC

Ezetimibe in


Ezetimibe in


Cmax (µg/ml)

8.19 ±0.11

18.20 ±0.05 *

17.5 ± 0.29 *

7.75 ± 0.12

18.08 ± 0.13*

17.9 ± 0.13*

Tmax (h)

1.58 ±0.33

0.2 ±0.05 *

0.28 ± 0.06 *

1.5 ± 0.21

0.2 ± 0.01*

0.29 ± 0.03*

t1/2 (h)

5.79 ±0.35

9.5 ±0.12 *

9.38 ± 0.25 *

5.3 ± 0.33

8.9 ± 0.24*

8.7 ± 0.13*

AUC0- (µg.h/ml)

59.04 ±1.07

209.56 ±2.28 *

202.9 ± 1.67 *

48.7 ± 1.95


179.70± 1.69*

Each value expressed as the mean ± SD (n=3),* significant at p < 0.001 compared to drug suspension.


In-vitro release studies:

In vitro release profiles of S-SNEDDs were compared with that of both L-SNEDDs and pure drugs (Fig. 4). In case of SNEDDs, the newly formed surface between the oily phase and aqueous phase requires very low free energy and it is proposed that the system components swell effectively with water which results in decreasing the droplet size and increase the rate of drug release. The release of atorvastatin and ezetimibe from S-SNEDDs was higher compared with that of pure drugs (more than 90% of drugs released within 30 minutes). However, the release rate L-SNEDDs was slightly higher than that of the S-SNEDDs within the first 40 minutes.


In-vivo studies:

Pharmacokinetic activity:

Time profiles of mean plasma concentration of ATV/EZT after single dose oral administration of L-SNEDDs, S-SNEDDs and ATV/EZT suspension showed pharmacokinetic parameters demonstrated in Table (3).


Upon comparing the pharmacokinetic parameters of the optimized liquid/solid formula with these of the pure drugs suspension, the differences between the calculated means were found significant as presented in Table (1). The relative bioavailability of atorvastatin and ezetimibe from S-SNEDDs were 3.44 and 3.69-fold respectively higher than drugs suspension proving the improvement of oral delivery/bioavailability of atorvastatin /ezetimibe when formulated in the form of SNEDDs. Although, the pharmacokinetic parameters of spray dried powder showed no significant difference from that of liquid formula, both liquid and solid SNEDDs showed significant difference compared to pure drugs suspension.


Pharmacodynamic study:

Since the pharmacokinetic parameters are not sufficient for predicting the Pharmacodynamic activity and it was reported that the plasma concentration of atorvastatin did not correlate with the reduction in LDL- C52, both pharmacokinetics and Pharmacodynamics of optimized SNEDDs were evaluated .After the poloxamer injection in rats by 12 hrs into the rats, TC level in both treated and hyperlipidemic control groups increased by more than 4-fold while, the beneficial HDL-C level reduced to 0.78 – 0.81 folds as compared to normal group. After 12 hr of oral administration of drug suspension (group III) and optimized formulae (group IV and group V), TC and Non-HDL levels in hyperlipidemic control group were significantly higher as compared to the other treated groups (Fig.5). After 36 h of administration, TC level showed further increase in both hyperlipidemic control group and that treated with ATV/EZT suspension while, TC level in groups treated with optimized formulae showed significant reduction. After 60hr. of administration, TC level and Non-HDL decreased to highly significant level in group treated with the optimized liquid and solid formulae as compared to hyperlipidemic control group. As shown in (Fig.5.a, 5.b), it was proven that there was a significant difference between the efficiency of both atorvastatin/ezetimibe suspension and optimized SNEDDs (either in liquid or solid form) in controlling both TC and Non-HDL levels.



Fig. 5: Changes in levels of (a) TC and (b) Non-HDL in rats after the oral administration of pure Atorvastatin/Ezetimibe suspension and Atorvastatin /Ezetimibe S-SNEDDs over three days. *significant at p < 0.001 compared to drug suspension.



In the current study, the purpose was to develop S-SNEDDs of poorly water soluble antihyperlipidemic drugs combination (ATV/EZT), determine its physicochemical characteristics and evaluate its ability to maintain the self emulsifying properties of liquid SNEDDs with enhancing the oral bioavailability and efficient control of serum compared to the pure drug suspension. Aerosil 200 was used as a solid carrier in the spray drying process because of its high oil adsorption capacity which may be due to that it wide surface area of 200m2/g and pore volume of 1.6ml/g which have significant impact on oil adsorption capacity of solid carrier53. The smallest droplet size was observed upon using water as a solvent with liquid SNEDDs: Aerosil 200 ratio of (4:1) and our results were in complete agreement with findings of  Cho, H.Y. et al.,201654 who prepared S-SNEDDs containing Paclitaxel for lymphatic delivery and reported smaller droplet size upon using water as a solvent compared to ethanol and then investigated the effect of increasing solid carrier (Aerosil 200) amount. The spray dried S-SNEDDs showed good micrometric properties so; it can be further filled into hard gelatin capsule or even compressed to tablets to avoid the drawbacks of liquid SNEDDs including the lack of stability and irreversible drug precipitation. The promising micrometric behavior may be due the usage of Aerosil 200 as a solid carrier as reported in many previous studies. The improved flow properties of Aerosil 200 further indicates towards improvement of unit weight uniformity. The solidification process through spray drying technique did not affect the self emulsifying properties of the liquid formula as the spray dried powder disperse rapidly and spontaneously upon reconstitution to produce an emulsion of nano range. Also, the solidification process did not alter the functional groups of the included drugs as proven by the results of FTIR where, the characteristic peaks of ATV and EZT reported in pure drugs, physical mixture and solid formula were similar.


In-vitro release studies showed that the release rates of both included drugs were much greater compared to these of drugs suspension and this can be attributed to the existence of the drugs in molecularly dissolved state and rapid conversion of crystalline to amorphous state which has been extensively used for the delivery of poorly water soluble drugs via enhancing the drug solubility and subsequently improve the dissolution rate and bioavailability55. This may be due to that the high internal energy of material amorphous forms making it dissolves at much faster rate compared to crystalline form56. The presence of the involved drugs in the amorphous state was proven by the results of DSC and PXRD. Also, the results of SEM suggested that atorvastatin and ezetimibe were dissolved in the pores of S-SNEDDs matrix and transformation of drugs from crystalline to amorphous form leading to obvious improvement of drugs dissolution rate57. The slower release rate of included drugs from S-SNEDDs compared to that of L-SNEDDs may be due to that the porous carrier (Aerosil 200) has a strong interaction with the adsorbed SNEDDs and smaller surface area because of the slight increase of droplet size after the reconstitution58 which impairing the release of both drugs. As general, Aerosil 200 which is hydrophilic fumed silica dispersed easily in buffer and has no marked impact on cumulative drugs release as at the end of dissolution study more than of 99% of incorporated drugs released from both L-SNEDDs and S-SNEDDs. This proved that the solidification process by spray drying didn't affect drug release.


In-vivo studies results showed the development of pharmacokinetic parameters (oral bioavailability) of involved drugs in S-SNEDDs compared to pure drug suspension because of the improvement of the systemic absorption of the involved drugs from S-SNEDDs may be due to transformation of drugs from crystalline form to the amorphous form which enhance the drugs solubilization, facilitated intestinal absorption because of higher dispersibility and nano range of the droplet size (< 200nm) that provides large surface area and the avoidance of pre-systemic hepatic effect by the enhancement of lymphatic transport through transcellular pathway59 and its surfactant content enhances drug permeability through disturbing the cell membrane. The surfactant content (Tween 80/ Cremophor RH 40 mixture) of optimized SNEDDs formula is a critical factor in enhancing oral bioavailability as it facilitates permeation through disturbing the cell membrane. Such bioenhancer surfactants were reported to improve the bioavailability of absorbed compounds by: facilitating transcellular/ paracellular absorption and decreasing the intestinal efflux and drug biotransformation through inhibiting p glycoprotein and/or CYP450 enzymes60. Our findings are in complete agreement with the work of (Kassem, A. M., etal., 2017)42 who investigated the efficacy of (Tween 80/Cremophor EL) in preparation of atorvastatin loaded SNEDDs and reported that the Cmax of optimized formula was about 1.71 folds higher than that of atorvastatin suspension. The superior lipid lowering activity of optimized SNEDDs compared to pure drug suspension may be owed to the very small particle size which increase absorption, dissolution rate and thus, enhance the oral bioavailability of included drugs61. In-vivo study results showed no significant difference in absorption and lipid lowering efficiency between optimized solid and liquid formulations proving that the solidification process did not affect the efficiency of liquid optimized formula.



The authors declare no conflict of interest.



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Received on 18.01.2021           Modified on 27.08.2021

Accepted on 12.12.2021         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(6):2459-2469.

DOI: 10.52711/0974-360X.2022.00411