Advanced Oral Formulation of Atorvastatin and Fenofibrate: Solubility Enhancement and Bioavailability Optimization using Nanosuspension

Ramesh Balasaheb Nawale*, Sanjeev Kumar Sahu, Manish Vyas

Department of Pharmaceutical Chemistry, Lovely Professional University, Phagwara, Punjab, India.

*Corresponding Author E-mail: nawale_ramesh@yahoo.co.in

ABSTRACT:

The study aims to enhance the oral bioavailability and solubility of Atorvastatin, and Fenofibrate classified as a class (II) based on the Biopharmaceutical Classification System (BCS) used for treating hypertension. Hence, a combination nano suspension was developed using a high-speed homogenization. The selection of stabilizers was initially done using a One-Factor-At-A-Time method, considering particle size and potential. A Box-Behnken design with 3 factors and 3 levels was employed in 15 experimental trials to study how stabilizer concentration, homogenization speed and homogenization duration is impacting Zeta potential, Poly dispersity Index and Particle size. The combination of Poloxamer 407 and Polyvinylpyrrolidone were identified as the optimal stabilizer mix. The resulting nano suspension displayed a reduced particle size of 183.9nm and a zeta potential of -22.9mV. SEM images showed that the particles were spherical in shape. Further investigation with FT-IR, DSC, and X-ray diffraction studies substantiated the compatibility between the drugs and stabilizers used. The optimal formulation was used and yielded an expected solubility of the drug of 91.26±8.24µg/mL and 74.32±9.11 with the combination stabilizer with more than 90% drug release within the first hour. The formulation also showed C_max (2.97-fold) and AUC0-24h(2.49-fold) for Atorvastatin nano formulation, while for FNF nano formulation an increase in the value of C_max (3.74-fold) and AUC0-24h (3.17-fold) was evident, highlighting the effectiveness of the nano suspension in improving solubility, absorption, and bioavailability due to lesser particle size with a higher effective surface area that may facilitate solubility and possibly improve release.

KEYWORDS: Poloxamer 407, Polyvinyl pyrrolidone, Atorvastatin (ATR), Fenofibrate (FEN), Nano suspension (NS), Box-Behnken design (BBD), High speed homogenization, Pharmacokinetics.

INTRODUCTION:

Multi-drug products with fixed-dose combinations are commonly used in chronic conditions notably in cardiovascular diseases, lipid disorders, and metabolic syndrome, because they simplify drug regimens for the patients and thereby enhance adherence to treatment. fixed-dose combinations thus maximize medication efficiency and help to lower healthcare concerns. A good number of older persons with type 2 diabetes and a metabolic disorder also commonly face high cholesterol levels.

This cohort often has the lipid disorders of hypertriglyceridemia, high LDL-C levels, and low HDL-C levels¹. In these conditions, fibrates might be employed in combination therapy when the statin fails. Fenofibrate (FEN) is favored as an add-on agent when statins alone are inadequately efficacious, as it carries a lesser risk of inducing myopathy². In clinical trials, combination medications have been demonstrated to lower triglycerides, total cholesterol, and low-density lipoproteins better than statin medications alone³. Despite the existence of SIM and FEN fixed-dose combinations in the market these drugs have poor solubility in aqueous media of less than 0.1g/mL in case of FEN and SIM of 0.0013 to 0.0015mg/mL making the drug to show poor oral bioavailability⁴. In similar for drugs like Fenofibrate and Atorvastatin fixed-dose combinations are available in the market. ATR also suffers from poor solubility and hence has low bioavailability issue leading to increased dose of the drug and its related side effects. ATR has a low solubility, highly crystalline, and it can be assumed that dissolution may be rate limiting step for poor absorption and low bioavailability^5-6. Hence, this study was taken up to evaluate the potential of combination nanosuspension of the selected fixed drug combination to enhance the solubility and dissolution, aiding in the development of an oral fixed-dose formulation with improved bioavailability and stability. Till date no study has been reported with the selected combination as nanosuspension.

Nanosuspensions provide a proficient colloidal drug delivery method that improves the bioavailability of poorly soluble pharmaceuticals. They enhance dissolution velocity and saturation solubility, resulting in improved oral absorption. This approach provides both therapeutic benefits and economic efficiency.

MATERIAL AND METHODS:

Atorvastatin and Fenofibrate were given as gift samples from Innomedix Pharma Pvt Ltd. Jaipur, India. Poloxamer 407 and Polyvinylpyrrolidone K30 (PVP) were provided by TCI chemicals, India. All the solvents were of analytical grade.

FTIR (Bruker), DSC (Perkin Elmer) and XRD (Philips PW-1710) studies were carried out for ATR, FNF, stabilizers and L-NS. DSC was carried out by heating at a frequency of 5ºC/min over a 200ºC range and in case of XRD the samples were scanned at 2θ between 80° and 2°, with an average step size of 0.045° and a time between steps of 0.5 seconds.

Formulation of Nano suspension:

Sono-precipitation high speed homogenization method was used with slight modification. An accurately weighted amount of the selected stabilizer Poloxamer 407(P 407) and Polyvinylpyrrolidone (PVP) was taken and dissolved in deionized water (20mL) to form the non-solvent phase (NSP). Drugs were dissolved in ethanol to form the organic phase (2mL) and injected rapidly using a syringe (gauge 18) into NSP under a magnetic stirrer at 1000rpm. The mixture was subjected to homogenization at an rpm of 9000 for sixty minutes under ice cold conditions. The mixture was again placed on a magnetic stirrer at 1000rpm for the complete evaporation of OP. The NS was subjected to freeze drying to get a stable product as per the standard protocol⁷.

Optimization of the nano suspension:

A complete study of how several factors affect the final formulation is desirable. Therefore, to create fixed-dose combinations-loaded NS, we adopted the QbD strategy- a methodical approach with encoded goals. Making a QTPP, or quality-targeted product profile, is essential to the FbD process. A BBD was employed and the selected formulation and processing parameters (Table 1).

HPLC method development:

The concentrations of FNF and ATR were ascertained utilizing a Water HPLC System equipped with a UV-DAD detector with slight modification as reported by Jain et al. The mobile phase is a mixture of acetonitrile and water (70: 30, v/v) and a C18 column RP-18 (125 mm x 3mm, 5µ) was used for the study. The samples were eluted at a flow proportion of 1.0ml/min, with FNF spotted at wavelengths of 286nm and 238nm. The retention time of FNF and SIM was 3.4 and 2.3mins. The standard calibration curve in the range of 0.3 to 250 µg/mL and 0.2 to 200µg/mL with correlation values over 0.999 was plotted graphically.Regression coefficients (R²) of 0.999 and 0.998 for FNF and ATR, respectively, indicated that the concentration of the drug was linear within the tested ranges of 0.3 to 250µg/mL and 0.2 to 200µg/mL. Specifying specificity was determined by comparing chromatograms of the samples and blank⁸.

Nanosuspension Characterization:

Size, zeta potential measurement and SEM:

The particle size (PS), Polydispersity Index (PI) and Zeta potential (ZP) of the NS was measured by dynamic scattering techniques after diluting the sample by 10 times (Malvern nano S90). The measurement was conducted at 25°C at a scattered angle of 90 degrees. The ZP of the samples was also determined using a disposable cell for zeta potential analysis employing the principle of electrophoretic mobility technique. The size and topography of NS were captured by FESEM (field emission scanning electron microscopy) at an accelerating voltage of 10KV and before subjecting for analysis⁸ the samples were coated with a layer of platinum for 40 seconds at 20mA.

Drug release:

For determining drug release from NS and plain drug using USP dissolution (Type 2) was employed. F-NS and pure drugs (100mg of sample) were weighted accurately and added into the dissolution vessels containing 0.5% Sodium lauryl sulphate of 900ml (pH 7.4). The dissolution was carried out at 37±0.5ºC at an rpm of 50. At regular time intervals samples were taken and filtered and analyzed and calculated and plotted the percentage of drug release from L-NS and plain drugs respectively⁹.

Stability studies:

The optimal freeze-dried L-NS samples were stored for three months under three different temperature settings (4-8°C, 28±2°C, and 40°C). At specified intervals (0^th, 15^th, and 30 days), PS, PI, and ZP were analyzed using a standard zeta-sizer.

Exploration of pharmacokinetic parameters:

Male Wistar rats were supplied by the Nutritional National Institute (NIN) in Hyderabad, India, 4-5 weeks of age and weigh of an average 200±20g. The IAEC (Protocol No. 1447/PO/Re/S/11/CPCSEA-85/A) approved the study, which adhered to CPCSEA guidelines. After a week of getting used to the outside light and dark cycle, the animals were split into two groups of six by chance. Either NS (ATR of 08mg/kg and FNF of 20mg/kg BW) or 0.5% w/v sodium carboxymethylcellulose pure drugs suspension was given to the animals. At set times, every 0.25 to 12 hours, 200µL of blood was taken from the eye (orbital venous plexus), placed into EDTA-coated container tubes and centrifuged at 7500rpm for 10minutes. For detection of drugs in the plasma specimen the protein precipitation method was utilized. The drugs were separated from serum (50µL) by mixing 300µL of acetonitrile (ACN) by mixing the contents for 10 minutes, and then subjected to centrifugation for 12 minutes at 8000rpm before analysis by HPLC.Pharmacokinetic data were analyzed utilizing Kinetica software employing a non-compartmental approach with pharmacokinetic variables expressed as Mean±SD¹⁰.

RESULTS AND DISCUSSION:

Preparation of nano suspension (NS):

The sonoprecipitation homogenization technique was selected for developing NSs. Intense desolvation causes immediate precipitation of the drug, resulting in the formation of nanoparticulate drug particles. Based on the Ostwald-Miers theory, the system is said to be in a regime of supersaturation, whereby nucleation and crystallization occurs. This regime is reached once an AP is introduced to a solution saturated with drug. Afterward, progressive solvent evaporation leads to the formation of numerous nuclei conducive for further crystal growth. The screening tests were conducted by using the One-Factor-At-A-Time approach to determine the ratio of OP to NSP is necessary to prevent the aggregation of size and a ratio was set at 1:30 to assess the stabilizer's influence on particle size (PS) and poly dispersity index (PI) using 8 mg of ATR and 20mg of FENO in first trials. With PS and PI readings shown in Figure 1, several stabilizers were tried to find the most successful one. The stabilizing efficiency was in the following sequence: PVP K-30<Poloxamer 407< Poloxamer 188<SLS<PVA<Pluronics 338<HPMC E 15 <TPGS. With low PS and PI, PVP K-30 and Poloxamer 188 created a uniform nanosuspension. While Poloxamer 407 and PVP K-30 avoided this problem, stability testing showed cake formation in HPMC, TPGS, Pluronics 338, and SLS-containing formulations. While PVA-stabilized formulations showed clear particle segregation, HPMC E15 produced a hard, non-dispersible cake. Zeta potentials for Poloxamer 407 formulations varied from -20.9 to -23.3mV and stayed steady. Due to steric stabilization via a polymeric coating, PVP K-30 produced smaller particle sizes but had lower zeta potential (13.24 to 11.98mV. A mix of stabilizers was used to improve electrostatic stability. Because of their hydrophilic character, consistency, surface energy, and functional groups, poloxamers also known as Pluronics showed better stability These copolymers are of type A-B-A, with a hydrophobic PPO group center shielded by hydrophilic PEOarms¹¹.

Experimental design using BBD:

There were three center points among fifteen leads in the investigation (Table 1). The model selection technique employed R₂, projected R², adjusted R², and coefficient of variation (C.V). ANOVA was also applied to assess how different factors affected the responses.

Table 1: Runs planned for the trails.

Run	Factor 1	Factor 2	Factor 3	Response 1	Response 2	Response 3
	A: Stabilizer to drug ratio	B: HS	C:HT	PS	PI	ZP
	% w/w	rpm	mins	nm		mV
1	0.5	12000	90	402.2	0.588	-36.45
2	1	9000	90	252.8	0.428	-36.45
3	1	9000	90	250.2	0.488	-33.45
4	1.5	6000	90	251.89	0.529	-49.88
5	1	9000	90	253.6	0.444	-33.35
6	1	12000	120	258.44	0.388	-29.78
7	1	6000	120	193.2	0.598	-30.69
8	1.5	9000	120	210	0.129	-48.06
9	0.5	6000	90	356.3	0.468	-16.43
10	1	12000	60	237.12	0.332	-37.92
11	1.5	9000	60	244.12	0.192	-39.44
12	1	9000	90	249.7	0.244	-38.35
13	1.5	12000	90	199.64	0.132	-29.11
14	1	9000	90	255.26	0.384	-36.65
15	1	6000	60	302.03	0.428	-37.89
16	0.5	9000	120	354	0.426	-16.46
17	0.5	9000	60	397.88	0.296	-32.46

Particle size (PS):

The particle size and PI extended from 193.2 to 402.2 nm after 15 rigorous trials. ANOVA data and the type of the model is mentioned in table 2 w.r.t to PS, PI and ZP. It was also observed that the typical effects (A, C, AB, BC, and A²) had a major consequence on the results with p-values under 0.05. Therefore, these variables are now regarded as significant; the subsequent regression equation is:

Particle size = +252.31 – 75.59 – 0.7525B – 20.69C – 24.54AB + 2.44AC +32.54BC + 52.00A² – 1.80B² – 2.81C²

Overall, increasing homogenization speed (B) and homogenization time (C) tends to decrease particle size. This is evident from the negative coefficients of B (-0.7525) and C (-20.69) in the equation, indicating that as these factors increase individually, the particle size reduces. The ultrasonic waves may be breaking down the particles, causing lower Tmix, which gives Da<1.

Polydispersity Index (PI):

The Polydispersity Index (PI) represents in a dimensionless way the range in the particle size distribution. It mostly lies in the range of 0 to 1.The p-values for the variables in the model A, B, AB, and B² were found to be under 0.050, suggesting that they had a significant influence and hence considered essential, and the equation of regression is given as below:

PI = +0.3976 – 0.0995A - 0.0729B + 0.0366C – 0.1293AB + 0.0483AC – 0.0285BC – 0.0721A² + 0.1037B² – 0.0648 C²

Higher A and C help in achieving a more uniform particle size, while excessive homogenization speed (B) may increase PI, leading to slight variability in particle sizes.

Zeta potential (ZP):

ZP is regulated by surface charges, which plays an imperative role in the stability of colloidal solution and the primary interaction of particles with the cell membrane to make them an essential component of any successful drug delivery system. In this work, the ZP values were in the array of -49.88 to -16.43 mV (Figure 2). The model's variables (A, C, AB, and AC) indicate a significant influence on the consequence. Thus, these terms are considered necessary, and the following formula for regression from above is:

Zeta Potential (ZP)

= -34.28 – 8.09A + 0.2037B + 2.84C + 10.20AB – 6.16AC + 0.235

Increasing A makes ZP more negative, while increasing C and the AB interaction make it less negative, affecting the stability of the particles. Lower zeta potential values further point towards good stability of the colloids, emphasizing that these parameters play a critical role in stabilizing the nanosuspensions. The 3D surface and contour plot of PS, PI and ZP is shown in Figure 2.

The ideal formulation that gave the highest desirability score (Fopt solution) was 0.856. The ideal conditions were 12000 rpm HS, 60 min HT, and a drug-to-stabilizer ratio of 1.48 (i.e., 250mg of the stabilizer for every 170 mg of the medications).Validation was performed using three checkpoints to confirm the model's robustness and the accuracy of the formulation. The observed mean values were 189.3nm PdI of 0.244 and -41.8mV, while the predicted mean standards for size, PI, and ZP were 184.54nm 0.103 and -48.8mV. The predicted values were almost similar with that of software and results from those formulations thus validating the proposed model (Figure 2D).

Figure 2: Response surface and contour plots graphically depicting how selected factors affect A) PS, B) PI, C) ZP and D) desirability and plot of overlay.

Nanosuspension Characterization:

Size, Zeta potential and SEM:

The parameters PS and PI of the formulation varied between 189.3±4.76nm and 0.245±0.142mV, indicating that the size of the particles is kept in a consistent range and hence indicates homogeneity of the nanosuspension. A polydispersity value less than 0.3 indicates a homogeneous system. Optimal formulation has ZP of 41.8±5.6mV. The increased ZP was due to the steric stabilization mechanism. The PS, ZP and SEM images of the drug and formulations are presented in Figure 3. The drug isshowing an irregular shape with enormous particle size range; however, the drug converted into a tiny spherical bead with a more uniform size range value between 118 and 229nm by the solvent-antisolvent precipitation-based nanosuspension. The study illustrates that the nanosizing effect achieved¹¹.

Drug release:

The dissolution contours of simple ATR, FNF and NS is depicted in supplementary Figure 4. Drug release endured for up to two h, for ATR and FNF (PD) of 48.66±11.15% and 52.8±9.44 % respectively. FNF and ATR showed drug releases of 98.66±11.44% and 99.23 ±12.06%, respectively, in case L-NS FNF, and ATR showed drug releases of 98.66±11.44% and 99.23± 12.06% in case L-NS. The L-NS data, at 15 minutes, showed that more than forty-five percent of drug release from the formulation was obviously enhanced. Increased drug release from NS further supports the Noyes-Whitney equation and Brunner-Nernst concept, which suggest that decreasing the particle size into the nano level increases the surface area which helps in dissolution¹².

Figure: 3: Graphical depiction of Images:I) PS, and ZP of the optimized NS.II)SEM pictures of A) and B) pure drug of ATR and FENO, C) and D) Nanosuspension of optimized formulation without and with scale.

Figure 4: In vitro release of ATR, FENO (FNF) and NS.

Figure 5A depicts the determination of component compatibility through IR spectra analysis of the nano-formulation, excipients, and drugs. The FTIR spectra of L-NS reflected a superimposition of bands from both drugs, and no additional bands or significant shifts were observed, confirming the absence of chemical interactions among the formulation components¹³. The thermal behavior of the optimized L-NS, the physical mixture, and the pure drugs according to differential scanning calorimetry (DSC) is portrayed (Figure 5B). Since no new additional peaks are observed it was confirmed that the drugs are chemically stable and no degradation products were formed. In case of XRD (Figure 5C) the pure FENO drug confirming the presence of solid peaks at different scattered 2θ angles of 6.03, 11.04, 11.6, 12.35, and 14.11°, while the ATR drug showed firm diffraction peaks at 2.97, 5.98, 9.01, 9.34, 10.13, 11.7, and 12.05°. Similar diffraction peaks for the studied material have also been reported previously. However, the characteristic diffraction peaks of L-NS were observed at scattering angles of 2 theta 2.9, 5.96, 6.15, 8.95, 9.29, and 10.07, which elucidated a reduction in peak intensity compared to the pure drugs, confirming involvement of solid-state interaction at a molecular level due to the inclusion of stabilizer encasing.

Figure 5: Compatibility studies: A) ATFT-IR of plain drugs (ATR, FNF), and NS along with PM and combination plain drugs; B) DSC overlay of plain drugs, combination drugs, PM and NS; C) XRD overlay of plain drugs, combination drugs, PM and NS; D) In vivo release studies in animals following oral administration of plain drugs( ATR,FNF) and NS.

Pharmacokinetic studies:

The plasma concentration versus time curve after an oral administration of the optimized nanosuspension and a commercial PD solution (0.25% w/v HPMC) is shown in Figure 5D. In comparison to unformulated pure drug, kinetics exhibited higher values of Cmax (2.97-fold) and AUC0-24h (2.49-fold) for ATR nanoformulation, while for FNF nanoformulation an increase in the value of Cmax (3.74-fold) and AUC0-24h (3.17-fold) was evident. It is the lesser particle size with a higher effective surface area that may facilitate solubility and possibly improveddrug release concentration gradient across the GIT and blood walls. The stability study of L-NS was conducted every 0, 30, 60, and 90 days on three different temperature conditions and the formulations found to be stable.

CONCLUSION:

In the current study, fixed combination NS of atorvastatin and fenofibrate was prepared using a sonoprecipitation high-speed homogenization technique using the design of experiments. Fifteen runs were conducted for the selected combination stabilizers of Pluronics 407 and PVP K30, speed of homogenizer and duration of homogenization. The optimized nanosuspension was found to have a particle size of 184.30 nm with a ZP of 41.0 mV. Compatibility studies evidenced that the drug was stable and tolerable with the selected stabilizers. The freeze-dried NS was found to be stable for up to 3 months. The in vitro release studies proved that NS has released more than 90 percent of the drug in less than 1 hour compared to the plain drug. A pharmacokinetic study revealed that the formulation displayed high bioavailability compared to the plain drug. The formulation as nanosuspension is a viable option for fixed dose combination drugs for improving the therapeutic benefits for the patient.

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Received on 05.12.2025 Revised on 19.02.2026

Accepted on 30.04.2026 Published on 20.05.2026

Available online from May 25, 2026

Research J. Pharmacy and Technology. 2026;19(5):2297-2302.

DOI: 10.52711/0974-360X.2026.00330

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Parameter	Model	R²	corrected R²	predicted R²	Lack of fit F-Value	Signal to noise ratio
PS	quadratic	0.9993	0.9983	0.9930	1058.90	101.474
PI	quadratic	0.8843	0.7355	0.6765	5.94	8.0646
ZP	linear	0. 948	0.9173,	0.8278	1.54	22.705