Enhancement in Dissolution Profile of Cilnidipine by Nanonization Technique: Optimization by Box-Behnken design

Nirmal Shah¹*, Dhiral Patel¹, Dipti Gohil¹, Avinash Kumar Seth¹, Dhaivat Parikh²

¹Department of Pharmacy, Sumandeep Vidyapeeth Deemed to be University,

Piparia - 391760, Vadodara, Gujarat, India.

²Department of Pharmaceutics, Institute of Pharmacy, Nirma University,

S. G. Highway, Ahmedabad – 382481, Gujarat, India.

*Corresponding Author E-mail: nimspharma@gmail.com

ABSTRACT:

Objectives: Cilnidipine has limited 25-30% of bioavailability due to BCS class II category drug. The aim of present work was to improve the solubility followed by dissolution rate of cilnidipine by formulation of nanosuspension. Materials and Methods: The nanosuspensionwas developed usingnanoprecipitation techniqueaidingultra-sonication with solvent (Dimethyl sulfoxide), antisolvent (water) and stabilizer (polyvinyl alcohol). The nano formulation was statistically optimized using Design Expert software by theBox-Behnken design. The concentration of Poly Vinyl Alcohol, Volume of Antisolvent and Stirring Time were considered as independent factors whereas Particle size and polydispersity index were considered as dependent factors. Results: The particle size of nanosuspension was found 178.57nm with narrow polydispersity index of 0.130. The microscopy study confirmed amorphous nature particles with rough surface. The zeta potential of 4.55 ±2.78 for optimized formulation finds stability of formulation during storage. The differential scanning calorimetry study and X-ray diffraction pattern showed the crystalline cilnidipine drug was converted to amorphous form upon precipitation into nanoparticles. Due to the nanonization of cilnidipine, the dissolution profile was noted as 92.41±3.56% which was significantly improved as compared with plain drug release data. The stability data showed satisfactory result for 2 months. Conclusion: It is summarized that the nanoprecipitation technique with ultra-sonication is approaching method for the formulation of homogenous nanosuspension with uniform sized dispersion of cilnidipine. Therefore, it can be assumed that the upon the enhancement in dissolution rate, the cilnidipine loaded nanosuspension may lead to improved absorption of drug from intestine followed by significant improvement in bioavailability.

KEYWORDS: Poor solubility, Nanosuspension, Nanosized particles, Improved dissolution rate, Bioavailability.

INTRODUCTION: 

Pharmaceutical industries are endlessly trying to achieve innovative approaches in order to obtain enough oral bioavailability, as nearby 40% of the new synthesized drug molecules are poorly water soluble. Due to less bioavailability and inconsistent absorption of such drugs, formulation with conventional dosage forms show lots ofissues.

Several new approaches are available to answer such issues but many of these approaches are not universally applicable to all drugs^1,2,3. Henceforth, to improve formulation efficacy and therapy there is anessentiality of some methods to handle the formulation problem with respect to pharmacoeconomics.

Cilnidipine possesses a slow and long-term vasodilating effect. Cilnidipine, a poorly soluble drug from BCS class-II category, is well absorbed from all segments of the intestine after the oral administration. It undergoes significant first pass metabolism that leads very low bioavailability (25-30%). Thus, it is a need to improve the solubility of drug followed by the dissolution rate which results in enhancingoral bioavailability^4,5. Since the bioavailability of the drug is dependent directly on the solubility, it is hypothesized that the drug may improve the bioavailability with nanosuspension formulation.^6,7,8

The nanosuspension is identical and excellently dispersed solid particles of drug in an aqueous media, with reduced particle size between 200-600nm^2,3. That may be useful in enhancing dissolution and bioavailability of poorly soluble drugs^{9,10,11,12,13,14}.

Thus, the work is undertaken to improve the solubility of the cilnidipine by converting to nano sized particles followed by enhanced dissolution profile. This may also reduce the dose of the drug due to improvement in the bioavailability.^15,16,17

The objective of this work is to design and characterize cilnidipine loaded stable nanosuspension using statistical tool to sort out the issues related to low dissolution profile.

MATERIALS AND METHODS:

Materials:

Cilnidipine was gifted by Niksan pharmaceutical Pvt. Ltd., Ankleshwar, Gujarat, India. Polyvinyl alcohol, Methanol and Dimethyl Sulphoxide were obtained through Loba Chemie, Mumbai, India. Other materials utilized in the present work were purchased and all were of analytical grade.

Drug excipients compatibility studies:

The Fourier Transform Infra-Red spectrophotometer (FTIR) (IR Affinity-1: Shimadzu corporation, Japan) was used to check any interaction between drug and excipients. The FTIR spectrum was studied for the mixture (Sample: KBr, in ratio 1:300) in the recommended range (4000–400 cm^-1) using FTIR spectrophotometer^18,19.

Optimization of nanosuspension using Box-Behnken design:

Design Expert® software version 10.0.0.1 was employed for formulating statistical design for nanosuspension. Factorial design allows quantification of the effects made by independent variables and interactions between them. Such statistical tool is useful in saving time as well as materials by generating very less trials and making quality products.

A Box-Behnken statistical design for three factor and three level was used to discuss and optimize the main effects and the effects of the formulation ingredients on the performance of nanosuspension. The independent variables such as concentration of Poly Vinyl Alcohol (PVA), % (X₁), volume of antisolvent, ml (X₂) and stirring time, min (X₃) were considered as levels depicted in Table 1. The design showed a total of 17 formulation batches to check the effect of experimental parameters with dependent responses that is particle size in nm (Y₁) and polydispersity index (PDI)(Y₂). A relation between two responses were clearlypresented in contour plots as well as in 3D surface response plots. The reliability of model was analysed by check point examination^20,21,22.

Table 1: The statistical design for optimization

Independent variables	Levels
	Low (-1)	Middle (0)	High (1)
X1	0.5	1	1.5
X2	75	100	125
X3	40	60	80

X₁: Concentration of PVA (%), X₂: Volume of antisolvent (ml), X₃: Stirring time (min)

Formulation of nanosuspension by nanoprecipitation technique:

The nanoprecipitation technique was utilized to prepare cilnidipine nanosuspension^23,24. The appropriate quantity of drug was added in Dimethyl sulphoxide (DMSO). This prepared drug solution was dropwise addedwith constant stirring into the water that contained stabilizer (PVA). The resultant suspension was ultra-sonicated (Ultrasonicator, Aczet Private Limited)under cold condition for 15 min followed by homogenization with emulsifier (EQ-127, REMI Sales and Engineering Ltd., India.) for predefined time span^25,26.

Assessment of experimental responses:

Particle size and PDI:

The particle size and PDI of nanosuspension wereanalysed using Zetatrac instrument (Microtrac 10.6.1, Mumbai, India) after suitable dilution with distilled water.

Characterization of optimized formulation:

Drug Content:

The drug incorporatednanosuspension was completely dissolved in minimum quantity of methanol followed by appropriate dilution with phosphate buffer pH 7.4 (10 mL) with constant stirring. The mixture was analysed spectroscopically after appropriate dilutions in UV-visible spectrophotometer (UV-1800- Shimadzu corporation, Japan) at 247 nm^27,28.

Zeta potential:

The zeta potential ofdrug loaded nanosuspension wasanalysedusing Zetatrac instrument (Microtrac 10.6.1, Mumbai, India).

Differential Scanning Calorimetry (DSC):

The DSC study was conducted by using differential scanning calorimeter (DSC TA – 60, Shimadzu, Japan) to study the thermogram of sample. The study was completed under a nitrogen purge (100ml/min). The aluminium pans were used to carry appropriate volume of samples (about 2-4 mg).Thereafter, they were sealed with a pinhole-pierced cover. The condition of 40 °C to 250°C at scan rate of 10°C/min, was utilized to study the thermograms^29,30,31.

Scanning Electron Microscopy (SEM):

The SEM analysis was performed by using Scanning Electron Microscope (Jeol -JSM-5610LV, England) to study the surface of cilnidipine nanosuspension. The mixturewasplaced on metal stubs with double-sided tape previouslyplaced onto aluminium stubs followed by coating with gold under a vacuum. Thereafter, at different magnifications photographs were taken to study the surface morphology of sample^32,33.

X-Ray Diffraction (XRD) study:

The crystallinity or amorphous properties of drug and formulation samples were studied withpowder X-ray diffractometer with model Xpert Pro-MPD, Panalytical, Netherlands. The system with glass sample holder was operated at a fixed 45 Kv and 40 mA of voltage and current, respectively. The scanning was carried outat 2Ө in range from 5^o to 80^o32,33.

In-vitro dissolution study:

Thedissolution profile of nanosuspension formulation was studied with dialysis sacmethod^34,35. The Dialysis membrane have property of 135 Mol. cut off 12000 - 14000 Da and was purchased from Himedia Laboratories, Mumbai, India.

Treatment of dialysis sac: Dialysis tubing of desired length was boiled in 100ml of 2% sodium bicarbonate and 1mM Ethylene Diamine Tetra Acetic acid (EDTA) for 20 min followed by rinsing in hot water and boiled again for 10 min in 1mM EDTA solution. The solution was allowed to cool and stored in 50% ethanol. The tubing was washed with distilled water before use.

An accurately amount of formulation was placed into the one end of dialysis sac. Thereafter, the thread was used to close the ends of sac. The membrane was tied with the paddle of USP type – II paddle type dissolution apparatus. To check the drug release characteristics from nanosuspensions, 900 ml of phosphate buffer of pH 7.4 and a temperature rate of 37± 1°C and 50rpm was set aside for study. The fresh buffer was placed in dissolution apparatus after subsequent withdrawals of 5 mL aliquots at specific time intervals. The samples were analysed using UV spectrophotometer at 247nm after filtration by cellulose acetate membrane filter of 0.45μm pore size^34,35.

Stability study:

The formulationsplaced in glass vials wereplaced for specific stability conditions at 25±1°C/60±5% RH and 40±1°C/75 ± 5% RH in precise stability cabinet for the duration of two months. The formulations were analysed for physical behaviour (appearance), % drug release and % drug content at an interval of every 15 days^{36,37,38,39,40}.

Statistical analysis:

The statistical tool Analysis of Variance (ANOVA) was employed to verify the data using the Graph Pad Instat software of version 3.01. The P<0.05 was deliberated as statistical significance. All parameters were verified in triplicate^36,37,41,42.

RESULTS AND DISCUSSION:

Drug excipients compatibility studies:

The FT-IR spectrum of the formulation revealed no significantvariation in the peaks of cilnidipine in the nanosuspension when compared to pure drug. It shows the absence of any interaction. That data reveals the fact that the drug is compatible with the other excipient used for the optimization of nanosuspension.

Optimization of nanosuspension:

The Box-Behnken design was utilized to develop cilnidipine nanosuspension by nanoprecipitation method. It specifies the minimal experimental runs and consumes less time for development. It is also proven that the statistical tool are more efficient and less costly technique for formulation and development of any dosage forms.

The Table 2 showed entire 17 experimental batches with finalized three independent and two experimental parameters.

Table 2: The experimental design for cilnidipine nanosuspension

Formulation code	Independent variables			Dependent variables
	X1	X2	X3	Y1	Y2
CLN 1	0	1	-1	280 ± 30	0.422 ± 0.205
CLN 2	-1	-1	0	390 ± 38	0.519 ± 0.188
CLN 3	0	0	0	175 ± 19	0.103 ± 0.059
CLN 4	-1	1	0	354 ± 41	0.511 ± 0.290
CLN 5	0	0	0	179 ± 11	0.105 ± 0.101
CLN 6	0	0	0	181 ± 09	0.117 ± 0.094
CLN 7	1	1	0	300 ± 19	0.442 ± 0.199
CLN 8	0	0	0	191 ± 21	0.121 ± 0.120
CLN 9	1	-1	0	315 ± 37	0.628 ± 0.164
CLN 10	0	1	1	250 ± 22	0.363 ± 0.197
CLN 11	1	0	-1	334 ± 30	0.617 ± 0.084
CLN 12	-1	0	-1	420 ± 48	0.622 ± 0.136
CLN 13	-1	0	1	359 ± 31	0.558 ± 0.174
CLN 14	0	-1	-1	325 ± 32	0.513 ± 0.210
CLN 15	0	0	0	184 ± 17	0.116 ± 0.067
CLN 16	0	-1	1	260 ± 28	0.481 ± 0.124
CLN 17	1	0	1	303 ± 20	0.598 ± 0.081

Values are stated as average ± SD, n = 3; Y₁: Particle size in nm and Y₂: PDI

The PRESS value was utilized to identify suitable model for the fitness in this optimization. The reduced quadratic model was selected after proper statistical analysis for the prescribed dependent variables based on low PRESS value.  The experimental responses were achieved by conducting experiments systematically to get polynomial equations of the full model.

Y₁= 182.00 – 33.87X₁– 13.25 X₂– 23.383 +5.25 X₁ X₃+ 7.50 X₁ X₃ + 8.75 X₂ X₃+ 116.50 X₁²+ 41.25 X₂² +55.50 X₃²

Y₂= 0.1124 + 0.0094 X₁– 0.0504 X₂– 0.0218 X₃ - 0.0445 X₁ X₂+ 0.0112 X₁ X₃ - 0.0068 X₂ X₃ + 0.2833 X₁²+ 0.1293 X₂² + 0.2031 X₂²

Henceforth, the not affecting parameters were removed (p>0.05) to produce reduced model.

Y₁= 182.00 – 33.87 X₁– 13.25 X₂– 23.38 X₃ + 7.50 X₁ X₃ + 8.75 X₂ X₃ + 116.50 X₁²+ 41.25 X₂² + 55.50 X₃²

Y₂= 0.1124 + 0.0094 X₁– 0.0504 X₂– 0.0218 X₃ - 0.0445 X₁ X₂+ 0.0112 X₁ X₃ + 0.2833 X₁²+ 0.1293 X₂² + 0.2031 X₃²

On the basis of p value, X₁ X₂and X₂ X₃ parameters were found insignificant and removed from experiment. The F value for particle size and PDI were found very less as compared to tabular F value thus it is endorsed that the absent terms are not contributing expressively to the assessment of responses. The higher coefficient value for X₁, X₂ and X₃ discloses that they can affect significantly to both responses. Therefore, it can be said that all three experimental responses have contributing effects on development of nanosuspension formulation.

The Contour plots (Data not shown) and 3D surface response plots were drawn for the experimental responses. All factors are crucial aspects for assessing the quality of cilnidipine nanosuspension. The 3D surface response in Fig. 1 showed that the low level of stirring speed and volume of antisolvent may results in an increases in particle size and PDI value that because of insufficient force and bulk required to achieve desired particle size and PDI. The plots also suggested that low and high levels of surfactant concentration do not provide desired results.

The check point investigation was executed to access the reliability of model. The formation of various batches are depicted in Table 3.As shown in Fig. 2, the overlay plot showed the composition of nanosuspension formulation (X₁ – 1.09%, X₂ – 99.77 ml and X₃ – 59.47 min) which was considered as optimized batch based on its reliable data of independent variables. This formulation showed low particle size and PDI that is 178.57nm and 0.130, respectively which are desirableasfar as the statistical analysis is required. The optimized nanosuspension formulation was further proceed for its characterization.

Fig. 1:3D Surface response plots for dependent parameters particle size (A, B and C) and PDI (D, E and F)

Table 3: Checkpoint analysis for optimization of nanosuspension

Formulation Code	Independent variables			Response	Predicted Value	Observed value	% Error
	X1 (%)	X2 (ml)	X3 (min)
NS 1	0.89	97.05	60.98	Y1 (nm)	195.70	190.87	-2.53
				Y2	0.129	0.134	3.73
NS 2	1.07	95.71	58.28	Y1(nm)	185.30	188.54	1.72
				Y2	0.130	0.136	4.41
NS 3	1.07	104.76	56.97	Y1(nm)	183.04	181.43	-0.89
				Y2	0.121	0.128	5.46
NS 4	1.11	111.23	67.05	Y1(nm)	184.12	186.48	1.27
				Y2	0.146	0.151	3.31
NS 5	1.09	99.77	59.47	Y1(nm)	180.49	178.57	-1.08
				Y2	0.126	0.130	3.08

Fig. 2: Overlay plot for optimization

Characterization of optimized formulation:

Drug Content:

The optimized nanosuspension showed an identical drug content of 97.54±1.21. This data showed that the technique utilized for the development of nanosuspension has a major potential in maximum drug loading.

Particle size and zeta potential:

A mean size of optimized formulation exhibited noticeably lesser size of 178.57nm with very lesser PDI of 0.130 which showed tapered distribution of particles within nanosuspension dispersion system. A zeta potential of nanosuspension was establish 4.55± 2.78mV that showed some stability of nanosuspension during storage. This steric stabilization was achieved due to the propery of PVA.

The DSC study:

This study is carried out to monitor endothermic and exothermic processes.  This may be utilized to study possible interactions in the formulation. Thermogram of pure drug cilnidipine in Fig.3(A) showed an endothermic peak at 110.18ºC which correlates with the standard melting point of drug. The Fig.3(B) of nanosuspension showed broad endothermic peak at 112.14ºC. This obtained data reveals no drug-excipients interaction with possible transformation of pure crystalline drug to amorphous nature in optimized nanosuspension.

Fig. 3:DSC thermograms of (A) Drug and (B) Optimized nanosuspension formulation

The SEM study:

This study is carried out to understand the surface morphology of the nano sized formulation. The Fig.4(A) depicted that the crystalline nature of pure drug whereas Fig.4(B) demonstrated the discrete particles with rough surface. 

Fig. 4:The SEM images of (A) Drug and (B) Optimized nanosuspension formulation

Table 4: Stability profile for optimized nanosuspension

Time (Days)	25 ºC ± 2^oC / 60% RH ± 5% RH			40 ºC ± 2^oC / 75% RH ± 5% RH
	Physical appearance	% Drug release	% Drug content	Physical appearance	% Drug release	% Drug content
0	No change	92.41 ± 3.56	97.54 ± 1.21	No change	92.41 ± 3.56	97.54 ± 1.21
15	No change	92.48 ± 1.45	98.24 ± 2.04	No change	92.28 ± 0.54	95.88 ± 2.34
30	No change	91.90 ± 2.37	96.88 ± 0.57	No change	92.30 ± 0.63	96.67 ± 0.25
45	No change	92.12 ± 0.83	97.70 ± 2.01	No change	91.93 ± 0.54	95.91 ± 0.18
60	No change	91.93 ± 1.45	96.76 ± 0.29	No change	91.57 ± 1.14	95.44 ± 1.08

Values are stated as average ±SD, n = 3

The XRD study:

This study was conducted to confirm the result obtained in DSC study in terms of crystalline behaviour of drug. The Fig. 5A depicts XRD pattern of cilnidipine which confirms crystallinity of drug due to presence of intense peak on scale. As compared to pure drug, the nanosuspension showed very less intense peaks as shown in Fig. 5B. This result may confirms the remarkable reduction in crystallinity of drug in formulation. Such amorphous conversion of drug may play important role in significant improvement of aqueous solubility.

Fig. 5: XRD study of (A) Drug and (B) Optimized nanosuspension formulation

In vitro dissolution study:

The optimized nanosuspension showed remarkable enhancement in dissolution rate of drug. The nanosuspension showed 92.41 ± 3.56 % drug release as compared to 48.54 ± 5.47 % of plain drug suspension. This showed the importance and vital role of nanonization techniques for the improvement in dissolution profile.

Stability Study:

The data prescribed in Table 4 showed insignificant changes as compared with the data obtained before the stability study. This confirms the reliability of nanonization technique in improving physicochemical properties of poorly soluble compounds.

CONCLUSION:

Cilnidipine lies under class II category in BCS. The less solubility in aqueous may results in lesser oral bioavailability. In this study, an attempt has been made to develop stable cilnidipine nanosuspension to enhance its aqueous solubility. The optimized formulation showed remarkably improved results in all aspects of characterization. From this research study, it has been proved that nanosuspension is a powerful tool to enhance the dissolution profile of drug that enhances the bioavailability of poorly water soluble compounds.

CONFLICT OF INTEREST:

The author stats no conflict of interest

ACKNOWLEDGMENTS:

Authors are thankful to Niksan Pharmaceutical Pvt. Ltd., Ankleshwar, Gujarat, India for gifting Cilnidipine. Authors are also thankful to the Sumandeep Vidyapeeth Deemed to be University, Piparia, Vadodara, Gujarat, India for providing all necessary infrastructure facility to perform this research study.

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Received on 28.07.2023            Modified on 16.10.2023

Accepted on 20.12.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2024; 17(4):1832-1838.

DOI: 10.52711/0974-360X.2024.00291