Formulation and Evaluation of Benidipine Nanosuspension

 

Sejal Patel*, Anita P. Patel

Dep. of Pharmaceutics, Nootan Pharmacy College, Faculty of Pharmacy,

Sankalchand Patel University, Visnagar, Gujarat, India.

 

ABSTRACT:

In the interest of administration of dosage form oral route is most desirable and preferred method. After oral administration to get maximum therapeutic effect, major challenge is their water solubility. Water insoluble drug indicate insufficient bioavailability as well dissolution resulting in fluctuating plasma level. Benidipine (BND) is poorly water soluble antihypertensive drug has lower bioavailability. To improve bioavailability of Benidipine HCL, BND nanosuspension was formulated using media milling technique. HPMC E5 was used to stabilize nanosuspension. The effect of different important process parameters e.g. selection of polymer concentration X₁(1.25 mg), stirring time X₂ (800 rpm), selection of zirconium beads size X₃ (0.4mm) were investigated by 2³ factorial design to accomplish desired particle size and saturation solubility. The optimized batch had 408 nm particle size Y₁, and showed in-vitro dissolution Y₂ 95±0.26 % in 30 mins and Zeta potential was -19.6. Differential scanning calorimetry (DSC) and FT-IR analysis was done to confirm there was no interaction between drug and polymer.

 

 

 

INTRODUCTION:

Drugs with poor aqueous solubility are increasingly posing challenges in the development of new drugs, since more numbers of poorly water-soluble drugs are introduced in the pharmaceutical era¹. Bioavailability of drug depends on its solubility and permeability as well dissolution in a medium². Bioavailability is a crucial challenge to the therapeutic efficacy of a drug regarding its route of administration³. Dissolution involves the process of transfer of solid drug particles into solution in the surrounding physiological fluid. The low aqueous solubility of Biopharmaceutics Classification System (BCS) class II drugs is a major issue for their clinical application⁴. To overcome this major obstacle some conventional methods such as micronization, chemical modification, solid dispersion, liposomes⁵, salt formation, pH adjustments, cyclodextrin complexation⁶, mucoadhesive microspheres, nanoparticles, nanosuspensions, micro emulsion^7,8, and self emulsifying systems are available to enhance the solubility, Dissolution rate and bioavailability of poorly soluble BCS Class II drugs, whereby the drug is formulated and maintained as nano size drug particle which are then suspended in a liquid usually water to form nanosuspensions⁹. Nanosuspensions have become a popular approach to improve the solubility. Nanosuspensions can be formulated either by particle size reduction of large drug particles to nanosized as top-down approach or by the precipitation of dissolved molecules into crystals as bottom up approach¹⁰. In media milling technique, top down approach, for the mechanical grinding of drug, zirconium beads and stabilizer was used to obtain nanosuspensions. Compared to other nano sizing techniques, media milling avoids organic solvents and is easy to scale up. For the formulation of nanosuspension polymers can be used like polyvinylpyrrolidone K30 (PVP K30), Poloxamer 188, Tween 80, Tween 20, HPMC E5, HPMC E15. BND is a dihydropyridine-derived calcium channel blocker having different mechanisms of action, that is, triple calcium channels (L, N, and T) blocking action. BND has relatively high vascular selectivity. The oral administration of BND is rapidly absorbed. But a dose of 8mg of BND in the fed state delay the rate of absorption and an increase in the area under the concentration curve when compared with the fasting state has been observed. The half life of BND is 5.3 hrs and its bioavailability is lower at 25-30%¹¹. Therefore Conventional tablets require to be given frequent dose in a single day daily. The released duration of drug is increased by using various polymers. Nanosuspension of BND has formulated to exhibit improved pharmacokinetic properties compared to non-nanoparticulate composition. The dosage form was preferred as nanosuspension to comprise only one stabilizer adsorbs or associates with the drug surface.

 

MATERIALS AND METHODS:

Materials:

BND was a gift from Prayosha Healthcare Pvt. Ltd (India). Zirconium oxide beads were gifted from Mahek Enterprise (India). Hydroxypropyl methyl cellulose E5, HPMC E15, PVP K20 was gifted from Prachin Chemical.

 

Method of analysis:

UV visible spectrophotometric method was used for analyzing BND. Instrument used for analysis was UV-visible spectrophotometer of Shimadzu AS, Japan; BND was measured at 357nm and methanol/water used as diluent.

 

Method for preparation of Nanosuspension:

BND nanosuspension was prepared by media milling technique; zirconium oxide beads (0.4 and 0.6mm) were used as milling media. In 20ml glass vial, accurately weighed quantities of zirconium oxide beads (6gms) were taken and 14ml distilled water was added in vial, polymer and drug were incorporated in it and carried out on magnetic stirrer for varied time period. The BND particles were fragmented into nanoparticles by the physical impact of the zirconium oxide beads at high stirring speed. BND nanosuspension was evaluated for various parameters and results of experimental design were analyzed to reduce systematic errors.

 

Characterization of Nanosuspensions:

Particle size of nanosuspensions^12,13:

The average particle diameter of the BND nanosuspension was determined using Malvern Zetasizer. Nanosuspension was suitably diluted with deionised water and sonicated for 2 minutes to reduce aggregation. Nanosuspension was placed in disposable sizing cuvettes and analyzed. Particle sizes were expressed by the 50% and 90% volume percentiles. Two samples per batch were analyzed, and the measurements were repeated three times for each sample.

 

Zeta potential:

Zeta potential indicates the stability of the suspension. Sample of BND nanosuspension was suspended with sufficient water, then sample was directly placed into cuvette and zeta potential was measured and expressed in mV.

 

Drug-excipients compatibility study:

Compatibility of drug and excipients were checked by DSC and FTIR by which interpretation has been observed.

 

FTIR study:

FTIR spectra of drug- polymer combination were compared with spectra of individual spectra of pure BND and polymer. There was no shifting of peaks found. There is no interaction between drug and polymer during preparation of nanosuspension and they are compatible with each other. This result indicates that the method used for preparation of BND nanosuspension does not affect any physico-chemical properties of the benidipine.

 

Differential scanning calorimetry:

DSC is a thermodynamic analytical technique used to evaluate the crystalline nature and thermal behavior of powders. This was obtained with a Differential Scanning Calorimeter. BND was heated in thematically sealed aluminum pans under air atmosphere at a scanning rate of 10°C/min from 30 to 300°C in an air atmosphere.

 

In - vitro dissolution:

Dissolution study of BND nanosuspension was carried out in modified diffusion cell apparatus. The drug release from BND nanosuspension was determined using a dialysis tube (donor compartment) in which the known quantity (10ml) of the nanosuspension was incorporated in a water-jacketed beaker containing 300ml of 0.1N HCl (pH 1.2) at 37±1°C for 30 mins. The beaker containing formulations were agitated on a magnetic stirrer. Samples were withdrawn periodically and replaced with an equal volume of fresh 0.1N HCl (pH 1.2). Samples taken were filtered through a filter paper (0.22µm) and assayed spectrophotometrically by UV-visible spectrophotometer at 357nm wavelength. Dissolution for each formulation was performed in triplicates.

 

Experimental Design¹⁴:

Interactive and polynomial terms were used to evaluate the responses in applied statistical model. The number of experiments required for these studies is dependent on the number of independent variables selected. The response (Y) is measured for each trial.

 

Y = β₀+β₁ X₁+β₂ X₂+β₃X₃+β₁₂ X₁ X₂+β₂₃ X₂X₃+β₁₃ X₁X₃+ε..........(1)

 

Where, Y is the dependent variable,

β₀ is the arithmetic mean response of the eight runs and

β is the estimated coefficient for the factor X

The main effects (X₁, X₂ and X3) indicate the average result of changing one factor at a time with varied ratio. The interaction terms (X1X2, X₂X₃ and X₁X₃) show how the response changes when two factors are simultaneously changed. A 2³ full factorial design was incorporatedin the present study. The three factors, X₁- concentration of polymer (HPMC E5), X₂- Zirconium oxide bead size(mm), X₃- Stirring speed(rpm)were varied. Particle size (nm) and % drug release in 30 min were taken as the response variable. In this present study 3 factors and two levels were evaluated. 8 possible experimental trials were performed. Statistical analysis of 2³design was performed by multiple regression analysis using Microsoft excel 2007.

 

Table 1: Variable level of 2³ factorial design for benidipine nanosuspension

Coded value	Actual value
	Conc. of Polymer (gm) (X1)	Zirconium oxide bead size (mm) (X2)	Stirring speed(rpm) (X3)
-1	0.75	0.4	400
+1	1.25	0.6	800

 

RESULT:

Milling of BND is crucial process of this invention to formulate nanosuspension. The different formulative variables (1) amount of polymer (2) Zirconium oxide bead size (3) stirring speed were contribute towards the change in particle size in nanosuspension preparation. Nanosuspension of BND was prepared by as formulation shown in table 2. F₁ and F₂ formulations were containing different concentration of polymer (HPMC E5) with same Zirconium oxide beads size (0.4mm) on slow speed stirrer at 400rpm. F₃ and F₄ formulations were containing different concentration of polymer (HPMC E5) with same Zirconium oxide beads size (0.6mm) on slow speed stirrer at 400rpm. F₁-F₄ formulations were containing different polymer concentration with different Zirconium oxide beads size of 0.4mm and 0.6mm on same stirring speed at 400rpm and observed no major change in particle size reduction, Whereas F₅-F₈ formulations were containing different polymer concentration with different Zirconium oxide beads size of 0.4mm and 0.6mm on same stirring speed at 800rpm and observed change in particle size reduction. F₅ and F₆ formulations were containing different concentration of polymer (HPMC E5) with same Zirconium oxide beads size (0.4mm) on high speed stirrer at 800rpm. Among F₅-F₈ increasing the polymer concentration (e.g. F6, F8) reduces the particle size while, reducing the X₂ reduces the particle size on 800rpm stirring speed in F5 and F6. F7 and F8 formulation shows maximum reduction in particle reduction but have been observed physically unstable nanosuspension. F1-F8 formulations shows that increasing the polymer concentration (1.25gm) and lesser the Zirconium oxide beads size (0.4mm) with high stirring speed (800 rpm) desired particle size can be obtained.

 

Dissolution studies of prepared BND nanosuspension (F₁-F₈) were carried out in 0.1N HCl (pH 1.2). Different concentration of polymer, Zirconium oxide beads size and stirring speed formulate different particle sizes which are correlated with dissolution of nanoparticles. The dissolution rate was markedly enhanced by the nanoparticles. On the basis of the experimental trials, a 2³ full factorial design was employed to study the effect of independent variables (i.e., concentration of polymer (X₁), Zirconium oxide beads size (X₂) and the stirring speed (X₃) on dependent variables particle size, and cumulative percentage release of BND after 30 min. From the experimentation, higher variability was found for the amounts of drug released from the smaller particle size than from the larger ones.

 

Table 2: Formulation and dissolution characteristics of 2³factorial design batches

Batch Code	X1	X2	X3	Y1 particle size (nm)	Y2 % Drug release in 30 min (Q30)
F1	-1	-1	-1	685	60 ± 0.46
F2	1	-1	-1	710	77 ± 0.62
F3	-1	1	-1	735	57 ± 0.79
F4	1	1	-1	750	65 ± 1.76
F5	-1	-1	1	512	88 ± 0.12
F6	1	-1	1	408	95 ± 0.26
F7	-1	1	1	395	84 ± 0.75
F8	1	1	1	275	80 ± 1.75

 

Accordingly, in order to reduce this variation, optimization was performed using a desirability function to obtain the levels of X₁, X₂ and X₃ which close to 400 nm, 95 ± 0.26 % for Y₁, Y₂ respectively. The observed responses for the 8 formulations are given in Table 2. In order to investigate the factors systematically, a factorial design was employed. As shown in equation 1, a statistical model incorporating interactive and polynomial terms was used to evaluate the responses.

 

Y = β₀+β₁ X₁+β₂ X₂+β₃X₃+β₁₂ X₁ X₂+β₂₃ X₂X₃+β₁₃ X₁X₃+ε .............(1)

 

The polynomial equations can be used to conclude after applied the magnitude of coefficient and the mathematical sign it carries (i.e., positive or negative). A coefficient with positive sign appears a synergistic effect of the factor on the response, while a negative sign indicates an antagonistic effect. The mathematical relationship in the form of factor’s coefficients, its corresponding P-values for the measured responses and correlation coefficient are listed in table 3. Concerning particle size, the results of multiple linear regression analysis showed that all the coefficients b_1, b₂ and b₃ bear a negative sign (R²=0.999).

 

Table 3: Summary of regression analysis for measured responses

Co- efficients	bo	b1	b2	b3	b12	b23	b13	R2
Y1	559.125	- 23.37	-19.62	-160.87	-3.62	-42.12	-33.37	0.999
P-Value	0.001	0.030	0.036	0.004	0.191	0.016	0.121
Y2	75.75	3.5	-4.25	11	2.5	0.5	2.75	0.999
P-Value	0.002	0.045	0.037	0.014	0.063	0.295	0.057

 

It can be concluded from the equation (2) that X₂ (Zirconium oxide beads size) showed the largest effect compare to X₁(conc. of polymer and X₃ (stirring speed) and negatively impacted.

The coefficients b₁, b₂, and b₃, b_23, b₁₃ were found significant at P < 0.05 (table 3).

 

Y₁=559.12-23.37 X₁-19.62X₂-160.87 X₃-3.62 X₁ X₂-42.12 X₂X₃-33.37 X₁X₃……(2)

 

Therefore increasing the concentration of polymer shows particle size reduction mean while as the stirring speed is increased, the effect observed much more prominent with stirring speed than amount of polymer and size of Zirconium oxide beads^. Figure 2 shows by contour plot a nearly linear ascending pattern for the particle size, as the concentration of polymer and stirring speed is increased, and the size of Zirconium oxide beads decreased.

 

Y₂=75.75+3.5 X₁-4.25X₂+11 X₃-2.5 X₁ X₂-0.5 X₂X₃-2.75 X₁X₃…....(3)

 

It can be concluded from the equation (3) that X₁ showed the more effective than X₂ and X₃ showed the more effective than X₁. During the dissolution experiments, it was noticed that more amount of polymer were suppose to favor the drug release. Increasing size of Zirconium oxide beads retard the dissolution rate, as well increasing the speed of stirring shows desired dissolution (figure 3). The coefficients b₁, b₂, and b ₃, b_12, b₁₃ were found significant at P< .05

 

From the multiple regression analysis, both the coefficients b₁ and b₃ bear a positive sign for BND release after 30 min.

 

Particle size determination:

 

Figure 1: Particle size determination of optimized nanosuspension

 

Figure 2: Contour plot showing Effect of X₁ and ZnO₂ beads size X₂ (A), X₂ and stirring speed (B), Effect of X₃ and X₁ (C) on particle size Y₁

 

Figure 3: Contour plot showing Effect of X₁ and X₂ (A), Effect of X₂ and X₃ (B), Effect of X₃ and X₁ (C) on Y₂.

 

Zeta potential:

HPMC E5 was used as stabilizer which provides steric stabilization. So, both negative Zeta potential is attributed to Nanosuspension. In general, zeta potential value of ± 30mV is sufficient for stability of nanosuspension. Zeta potential of optimized BND nanosuspension was observed -19.6.

 

Figure 4: Zeta potential of optimized BND nanosuspension

 

FTIR study:

FTIR spectroscopy was done with BND and mixture (BND+HPMC). Comparative spectra of them are presented in Figure: 5 showing Drug and Polymer’s compatibility.

 

Figure 5: FTIR spectroscopy of BND and mixture (BND+HPMC)

 

DSC Study:

DSC thermo gram corresponding to benidipine and mixture (BND + HPMC E5) both has been shown in Figure: 6 Benidipine shows a characteristic endothermic peak at 222.87°C corresponding to its melting point. A sharp endothermic peak at was observed for mixture of BND and HPMC E5.

 

Figure 6: DSC thermogram of benidipine and mixture (BND + HPMC E5)

 

In –vitro dissolution study:

In order to observation the achievement of improving the rate of dissolution of benidipine is accomplished by formulating nanosuspension by media milling, the results of in vitro dissolution of different benidipine samples are shown in Figure: 7 The rate of dissolution of unmilled benidipine seems very low (48%). In account of the factorial design, optimized formulation of benidipine nanosuspension F6 shows improved the dissolution rate, since almost 95 ± 0.26% of the drug was dissolved in the first 30 mins.

 

Figure 7: In vitro dissolution study

 

CONCLUSION:

From the results of this study it may be concluded that nanosuspensions of poorly soluble drugs such as benidipine are easy to prepare and enhance the solubility as well dissolution profile of the same. With help of factorial design and desirability function, amount of stabilizer, zirconium oxide bead size and stirring rate was identified as influencing parameters affect on particle size, drug release after 30min (Q₃₀). The drug particle size decreases with the amount of stabilizers, maintain the beads size and increasing of the stirring rate. Dissolution study in 0.1N HCl exhibit that nanosuspension shows higher drug release compared to the pure drug. Consequently nanosuspensions represent a promising alternative to current delivery systems aiming to improve the biopharmaceutical performance of drugs with low water solubility and low dissolution rate.

 

ACKNOWLEDGMENT:

The Author and co-author, thanks to all for providing the necessary facility to accomplish the work we also humble gratitude to our colleague and non-teaching staff for their support during the work.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Received on 04.08.2020           Modified on 17.09.2020

Accepted on 19.10.2020         © RJPT All right reserved