1. INTRODUCTION:

The principal advantages for getting controlled release dose formulations is to get a more uniform plasma drug profile and it can be achieved by formulating extended release dose formulations in the treatment of hypertension in order to achieve 24 hr control of blood pressure (BP) which provide more consistent 24 hour BP lowering activity, with attenuation of early morning BP surges.

In the present paper BH used as anti-hypertensive drug. One of the most important factors contributing to myocardial ischemia - reperfusion injury is believed to be an overload of intracellular Ca ²⁺, which may arise from a Ca ²⁺ current via Na⁺ Ca ²⁺ exchange, leakage of Ca ²⁺ from the sarcoplasmic reticulum or mitochondria, release of Ca ²⁺ from Ca ²⁺ binding proteins or Ca ²⁺ influx through Ca ²⁺ channels ^1-6. Numerous attempts have been made to demonstrate the cardio protective effects of Ca2+ antagonists in ischemia–reperfusion injury using animal models. The 1, 4-dihydropyridine derivative Ca ²⁺ channel antagonists are widely used as anti-hypertensive or anti-anginal agents by preventing the rapid vasodilatation that produces a reflex increase in sympathetic nerve activity, which in turn results in the cardiac events leading to coronary artery disease ^7-8. Benidipine hydrochlorides, which belong to 1, 4-dihydropyridine derivative group, have a slow onset and long-lasting effects.

Polymers which are use in development of extended release tablet are mainly classified in combination of non-ionic polymers, second type is combination of non-ionic and anionic polymers and third type is the combination of cationic and anionic polymers e.g., chitosan (CS)– sodium alginate (SA), and CS- xantham gum (XG) . Polymers play an important role by controlling drug release pattern in extended release tablets ^9-11. From our previous research outcomes ¹² we have found that the polymers like Eudragit, HPMC, and Chitosan have the most profound influence on the dissolution profiles of Benidipine Hydrochloride and thus extended drug release of prepared matrix tablet. So based on this outcomes we have formulated two most suitable batch of 250 mg tablet containing 12 mg of BH once daily tablet for extending drug release in spite of taking 4 - 6 mg of tablet for 2 to 3 times daily, as frequent administration of drug causes side effects like dizziness, palpitation etc. to the patient and some time poor patient compliance also increase the symptoms of high blood pressure. Patients suffering from high BP, use of orally treated with maximum dose of 12 mg Benidipine causes blood pressure to reduced and which is maintained up to 24 hr showing its long-lasting pharmacological activity ^13-14

2. MATERIALS AND METHODS:

2.1 Materials:

BH was procured from Ranbaxy Laboratories, Bangalore. Chitosan (50kDa) and different Eudragit grades were procured from Merck Chemicals Ltd., Germany. Other ingredients like HPMC K 100 M, MCC, Lactose obtained from as gift sample from Degussa India Pvt., Ltd.. In our preparation we have taken glidants as aerosil (which is Colloidal SiO₂)_,and has been procured along with other chemicals like Glecyryl Behenate and magnesium stearate from S.D. Fine chemicals, Mumbai.

2.2 Preparation of Matrix Tablets:

Tablets are prepared by direct compression method by accurately weighing quantity of drug and excipients which were passed through sieve no. 20 and 40 respectively. Then the mixture was blended thoroughly for 15 min by adding glidants. The above formula then compressed in 10 mm punch tablet machine karnavati tablet press punching machine preparing tablets of weight 250 mg. As per formula two batches F1 and F2 were prepared. The composition was given in Table 1. The tablets were round and flat with a thickness of 3.1 ± 0.2 mm.

Table 1: Compositions of 250 Mg Benidipine Hydrochloride (BH) Matrix Tablet

Ingredients (mg)	F1	F2
Drug (BH)	12	12
HPMC K 100M	88	108
Eudragit RL100	30
Eudragit RS100		30
Chitosan	60	40
MCC	55
Lactose		55
Aerosil (Colloidal SiO₂)	2.5	2.5
Glyceryl Behenate	2.5	2.5

2.3 Evaluation of tablets:

All the post compression parameters are evaluated such as thickness, hardness, friability, weight variation, drug content and diameter of tablet etc

2.4 In vitro dissolution studies:

In vitro dissolution test was carried out by using USP type II (paddle type) apparatus. The tablet is kept in 900 ml of dissolution fluid of 0.1N HCl (pH 1.2), and stirrer rotating with 75 rpm, and maintaining the temperature 37±0.5°C of dissolution media for first 2 hr then dissolution fluid is changed to phosphate buffer pH 6.8 maintaining same condition for remaining hour. At predetermined time intervals of 5 ml sample were withdrawn with syringe replacing the fresh media was replaced with the fresh media to maintain an ideal sink condition. The percentage drug release was calculated. The cumulative percentage of drug released was plotted against time in order to obtain the release profile.

2.4 Drug release mechanism using various kinetic models:

Drug release data were analyzed by various mathematical models to check drug release mechanism including zero order, first order ¹⁵, Higuchi ^16, Ritgere Peppas ¹⁷, Hixsone Crowell ¹⁸.

Drug dissolution from dosage forms that do not disaggregate and release the drug slowly shows zero order and can be represented by the equation:

Q₀- Q_t = K₀t

Where Qt is the amount of drug dissolved in time t,

Q₀ is the initial amount of drug in the solution (most times, Q₀ = 0)

K₀ is the zero order release constant expressed in units of concentration/time.

The release of the drug which followed first order kinetics can be expressed by the equation can be expressed as:

log C = log C₀ - K_t / 2.303

K_t is the zero order release constant expressed in units of concentration/time.

Accordingly, Ficks model expression is given by the equation:

f_t = Q = A √D(2C -Cs) Cs t

Where C is the drug initial concentration, Cs is the drug solubility in the matrix media. D is the diffusivity of the drug molecules (diffusion coefficient) in the matrix substance and Q is the amount of drug released in time t per unit area A, and.

Hixson and Crowell recognized that the particle size regular area is proportional to the cube root of its volume. They derived the equation:

W₀ ^1/3 -W_t ^1/3 = K _t

where W₀ is the initial amount of drug in the pharmaceutical dosage form, Wt is the remaining amount of drug in the pharmaceutical dosage form at time t and K is a constant incorporating the surface volume relation.

Ritgere Peppas derived an equation to determine the drug release mechanism as shown in equation.

M_t/ M₀ = K tⁿ (4)

Where K is the kinetic constant, M_t/ M₀is the fraction of drug released at time t, and n is the diffusion exponent, indicative of the mechanism of the drug release. The equation generally holds for Mt/M₀ > 70% of drug release.

n = 0.45 or 0.45 < n < 0.89 or n > 0.89, indicates Fickian diffusion or anomalous transport or Case ‘‘II’’ transport kinetics respectively.

Regression analysis was performed to obtain the release rate constant and the values of coefficient of regression (R²). In this research work we have used Kinetic DS3 software ¹⁹. Kinetic DS3 is simple software for curve fitting particularly for description of a cumulative dissolution curve by simple equation. Models parameters are established by linear and non-linear regression (simplex method). The model with the highest correlation coefficient (R) was considered to be the best fit for the designated kinetic release.

2.5 In vivo pharmacokinetic study:

The analytical method used for the estimation of Benidipine Hydrochloride in rabbit plasma is done by HPLC method. The HPLC instrument used for this analysis was of Waters HPLC 2965 SYSTEM with self regulating injector and PDA detector. The Empower 2 Software was used for this instrument. For measuring absorbance of Benidipine Hydrochloride solution UV-Visible spectrophotometer was used. Different pharmacokinetics data are calculated using PK Solver software, which is an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel.

The in-vivo study was conducted in accordance with Animal Ethical Guidelines for investigations in laboratory with Committee for the Purpose of Control and Supervision of Experiments on Animals with Registration number CPCSEA/IAEC/JLS/01/09/18/03. In vivo pharmacokinetic parameters for the optimized formulation BH extended release matrix tablets batch F1is performed in New Zealand white rabbits by following parallel design method. Six rabbits are selected and divided in 2 groups each having 3 rabbits. To the one group of rabbit marketed tablets is given in powder form with water, so that it can reach to stomach, where as to the other group optimized selected batch is given. After different time interval, blood samples is withdrawn from marginal ear vein of rabbit and collected samples are centrifuged for 30 min which separates plasma and clear fluid. Fluid is collected and diluted with mobile phase. The mobile phase used is mixture of 65ml Phosphate Buffer solution of pH 4 and 35ml of methanol. The flow rate was adjusted to 1ml/min. The detection was carried out at 272nm with UV- spectroscopic detector with run time of 10-20minute. Then obtained peak area values to corresponding concentration were plotted for preparation of standard calibration curve of Benidipine Hydrochloride by HPLC analysis. For the HPLC instrument Inertsil ODS 3V C₁₈Column (150mm X 4.5 mm, 5µ) was used for the analysis.

2.6 HPLC method validation for determination plasma samples:

The in vivo estimation of drug in rabbit plasma was carried out by HPLC method. The peak area values to corresponding concentration of Benidipine Hydrochloride. The Peak area values to corresponding concentration of BH by HPLC analysis shown in table 2 and the assay of validation is shown in Table 3, whereas Calibration curve of BH by HPLC method in shown in figure 1.

Table 2: Peak area values to corresponding concentration of BH for standard calibration curve (HPLC Analysis)

Standard Concentration(ng/ml)	Peak Area	Found Concentration	Recovery (%)
60	838231	61.90856	103.1809
120	1617125	125.6166	104.6805
180	2335973	184.4132	102.4518
240	3014624	239.9221	99.96752
300	3685182	294.7689	98.25631
Mean(n=5)			101.7074
SD			2.573989

Table 3: Assay Validation Sheet corresponding concentration of BH for standard calibration curve (HPLC Analysis)

Parameters	Value
Accuracy	101.7074±2.573
Slope	12226
Intercept	81337
Linearity range	60-300(µg/ml)
Correlation Coefficient (r2)	0.998

Figure 1: Calibration curve of BH by HPLC method

3. RESULTS:

The physical characterization of the both batches of tablets revealed that the weight of the tablets was confined within ±4% of the average weight, thickness varied from 3.2 to 3.8 mm with 8mm of tablet diameter. The % friability is in the less than 0.5% and the drug content varied within ±5% of the labeled amount. Hardness was within the range of 8-9 kg/cm^2. All these variation were found to comply with the requirements of official Compendium of Indian Pharmacopoeia.

DSC thermogram of pure drug, and physical mixture of excipients used for formulations were obtained and shown in figure 2 and 3.

Figure 1: DSC Thermogram of Drug Benidipine Hydrochloride

Figure 2: DSC Thermogram of Tablet

The in vitro drug release pattern explain linear curve. Tablets of both batches were subjected for invitro release studies and % drug release pattern is shown in Figure 4 and the drug release mechanism is represented in Table 4.

Figure 4: In vitro dissolution study of BH matrix Tablets formulation batch F1&F2

Table 4: Drug release mechanism parameters of Optimized formulation F2

Kinetic model	R²	n(slope)
Zero order	0.9657	7.873
First order	0.985	-0.0687
Higuchi	0.998	37.563
Korsmeyer-Peppas	0.942	0.720

The stability studies on selected formulations were carried out for 6 months as per ICH guidelines. Under the specific storage conditions, no significant changes observed in the prepared tablets.

To validate the accuracy of method, recovery studies were performed where to the pre analysed sample solution, a definite concentration of standard drug was added and then its recovery was analyzed and the results shows the method was found to be accurate as % recovery is between ranges of 100 ± 15%.

During In vivo the plasma drug concentration of each rabbit measured at different time interval for marketed Beniduce Tablet and optimized formulation were presented in Table 5 and 6 respectively where as the pharmacokinetic parameter comparison data is represented in Table 7. Different pharmacokinetics data are calculated using PK Solver ²⁰. The maximum plasma concentration of (C_max) and the time to reach C_max (T_max) were calculated by using PK solver software directly from the plasma concentration versus time data. The elimination rate constant (k_e) was the slope of the terminal four points in plasma concentration-time curve, and the half-life of the preparation (t _1/2) was calculated by 0.693/ k_e. Plasma drug concentration and time profile graphs for marketed and selected formulation batch F2 shown in figure 5.

Table 5: Plasma drug concentration-time profile of marketed formulation

Time (in hr)	Plasma drug concentration (µg/ml) in Rabbit For marketed Tablet
1	460.0613
2	740.89
3	830.986
4	700.4456
5	440.9874
6	300.8965
12	160.9086
18	14.17654
20

Table 6: Plasma drug concentration-time profile of optimized formulation (F1)

Time (in hr)	Plasma drug concentration (µg/ml) in Rabbit For F2 Tablet batch
1	308.06
2	708.83
4	680.83
8	638.57
12	460.83
16	339.28
20	289.24
24	240.7
36	159.98

Figure 5: Comparison of plasma drug concentration-time profile of marketed tablet, and formulation (F2)

Table 7: Comparison of pharmacokinetic parameters of marketed and optimized formulation

Parameters	Marketed (Beniduce Tablet)	Batch (F2)
C_max(ng/ml)	830.986	708.83
T_max(hr)	2	1.5
t_1/2(hr)	1.9224	2.7224
K_el (hr ^-1)	0.36049	0.25456
AUC_0-t	7256.527	10461.681
AUMC_(0-t)	44752.09575	139634.373
MRT(hr)	6.1671	16.8115

4. DISCUSSION:

The present study was undertaken to formulate BH extended release matrix tablets and to evaluate different pharmacokinetics parameters from in vivo study. DSC study provides the information on the physical characteristics of the sample. Any significant changes in the thermal behaviour of either a drug or a polymer demonstrate the possible interaction of the drug and the polymer. DSC thermo gram showed an endothermic peak at 176.85⁰C which is corresponding melting point of drug. DSC thermo gram of tablet showed an endothermic peak at 158.55⁰C. So based on this report we may conclude there was compatibility with the drug showing that the formulation is thermodynamically stable.

Drug release pattern in formulation F1 release extends up to more than 17 hr which may be due to poly electrolyte complex between Eudragit RL100 and Chitosan ²¹ and also HPMC K 100M makes the tablet swell and extended drug release. Generally when a tablet is placed in the dissolution media, wetting of the surface occurs first, followed by hydration causing the formation of pores due to dissolve of the excipients which increases its porosity strength by which entry of the liquid through the microscopic pores increase. But when polymer is added to formulation, polymer content block microscopic pores, leading to reduced drug release rate, due to the formation of a thick and turbid gel, which is resistant to erosion and diffusion. Another reason for extending drug release is due to presence of MCC as various research reports indicated that MCC has strong tablet binding properties, which decrease the tablet porosity ²² and helps in extending drug release from the matrix tablets ^23-24.

Similarly Formulation F2 contain HPMC K 100 M polymer along with Eudragit RS 100. And the drug release we found tablets of this batch shows extended of drug release due to swelling of HPMC K 100M polymer and release pattern may extend more than 18 hr. Another reason for extended drug release may be due to presence of lactose in this formulation, which is a water-soluble excipients, it increases the hydration rate and relaxation of the polymer chains, resulting in slow release of drug by diffusion from the matrix tablet layer ²⁵.

Due to better drug release pattern F2 is selected as the most suitable batch for further study. Regression analysis was performed by using Kinetic DS3 software to obtain the release rate constant and the values of coefficient of regression (R²). From the drug release data the software predicts the tablet showed the Higuchi square root model and R² values for batch F1 is (R² =0.998) indicates that the drug released by diffusion mechanism and from Peppas equation n value 0.720 (range 0.89-1) indicates that diffusion– erosion mechanisms resulting from swelling and hydration behavior of Chitosan and anionic polymers forming a Complex in extending drug release. The in vitro dissolution data of optimized formulation F1 were fitted in various kinetic models. The R² values for zero order was 0.957 and for first order was 0.985.The R² values for Higuchi model was found maximum 0.998. Hence it follows Higuchi kinetics.

The in vivo pharmacokinetic parameters for the optimized BH tablets were performed in New Zealand white rabbits. The formulation batch F2 was selected for in vivo pharmacokinetic studies along with the available marketed conventional formulation. From the plasma concentration versus time data the results of in vivo evaluation parameters are calculated by using PK solver software which shows that marketed conventional BH tablet administered as plain drug reached peak plasma concentration of 830.986 µg/ml after 2 hr of administration. But optimized and selected prepared tablets reached the maximum concentration of 708.83 µg/ml after 1 hr of administration, but it continues to release drug. The results proved that drug is released slowly for prolonged period of time and achieving better BP control. So based on this research outcome and results, we may conclude our objective is achieve by extending drug release pattern of BH. The results showed that more C_max for test formulation indicates prolonging of drug release from dosage form and maintain the plasma drug concentration within therapeutic range up to 36 h. Higher value of both the AUC_0-t and AUC last for optimized formulation denoted the extent of absorption is more, but more value of plasma half life and MRT indicated rapid elimination of the drug.

5. CONCLUSION:

So based on this outcomes combination of polymers could be successfully employed for developing extended release matrix tablets of BH. In vivo pharmacokinetic study proves that the our prepared tablets show prolonged release and may be able to sustain the therapeutic effect and capable of maintaining constant plasma concentration through which reduce the frequency of administration and decrease the dose-dependent side effects associated with repeated administration of conventional BH tablets by decreasing side effects like dizziness, palpitation etc. to the patient and some time poor patient compliance also increase the symptoms of high blood pressure.

6. ACKNOWLEDGEMENT:

The authors are also thankful to the principal and management of College of Pharmaceutical Sciences, Puri for providing us with the facility for carrying out the research work.

7. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 22.08.2021 Modified on 14.10.2021

Research J. Pharm. and Tech 2022; 15(11):4924-4930.

DOI: 10.52711/0974-360X.2022.00827