In Vitro and In Vivo Evaluation of Floating Microspheres of Prazosin Hydrochloride

Vanitha Kondi, Ramesh Alluri

Department of Pharmaceutics, Vishnu Institute of Pharmaceutical Education and Research, Narsapur,

Medak (DT), Telangana – 502313, India.

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

ABSTRACT:

The concept in the development of controlled oral release floating dosage forms is not just to prolong the delivery of drugs but also to prolong the presence of dosage forms in the stomach in order to improve the bioavailability of drugs with a ‘narrow absorption window’. Prazosin hydrochloride is a selective α-1-adrenergic receptor antagonist used to treat hypertension. It has a mean plasma half -life of 2-3 hour.Prazosin has a shorthalflife and low bioavailability in the upper part of the GIT hence it is suitable for gastro-retentive system. In the present study, an antihypertensive drug, Prazosin hydrochloride, is delivered through a gastroretentive microparticulate system capable of floating on simulated gastric fluid for 24 h. Microspheres are prepared by solvent evaporation technique. Microspheres showed excellent buoyancy and a controlled release pattern with 24h. Showed 99.87% drug release at the end of 24 hours. In vivo bioavailability studies performed on albino rats and Tmax, Cmax, AUC and Kel were calculated and confirmed significant improvement in bioavailability. The data obtained thus suggests that floating microspheres can be successfully designed to give controlled drug delivery, improved oral bioavailability.

KEY WORDS: floating microspheres, prazosin, in vivo studies, bioavilability studies, pharmacokinetic parameters.

INTRODUCTION:

The most convenient and commonly employed route of drug delivery has historically been by oral ingestion¹. Drugs that are easily absorbed from the gastrointestinal tract and having a short half life are eliminated quickly from the blood circulation. To avoid these problems, oral controlled release formulations have been developed, as these release the drug slowly into the gastrointestinal tract and maintain a constant drug concentration in the serum for longer periods of time^{2, 3}.

However, incomplete release of drug and a shorter residence time of dosage forms in the upper gastrointestinal tract, a prominent site for absorption of many drugs, will lead to lower bioavailability. Efforts to improve oral drug bioavailability have grown in parallel with the pharmaceutical industry. As the number and chemical diversity of drugs has increased, new strategies are required to develop orally active therapeutics. The past two decades have been characterized by an increased understanding of the causes of low bioavailability and a great deal of innovation in oral delivery technologies, marked by an unprecedented growth of the drug delivery industry⁴. Thus, gastroretentive dosage forms which prolong the residence time of the drugs in the stomach and improve their bioavailability have been developed ^5,6.Floating microspheres are non-effervescent gastro retentive drug delivery systems. These microspheres having a size less than 200 μm, free flowing powders and remain buoyant over gastric contents and for prolonged period of time. The drug is released slowly from the floating system at the desired rate, resulting increased gastric retention and reduced fluctuations in plasma drug concentration^7,8. Prazosin is a selective α-1-adrenergic receptor antagonist used to treat hypertension. Prazosin acts by inhibiting the postsynaptic alpha1-adrenoceptors on vascular smooth muscle. It inhibits the vasoconstrictor effect of catecholamines (epinephrine and norepinephrine), resulting in peripheral vasodilation. It has a mean plasma half -life of 2-3 hour. Prazosin has a short halflife and low bioavailability in the upper part of the GIT hence it is suitable for gastro-retentive system ⁹. The aim of the present work was to develop a new drug delivery system that provides gastric retention, increase the bioavilability and reduces the side effects like postural hypotension which may lead to precipitation of congestive heart failure.

MATERIALS AND METHODS:

Prazosin hydrochloride (PH) was obtained as a gift sample from Synthokem labs, Hyderabad. HPMC K100M, ethyl cellulose and Eudragit were provided by Dr. Reddy’s laboratories, Hyderabad and all solvents used were of analytical grade. Albino rats were procured from animal house, Vishnu Institute of Pharmaceutical Education and Research.

Preparation Of Microspheres:

Microspheres containing Prazosin hydrochloride as a core material were prepared by a Non-aqueous Solvent Evaporation method^10,11. Drug and polymers (HPMC, ethyl cellulose and eudragit) were mixed in dichloromethane and chloroform at various ratios showed in Table no. 1 The slurry was slowly introduced into 100ml of liquid paraffin containing 1% of Tween80 as emulsifying agent while being stirred at various rpm by a mechanical stirrer with a three bladed propeller at room temperature. The solution was stirred for 2 hours for complete evaporation of solvent and the microspheres were collected by filtration. The microspheres were washed three times with n-hexane and three times with 180 ml petroleum ether to remove the remaining oily phase and then dried overnight at room temperature for 24 hours and subsequently stored in a desiccator.

Table1: Formulation of Floating microspheres of Prazosin HCl

Ingredients	F1	F2	F3	F4	F5	F6	F7	F8	F9	F10
Prazosin HCL(mg)	100	100	100	100	100	100	100	100	100	100
HPMC(mg)	100	100	100	100	100	100	100	100	100	100
EC(mg)	50	100	50	100	50	100	50	100	75	75
Eudragit(mg)	50	100	50	100	50	100	50	100	75	75
Heavyliquid Paraffin(ml)	100	100	100	100	100	100	100	100	100	100
Dichloromethane(ml)	10	10	10	10	10	10	10	10	10	10
Ethanol(ml)	15	15	20	20	15	15	20	20	17.5	17.5
Tween 80(%)	1	1	1	1	1	1	1	1	1	1
Petroleum ether(ml)	180	180	180	180	180	180	180	180	180	180

In vitro Dissolution studies:

Dissolution test was performed in USP XXIII dissolution test apparatus by paddle method. The dissolution media used was 500ml of simulated gastric fluid maintained at 37±0.5⁰C and rotated at 50 rpm/min. Aliquots samples were withdrawn at specified time intervals and replaced with the same volume of fresh media, filtered and analyzed spectrophotometrically at 254 nm for cumulative percentage drug release^12,13.

In-vivo studies:

In vivo bioavailability studies were conducted in healthy albino rats weighing 200-250gms. Six rats were divided into three groups and fasted for 24 h¹⁴. The animal experiments were performed after prior approval by the Institutional Animal Ethics Committee for animal research (IAEC, 1358/ac/10/CPCSEA) Vishnu Institute of Pharmaceutical Education and Research, Medak, Andhra Pradesh, India. The study was conducted in accordance with the guidelines provided by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Study design¹⁵:

Pharmacokinetics study in normal rats:

Rats were divided into 3 groups (n=6).

Group 1 - (Control) - Rats were orally administered with 0.9%w/v NaCl

Group 2 - (Standard) - Rats were fasted overnight and were orally administered with pure drug at dose of 1.54 mg/kg.

Group 3 - (Test) - Rats were fed orally with prazosin microspheres at dose of 1.54 mg/kg

Dose calculations:

Human Equivalent Dose (HED in mg/kg) = Animal Dose (mg/kg) × Animal K_m ÷ Human K_m

For a typical adult (body weight 60 kg, body surface area 1.6 _m²), K_mis 37

Rat K_mis 6

Sample collection:

Blood samples were collected from retro-orbital plexus. About 0.3 ml of blood is collected from Retro Orbital at 1,2,4,8,10 and 12 hrs in Eppendorf tubes and Serum is separated by centrifugation at 2500 rpm for 15 minutes and stored at refrigerated conditions until analysis¹⁶.

Accelerated stability studies:

In order to determine the change in evaluation parameters and in vitro release profile on storage, stability study of optimized batch was carried out as per ICH guidelines at temperature 40° ± 2°C/75% ± 5% RH in a humidity chamber for 2 months.

RESULTS:

In Vitro Drug Release Studies:

In vitro release of drug from the various Prazosin hcl floating microspheres was presented in Table 2. It was observed that the drug release was dependent on the drug to polymer ratio.

Table 2: In vitro drug release profile of Prazosin hcl floating Microspheres (F1-F18)

Formulations				TIME(hrs)
Formulations	0	0.5	1	2	4	8	12	16	20	24
F1	0	15.38	27.09	59.87	72.97	89.04	99.23
F2	0	17.64	25.04	59.54	78.54	98.23
F3	0	12.14	26.38	67.05	73.13	86.40	99.21
F4	0	15.96	24.38	58.42	83.77	98.44
F5	0	14.50	20.38	55.36	76.08	87.21	95.36
F6	0	24.32	45.02	63.46	88.06	99.57
F7	0	11.08	24.67	59.47	65.48	87.90	95.47
F8	0	19.39	49.09	70.68	85.96	99.12
F9	0	12.01	21.05	39.87	46.67	55.59	87.64	97.32
F10	0	15.02	29.32	35.43	57.22	71.89	81.58	97.01
Optimized formulation	0	13.62	22.76	37.63	44.57	58.39	66.50	79.08	87.31	99.87

CALCULATION OF BIOAVAILABILITY¹⁹:

Bioavailability is the amount of drug that available in the systemic circulation after administration of the drug. It is calculated by the following formula. Calculated mean pharmacokinetic parameters of Prazosin pure drug and microspheres shown in Table No. 3.

Percentage increase in Bioavailability = (AUC of test –AUC of standard)/(AUC of standard) x 100

Percentage increase in Bioavailability of Prazosin = (70.73 –45.10)/(45.10)x 100

Percentage increase in Bioavailability of Prazosin = 56.82%

TableNo.3: Mean Pharmacokinetic parameters of Prazosin pure drug and Prazosin microspheres in Normal rats

Pharmacokinetic Parameters	Group 2	Group 3
Cmax (μg/ml)	3.42±1.20	4.78±1.69
Tmax(h)	3.0±0.05	5.5±0.24
Kel(ml/h)	0.220±0.005	0.066±0.0001
T½(h)	2.88±0.56	7.5±0.015**
AUC_0-12(μg-h/ml)	45.10±12.03	70.73±17.21**

Mean ±SD (n=6);

** Significant at P<0.001 compared to Prazosin control.

Data analysis was done by using statistical Program software Prism Graph pad and the significance was determined by students paired ‘t’ test.

In vivo bioavailability studies:

The in vivo evaluation of the floating microspheres of Prazosin hydrochloride was conducted in albino rats²⁰. The mean pharmacokinetic parameters of the pure drug and the formulation were estimated and are given in Table 3.Assessment of the AUC showed that the bioavailability was lesser for the pure drug (normal oral bioavailability being only 56%) and increased to2times with formulation. The elimination was also decreased with the microparticulate formulation when compared to the free drug. Thus, the bioavailability of an antihypertensive drug Prazosin hydrochloride has been significantly increased by formulating it into a floating microspheres.

Accelerated stability studies:

Optimized formulation showed 99.87% of drug release in initial month. Evidently, a slight increase in drug release at the end of 2 months was observed on comparing the fresh microspheres to the stored microspheres shown in figure No. 2. However, even with this increment, the stored microspheres compiled with the reported specifications of sustained-release products. This indicates that the optimized formulation was fairly stable at accelerated storage condition.

Figure No. 2: Cumulative percent drug release of optimized formulation at Accelerated stability studies

DISCUSSION:

Pharmacokinetic parameters were calculated by Method of residuals and the AUC is calculated by trapezoidal rule. It was observed that there was a significant increase in Prazosin Cmax, t1/2, AUC and in the Group 3( prazosin microspheres)when compared to Group2 (Prazosin pure drug).The in vivo studies revealed that the oral bioavailability of the drug increased by more than twice by formulating it into microspheres. Further pre-clinical and clinical trials are required to use of the dosage form in human beings. Detailed in vivo studies in human beings need to be done to correlate the in vivo data within vitro values. Thus, the formulated floating microspheres seem to be a potential candidate as an oral gastro retentive controlled drug delivery system.

ACKNOWLEDGMENTS:

We acknowledge with thanks to Department of Science and Technology, New Delhi for sponsoring this project under (WOS-A) with file no. SR/WOS-A/LS-496/2012

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Received on 25.06.2016 Accepted on 19.07.2015

Asian J. Pharm. Tech. 2016; 6(4): 202-206.

DOI: 10.5958/2231-5713.2016.00030.1