FORMULATION AND EVALUATION OF GASTRORETENTIVE HYDROCHLOROTHIAZIDE FLOATING MICROSPHERES: STATISTICAL ANALYSIS

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ABOUT AUTHORS:
Y. Veda Pravallika* , K.Rajyalakshmi
Bapatla college of pharmacy,
Bapatla, Andhra Pradesh
yadlapallivedapravallika@gmail.com

ABSTRACT:
The main objective of this work is to formulate gastroretentive floating microspheres of hydrochlorothiazide and to study the effect of formulation variables like drug to polymer ratio and concentration of polymer dispersion. The hydrochlorothiazide floating microspheres were formulated by using orifice-ionic gelation technique. These microspheres were formulated by using sodium alginate as sustained release polymer, sodium bicarbonate as gas generating agent and calcium chloride as a cross linking agent. The rheological properties of the polymer dispersion were studied. The microspheres were formulated by different drug to polymer ratio’s at various concentrations of sodium alginate dispersion. Thus formulated microspheres were evaluated for floating behaviour, drug entrapment efficiency, drug content, micromeritic properties, particle size, swelling index, invitro drug release studies and release kinetics. The better formulation was subjected for stability studies and SEM analysis. The effect of formulation variables on the entrapment efficiency and release rate constant was studied by statistical analysis. The hydrochlorothiazide microspheres formulated with 1:3 drug to polymer ratio and 3.5% sodium alginate dispersion concentration was selected as optimized formulation based on the results.

REFERENCE ID: PHARMATUTOR-ART-2386

INTRODUCTION:
Gastro retentive drug delivery systems (GRDDS) are suitable for drugs with an absorption window in the stomach or the upper small intestine^[1], for drugs which act locally in the stomach^[2] and for drugs that are poorly soluble or unstable in the stomach and for drugs that are poorly soluble or unstable in the intestinal fluid^[3]. Various approaches have been made to promote the retention of an oral dosage form in the stomach, like floating drug delivery systems (FDDS), expanding and swelling systems, bioadhesive systems, high density systems, modified shape systems and other delayed gastric emptying devices. FDDS or hydrodynamically balanced systems have bulk density lower than gastric fluid and thus remain buoyant in the stomach without affecting the gastric emptying rate for prolonged period of time^[4].

In multi particulate drug delivery systems, dosage form of drug is devided into several discrete delivery entities in contrast to single entity dosage forms. The microspheres are having high degree of dispersion in the digestive tract there by decreases absorption variability and avoid dose dumping. The use of microspheres in pharmaceuticals have a number of advantages like taste and odour masking, protection of drugs against environment, production of sustained release, controlled release and targeted medications^[5].

Hydrochlorothiazide is one of the best thiazide diuretic. It acts orally and the dosage used for treatment of congestive heart failure and hypertension ranges from 25 to 50 g daily alone or combination with other antihypertensive drugs upto 100 mg if necessary. Hydrochlorothiazide has half-life approx. 2.5 hours and oral bioavailability 70%^[6]. It is only absorbed from the upper part of the duodenum and once it passes this absorption site, little or no absorption takes place^[7].

Thus The objective of the present study was to formulate the floating microspheres of hydrochlorothiazide in order to retain the formulation in the stomach for better absorption and study the effect of polymer, polymerconcentration and viscosity of polymer dispersion on drug release behaviorand the buoyancy properties and Micromeritic properties of prepared formulations.

MATERIALS AND METHODS:

Materials
Hydrochlorothiazide is gift sample from NATCO pharmaceuticals Pvt. Ltd, Sodium alginate, sodium bicarbonate, Calcium chloride and glacial acetic acid collected from SD fine chemicals Pvt. Ltd, Mumbai.

Methods

Drug-excipient compatability studies by FT-IR:
The physico-chemical compatability between the selected drug (Hydrochlorothiazide) and the excipients used un the research was tested by IR spectroscopy. The samples were scanned under diffuse reflectance using KBr pellet technique. The spectra were recorded for pure drug, pure polymer and drug-polymer mixture using FT-IR. Samples were prepared in KBr desks (2mg sample in 200mg KBr). The scanning range was 400-4000 cm^-1 and the resolution was 2 cm^-1.

Preparation of microspheres:
The floating microspheres containing Hydrochlorothiazide were prepared by orifice ionic gelation technique. Sodium alginate and gas forming agent sodium bi carbonate was dispersed in distilled water to form a homogenous polymer mixture. The drug hydrochlorothiazide was added to the polymer dispersion and mixed thoroughly on a magnetic stirrer to form a homogenous dispersion. The gelation medium was prepared by dissolving calcium chloride in 1.5% glacial acetic acid solution. The homogenous alginate solution was extruded using 24G syringe needle into the gelation medium. The distance between the edge of the needle and surface of gelation medium was about 10 centimetres. The gel microspheres formed were left in the solutionfor 30 minutes with gentle stirring room temperature to improve mechanical strength. After that the microspheres was collected and washed with distilled water twice, dried at 60⁰c until get constant weight of microspheres.^[8] The composition and conditions maintained during the preparation of microspheres were showed in table-1.

Table-1: Composition of Hydrochlorothiazide floating microspheres

S. No.	Formulation code	Drug (mg)	Polymer (mg)	Concentration of sodium alginate (%)
1	F1	250	250	3.5
2	F2	250	500	3.5
3	F3	250	750	3.5
4	F4	250	1000	3.5
5	F5	250	1250	3.5
6	F6	250	750	3.0
7	F7	250	750	4.0
8	F8	250	750	4.5

Studies on influence of sodium alginate dispersion viscosity:
Accurately weighed quantity of Sodiumalginate and sodium bicarbonate was dissolved in distilled water to get 3, 3.5, 4.0, 4.5 and 5% w/v solutions. The viscosity of the prepared polymer solutions were measured by using Brook field viscometer at shear rate of 0.13 to 0.66 and torque 10-50%. The viscosity of the polymer solution was noted as per Newtonian, Bingham and power law equations.

Newtonian equation: 
σ = ηγ
Where η = Viscosity in cps & γ = Shear rate in sec^-1.

Bingham equation:
σ = σ_γ + η_pγ
Where  η_p = Plastic viscosity & σ_γ = Bingham yeild stress.

Power law: 
σⁿ = ηγ
Where η = Viscosity coefficient and Exponent n= Index of pseudo plasticity.

Micromeritic properties of hydrochlorothiazide floating microspheres^[9]:
The flow properties of prepared microspheres were investigated by measuring the bulk density, tapped density, Carr’s index Hausner’s ratio. The bulk and tapped densities were measured in a 10 ml graduated measuring cylinder. The sample contained  measuring cylinder was tapped mechanically by means of constant velocity rotating cam. The initial bulk volume and final tapped volume were noted from which, their respective densities were calculated.

Drug content and entrapment efficacy of floating microspheres^[10]:
10mg of drug equivalent formulations was dissolved in 100 ml of 0.1N HCl.  The samples were assayed for drug content by UV- spectrophotometer by making suitable dilutions at 272 nm and the drug content was calculated. The percentage entrapment efficiency was calculated by using following formula.

% Drug entrapment = (Calculated drug content/ Theoretical drug content) x 100.

In vitro drug release studies^[11]:
The drug release studies were carried out using  dissolution apparatus USP type II. A weight of floating microspheres corresponding to 25mg of drug was placed in basket. The dissolution medium used was 900 ml of 0.1N hydrochloric acid at 37°C and 100 rpm. At specific time intervals, 5 ml aliquots were withdrawn and analysed by UV spectrophotometer at the respective λmax value 272 nm after suitable dilution against suitable blank. The withdrawn volume was replaced with an equal volume of fresh 0.1 N hydrochloric acid.

In vitro drug release kinetic studies:
In order to study the exact mechanism of drug release from the alginate microspheres, drug release data was analysed according to zero order, first order, Higuchi and Korsmeyer – Peppas equations. The order of drug release from alginate microspheres was described by using zero order kinetics or first order kinetics. The mechanism of drug release from matrix systems was studied by using Higuchi equation, and Korsmeyer drug release.

Floating behavior^[12]:
Three hundred milligrams of the microspheres were placed in 900 ml of 0.1 N hydrochloric acid. The mixture was stirred at 100 rpm in a dissolution apparatus for 12 h. After 12 h, the layer of buoyant microspheres was pipetted and separated by filtration. Particles in the sinking particulate layer were separated by filtration. Particles of both types were dried in a desiccator until constant weight was obtained. Both the fractions of microspheres were weighed and buoyancy was determined by the weight ratio of floating particles to the sum of floating and sinking particles.

% Buoyancy = [Wf / Wf + Ws)] x 100

where Wf and Ws are the weights of the floating and settled microspheres.

Swelling index^[13]:
50 mg of microspheres were weighed and transferred to a petri plate containing 10ml of 0.1N Hydrochloric acid maintained at 37⁰c. The microspheres were withdrawn at 1hr intervals upto 4 hours. The swelling index was calculated by using the Formula.

Swelling index = (W_t – W₀ / W_t) x 100

Where W₀ = Initial weight & W_t = weight of microspheres at time t.

Particle size analysis^[14]:
The particle sizes of the drug loaded formulations were measured by an optical microscope fitted with an ocular and stage micrometer and particle size distribution was calculated. The Olympus model (SZX-12) having resolution of 10 xs was used for this purpose. The instrument was calibrated at 1unit of eyepiece micrometer was equal to 1/10mm (10 μm). In all measurements at least 100 particles in five different fields were examined . Each experiment was carried out in triplicate.

Scanning electron microscopy analysis (SEM)^[15]:
The shape and surface characteristics were determined by scanning electron microscopy. A working distance of 20nm, a tilt of zero-degree and accelerating voltage of 15kv were the operating parameters. Photographs were taken within a range of 50-100 magnifications.

Stability studies^[16]:
The optimized formulation was kept at room temperature for 3 months. Then the microspheres are evaluated for percent drug entrapment and dissolution study.

Statistical analysis^[17]:
All the means are presented with their standard deviation (mean± standard deviation). One way Anova and un paired student’s t-test was used to compare the effect of drug to polymer ratio and concentration of sodium alginate dispersion on the entrapment efficiency and release rate constant. A p value <0.05 was considered significant.

Table-2: Rheological properties of polymer dispersions

S.No.	Concentration of polymer dispersion (%)	Viscosity of polymer dispersion (cps)	Correlation coefficient
			Newtonian	Bingham	power law
1	3	436.15	0.9965	0.9462	0.9521
2	3.5	567.32	0.9979	0.9512	0.9310
3	4	652.40	0.9976	0.9126	0.9462
4	4.5	736.54	0.9943	0.9360	0.9517
5	5	861.30	0.9916	0.9398	0.9623

Fig-1: Rheogram of 3.5% sodium alginate dispersion

Fig-2: FT-IR graph of Hydrochlorothiazide

Fig-3: FT-IR graph of Sodium alginate

Fig-4: FT-IR graph of formulation containing Hydrochlorothiazide, sodium alginate and sodium bicarbonate

Table-3: Micromeritic properties of the Hydrochlorothiazide floating microspheres

Formulation	Bulk density (gm/ml)	Tapped density (gm/ml)	Hausner’s ratio	Carr’s index	Angle of repose (0)
F1 (1:1)	0.2339±0.23	0.2843±0.19	1.21±0.13	17.72±17	29.25±0.13
F2 (1:2)	0.2613±0.42	0.3134±0.33	1.19±0.19	16.64±23	27.34±0.44
F3 (1:3)	0.3148±0.17	0.3675±0.19	1.16±0.24	14.34±26	24.77±0.24
F4 (1:4)	0.3793±0.33	0.4324±0.35	1.13±0.33	12.28±37	22.93±0.32
F5 (1:5)	0.4496±0.28	0.5017±0.24	1.11±0.32	10.38±29	20.32±0.27
F6 (3%)	0.2873±0.13	0.3439±0.17	1.19±0.14	16.45±0.19	27.79±0.23
F7 (4.0%)	0.3379±0.37	0.3852±0.33	1.14±0.12	12.27±0.15	22.78±0.12
F 8(4.5%)	0.3624±0.43	0.4012±0.45	1.10±0.09	9.67±0.11	21.02±0.33

Table-4: Physico parameters of the Hydrochlorothiazide floating microspheres

Formulation	Dug entrapment efficiency (%)	Drug content (%)	Total floating time (hours)	Swelling index (%)	Average particle size (µm)
F1 (1:1)	53.84±0.36	26.92±0.34	>12	69.15±0.46	435.20±1.78
F2 (1:2)	76.55±0.44	25.51±0.42	>12	72.36±0.38	560.98±1.57
F3 (1:3)	92.30±0.32	23.07±0.33	>12	75.18±0.43	750.30±1.33
F4 (1:4)	92.15±0.27	18.43±0.25	>12	82.13±0.45	930.15±1.69
F5 (1:5)	91.54±0.36	15.25±0.35	>12	84.27±0.35	1036.45±1.54
F6 (3%)	84.59±0.42	21.14±0.39	>12	71.83±0.53	679.35±1.92
F7(4.0%)	91.79±0.28	22.94±0.32	>12	78.35±0.45	840.28±1.75
F8 (4.5%)	90.50±0.27	22.62±0.31	>12	80.20±0.39	915.40±1.56

Fig-5: Drug release profile plot of Hydrochlorothiazide floating microspheres

Fig-6: First order profile plot of Hydrochlorothiazide floating microspheres

Fig-7: Peppas plot of Hydrochlorothiazide floating microspheres

Table-5: Kinetic profile of all formulations

Formulation	Zero order (r2)	First order (r2)	Higuchi (r2)	Peppas (r2)	n	Release rate constant K (hr-1)	T50% (hr)	T90% (hr)
F1 (1:1)	0.8586	0.9750	0.9913	0.9980	0.3532	0.3901	1.77	5.90
F2 (1:2)	0.8205	0.9861	0.9941	0.9946	0.4049	0.2279	3.04	10.10
F3 (1:3)	0.8231	0.9779	0.9873	0.9981	0.4384	0.1963	3.53	11.73
F4 (1:4)	0.9146	0.9924	0.9963	0.9980	0.4402	0.1312	5.28	17.55
F5 (1:5)	0.9483	0.9960	0.9896	0.9989	0.4667	0.1208	5.73	19.06
F6 (3%)	0.8748	0.9880	0.9973	0.9994	0.4307	0.3291	2.10	6.99
F7 (4.0%)	0.9320	0.9916	0.9936	0.9990	0.4501	0.1734	3.99	13.28
F8 (4.5%)	0.9658	0.9925	0.9822	0.9993	0.4809	0.1208	5.73	19.06

Stability studies:

Table-6: Drug release profile of  optimized formulation

Time (hours)	Cumulative percentage drug release
	Before storage	After storage
0	0.000	0.000
1	31.298±0.324

 

PharmaTutor (Print-ISSN: 2394 - 6679; e-ISSN: 2347 - 7881) Volume 4, Issue 1 Received On: 31/08/2015; Accepted On: 09/09/2015; Published On: 01/01/2016 How to cite this article: Pravallika YV, Rajyalakshmi K; Formulation and evaluation of Gastroretentive hydrochlorothiazide Floating Microspheres: Statistical Analysis; PharmaTutor; 2016; 4(1); 28-36