RESEARCH ARTICLE TITLE -

FORMULATION DEVELOPMENT AND EVALUATION OF GASTRORETENTIVE

RAMIPRIL MICROBEADS.

AUTHORS –

SURAJ S. MULAJE*1, SHIVPRASAD M. BIRAJDAR2, ANILKUMAR CHINCHOLE3

(*1, 2, 3 –Department of Quality Assurance, Maharashtra college of pharmacy, Nilanga.

Dist.-Latur (M.S.) India)

ADDRESS FOR CORRESPONDENCE-

Department Of Quality Assurance, Maharashtra College of Pharmacy, Nilanga. Pin-

413521 Dist.- Latur (M.S.) India.

E-mail address- surajmulje24@gmail.com, Contact No.- 9730371957

ABSTRACT-

New era of Novel Drug Delivery System oriented towards increasing safety &

efficacy of existing drug molecule through novel concepts like oral drug delivery system. The

focus of current investigation was to formulate & evaluate for gastroretentive Ramipril

microbeads for better patient compliance & quality. Ramipril is antihypertensive agent &

angiotensin converting enzyme (ACE) inhibiter. It was selected as a model drug for

investigation because of its suitable properties like Elimination half- life- 9 to18 (hr.) &

Tmax 0.53 to 1(hr.). As a part of preformulation, different aspects like determination of

melting point, pH and solubility. λmax ( in 0.1N HCL & in phosphate buffer pH 7.4) &

calibration curve of Ramipril determined using UV- Visible spectroscopy. The infrared

spectrum of pure drug was also recorded & Model drug was assessed by carrying out

differential scanning calorimetry (DSC). The calcium alginate floating microbeads were

formulated by ionotropic gelation method. Calcium alginate microbeads formed as alginate

undergoes ionotropic gelation by calcium ions and carbon dioxide gas developed from the

reaction of carbonate salt and acetic acid which is permeated through the alginate matrix,

leaving gas bubbles or pores which provide the buoyancy followed by dispersing (1%) of

Ramipril into polymeric solution. Ramipril loaded calcium alginate floating microbeads of

different size were prepared for different batches(F1 to F6). Floating microbeads were

evaluated for different evaluation parameters & results were determined. stability study was

also carried out for Present study as per the ICH(Q1) guideline.

Keywords- gastroretentive , microbeads, Ramipril, stability, ionotropic gelation

INTRODUCTION -

Historically, the oral route of administration has been used the most for both

conventional and novel drug delivery systems. There are a variety of both physicochemical

and biological factors that come into play in the design of sustained release systems.

Sustained release systems include any drug delivery system that "achieves slow release of

drug over an extended period of time". If the system can provide some control, whether this

is a temporal or spatial nature, in other words, if the system is successful in maintaining

constant drug levels in the target tissue or cells, it is considered a controlled release system.

[1-5]

Dosage forms that can be retained in stomach are called gastroretentive drug delivery

systems (GRDDS). GRDDS can improve the controlled delivery of drugs that have an

absorption window by continuously releasing the drug for a prolonged period of time before

it reaches its absorption site thus ensuring its optimal bioavailability.[6,7] The approaches

that have been pursued to increase the retention of an oral dosage form in the stomach include

bioadhesive systems, swelling and expanding systems, High density systems and Low density

(Floating) systems. Floating drug delivery systems (FDDS) have a bulk density less than

gastric fluids and so remain buoyant in the stomach without affecting the gastric emptying

rate for a prolonged period of time. .[8] While the system is floating on the gastric contents,

the drug is released slowly at the desired rate from the system. After release of drug, the

residual system is emptied from the stomach. This results in an increased GRT and a better

control of the fluctuations in plasma drug concentration. The main aim of gastric floating

drug delivery system is to achieve better bioavailability by releasing of the drug from specific

system to its absorption window and the high level of patient compliance due to the ease of

administration and handling of these forms. Oral route has been the commonly adopted and

the most convenient route for the drug administration [9,10]

Ionotropic gelation is based on the ability of polyelectrolyte’s to cross link in the

presence of counter ions to form gelispheres. Since, the use of Alginates, Gellan gum,

Chitosan and Carboxymethyl cellulose are widely used for the encapsulation of drug and

even cells. In this method cationic or anionic polymer forms meshwork structure due to by

combining with the polyvalent cations or anions. The gelispheres are produced by dropping a

drug-loaded polymeric solution into the aqueous solution of polyvalent cations or anions.

Biomolecules can also be loaded into these gelispheres under mild conditions to retain their

three dimensional structure.

Ramipril is antihypertensive agent & angiotensin converting enzyme (ACE) inhibiter

which prevents the formation of angiotensin II from angiotensin I and exhibits pharmacologic

effects that are similar to Captopril. It competitively inhibits angiotensin-converting enzyme

(ACE) from converting angiotensin I to angiotensin II resulting in increased plasma renin

activity and reduced aldosterone secretion. It also increases bradykinin levels. By these

mechanisms, ramipril produces a hypotensive effect and a beneficial effect in Cardiac heart

failure. The present study aimed to formulation development and evaluation of

gastroretentive Ramipril microbeads by using sodium alginate as natural biodegradable

polymer & evaluation of microbeads for various properties. [11-14]

MATERIALS AND METHODS –

Materials-

Ramipril & HPMC K100M were obtained as a gift sample from Glenmark Pvt. Ltd,

Mumbai, India. Other excipients like Sodium alginate, Calcium carbonate, Calcium chloride

& acetic acid were purchased from Vishal-chem, Mumbai.

Methods –

Melting point:

The melting point of Ramipril was determined by melting point apparatus

(Chemiline).

Determination of pH and solubility:

The pH of Ramipril in distilled water checked by using digital pH meter and the

reading was noted. Solubility study of Ramipril was determined by adding excessive

dug in solvent. The solubility study was carried out in distilled water, 0.1N HCl and

pH 7.4 phosphate buffers and absorbance taken at room temperature and after 24 hrs.

Equilibrium solubility was determined by taking supernatant and analyzing it by using

UV-Visible double beam spectrophotometer.

Determination of λ-Max:

UV spectrum of Ramipril was carried out in 0.1N HCL and pH 7.4 Phosphate buffer

& Wavelength for maximum absorbance was recorded.

Calibration curve by UV spectroscopy:

A stock solution containing 100 μg/ml of Ramipril in 0.1N HCl was prepared. Then

suitable aliquots of stock solution (0.2-2ml) of Ramipril were transferred into 10 ml

volumetric flask and diluted with 10 ml 0.1 N HCl to get solution of different

concentration of 2-20 μg/ml. The absorbance of all solutions was measured against 0.1N

HCl as a blank at 210 nm. Using this concentration-absorbance data, Beers-Lambert

graph was plotted. This standard curve was used to estimate Ramipril release from

floating hydrogel microbeads formulations in the dissolution fluid. Same process was

respectively used to prepare calibration curve of Ramipril in phosphate buffer pH 7.4

Determination of IR spectrum

The infrared spectrum of Ramipril (pure drug) was recorded by potassium bromide

dispersion technique in which mixture of drug and potassium bromide was placed in

sample holder and an infrared spectrum was recorded using FTIR Spectrophotometer

(Jasco-4100, Japan). The identified peaks were compared with the principle peaks of

reported IR spectrum of Ramipril and the sample was authenticated.

Determination of thermal behaviour by differential scanning calorimetry (DSC):

Ramipril was assessed by carrying out thermal analysis. The inert atmosphere was

maintained by purging nitrogen gas throughout the experiment. The samples (5mg)

were carefully transferred and heated in a crimped aluminium pan for accurate results.

The samples were heated from 30°C-250°C at the rate of 10°C/min.

Fourier transform infrared spectroscopy (FTIR):

The infrared spectrum of physical mixture of sodium alginate: Ramipril (1:1), HPMC

K100M: Ramipril (1:1), Calcium carbonate: Ramipril (1:1) and mixtures were

recorded by potassium bromide dispersion technique. The identified peaks were

compared with the principle peaks of reported IR spectrum of Ramipril and respective

polymers.

Formulation of calcium alginate floating microbeads:

The calcium alginate floating microbeads were formulated by ionotropic gelation

method. Calcium alginate microbeads formed as alginate undergoes ionotropic

gelation by calcium ions and carbon dioxide gas developed from the reaction of

carbonate salt and acetic acid which is permeated through the alginate matrix, leaving

gas bubbles or pores which provide the buoyancy

formulation of drug loaded calcium alginate microbeads:

The weighed amount (0.5%) of HPMC K100M was dissolved in water. The solution

was heated to 80˚C to dissolve HPMC K100M. After cooling, weighed amount(0.5-

3%) of sodium alginate was added, stirred for 1 hr. using mechanical stirrer (Remi).

The weighed amount (1%) of Ramipril was dispersed into polymeric solution and

stirred for 30 min. Weighed amount (0.5-1%) of Calcium carbonate was added to the

solution and then allowed to stand in sonicator till the removal of entrapped air

bubbles. After sonication, this solution was added drop wise from the distance 2.5 cm

using syringe fitted with needle (23G) into 50 ml calcium chloride solution containing

10 % acetic acid at room temperature. Microbeads were left for curing for specified

time (15 min) and after curing, microbeads were collected by filtration and washed

twice with distilled water and allowed to dry at room temperature for 24 hrs.[27,

28,32,37,38,39]. Ramipril loaded calcium alginate microbeads were evaluated for

microbeads size, sphericity, percent entrapment efficiency, floating lag time, floating

duration, percent swelling index and percent cumulative drug release for 12 hrs.

Ingredient(%) F1 F2 F3 F4 F5 F6

RAMIPRIL 1 1 1 1 1 1

Sodium Alginate 0.5 1 1.5 2 2.5 3

Calcium carbonate 0.5 0.5 0.5 1 1 1

HPMC 0.5 0.5 0.5 0.5 0.5 0.5

Calcium carbonate 1 1 1 1 1 1

Acetic acid 10 10 10 10 10 10

Table No. 1 : Formulation batches

Evaluation of floating microbeads

Microbeads size and sphericity: [15]

The microbeads size was measured by randomly picked 10 microbeads on a glass slide

under polarized light. The size of microbeads was calculated by measuring the number of

divisions of theocular micrometer covering the length and width of microspheres. The

stage micrometer was previously used to standardize the ocular micrometer. Microbeads

sphericity was observed through optical microscope

Floating lag time and floating duration: [16,17]

Floating properties of dry microbeads were evaluated in a USPXXIII dissolution test

apparatus II (paddle type) filled with 900ml of 0.1N HCl (pH1.2). Paddle rotation speed

of 100 rpm was kept constant. Temperature was maintained at 37 ± 0.2°. Hundred

microbeads were placed in the media and floating lag time and floating duration of

microbeads was measured by visual observation

Percent drug content and Percent entrapment efficiency for calcium alginate

floating microbeads: [18]

An accurately weighed quantity of 2.5 mg microbeads were taken and crushed in

mortar with pestle and added to 2.5 ml pH 7.4 phosphate buffers. Solution was

sonicated for 1-2 hrs using sonicator. Then in to this solution 7.5 ml 10 % acetic acid

was added and further sonicated for 1 hr. The resultant dispersion was filtered through

Whatman filter paper of 0.45 μ size to obtain clear solution and analysed for drug

content at 210 nm using UV spectrophotometry (LabIndia3000+). The experiments

were done in triplicate and results were calculated

Percent entrapment efficiency: [19]

The encapsulation efficiency was calculated according to the following relationship.

%Entrapment efficiency= AQ/TQ × 100

Where,

AQ - Actual amount of drug found in the microbeads.

TQ- Theoretical amount of drug found in the microbeads.

Equilibrium Swelling Studies: [20,21,22]

The accurately weighed (0.25 gm) dried microbeads were placed in USP dissolution

apparatus II containing 900 ml, 0.1 N HCl (pH 1.2) maintained at 37±20C and allowed to

swell up to constant weight i.e. 12 hr. The microbeads were removed, blotted with filter

paper, and changes in weight were measured. The experiments were carried out in

triplicate. The degree of swelling (Swelling index) was then calculated from the formula,

Swelling index= (Wg−Wo)Wo×100

Where,

o Wo- Initial weight of microbeads

o Wg- Weight of swelled microbeads in the medium after 12hrs

In- vitro Dissolution Studies: [23]

In-vitro dissolution studies were performed for all the formulation using USP

dissolution test apparatus I (basket type). The dissolution study performed in

replicates and results expressed were the mean of three experiments. Analysis of data

was done by using 'PCP Disso V-3' software, India.

Stability studies as per ICH guidelines for 3 months [24,25]

Present study was carried out as per the ICH guidelines for the stability study of the new

drug substances and products. Stability of a drug has been defined as the ability of a

particular formulation, in a specific container, to remain within its physical, chemical,

therapeutic and toxicological specifications. The tablets from the selected were studied

for stability and kept under the three months. After one month, the formulation was

observed for changes in physical appearance and chemical analyses were done after every

one month up to three months. The results were illustrated.

RESULTS:

Melting point:

Table No.2: standard and practical melting point of Ramipril

Sample Name Standard Practical

Ramipril 106-1100C 108-1100C

pH study:

Table No. 3: pH of Ramipril in distilled water

Sample Name

pH

Ramipril

3.5-5

Solubility study:

Table No. 4: Solubility study of Ramipril at room temperature

Ultraviolet absorbance spectroscopy:

Table No. 5: λ maxvalues of Ramipril in 0.1N HCl and Phosphate buffer (pH 7.4)

Sr. No

Solvent λmax in nm

1 0.1N HCl 210

Ramipril Solubility (mg/ml) Solubility after 24Hrs.(mg/ml)

Distilled Water 3.5 4.1

0.1N HCl 4.0 4.5

PH 7.4 Phosphate buffer 1.5 2.5

2 Phosphate buffer (pH 7.4) 211

Figure No.1: U.V. absorption spectrum of Ramipril in 0.1N HCl

Figure No.2: U.V. absorption spectrum of Ramipril in Phosphate buffer 7.4

Calibration curves of Ramipril :

Figure No.3: Calibration curve of Ramipril in phosphate buffer pH 7.4

Figure No.4: IR spectrum of Ramipril

Differential Scanning Calorimetry

Figure No. 5 : Differential scanning colorimetry of Ramipril

Compatibility studies –

Figure No.6: FTIR Spectra for compatibility study: Ramipril and mixture

Size and Sphericity:

Table No. 6: Size and sphericity of batches of Ramipril loaded calcium alginate floating microbeads

Batch Code Microbeads size (μm)* Sphericity

F1 683.25±4.56 Spherical with porous F2 667.59±9.6

1Spherical with porous

F3 630.65±6.48

Spherical with porous

F4 865.54±4.56 Spherical but without porous F5 850.21±8.12 Slightly irregular with porous F6 871.45±4.36 Spherical but without porous

Floating lag time and floating duration:

Table No. 7: In-vitro buoyancy study

Batch Code Floating lag time (Sec) Floating duration (hrs.)

F1 <5 8-12

F2 <5

8-12

F3 <5 >12>12 F4 <5 >12

F5 <5 >12

F6 <5 >12

Percent entrapment efficiency:

Table No. 8: Percent entrapment efficiency study

*All values are expressed as Mean ±SD,

n = 3

Batch Code % Entrapment Efficiency*

F1

67.93±0.21

F2

71.98±0.60

F3 75.29±0.27

F4 66.78±0.20

F5 71.12±0.18

F6 73.14±0.17

Percent Swelling Index:

Table No. 9: Swelling index study

*All values are expressed as Mean ±SD, n = 3

In-vitro percent Cumulative Drug release:

Table No. 10: In-vitro percent cumulative drug release study

*All values are expressed as Mean ±SD, n = 3

Batch Code % swelling index

(After 12 hr.)*

F1 1.50±0.09

F2 1.65±0.07

F3 1.35±0.06

F4 2.55±0.19

F5 2.18±0.16

F

6

2.05±0.01

Batch Code Percent Cumulative Drug

release (after 12 hrs.)*

F1 85.14±0.22

F2 82.97±0.33

F3 81.1±0.56

F4 99.24±0.33

F5 95.66±0.35

F

6

90.05±0.12

Dissolution study:

Table No. 11: Average*(± SD) cumulative percent drug release from calcium alginate floating microbeads (F1 to F6)

Time (hr) F1 F2 F3 F4 F5 F6

0 0 0 0 0 0 0

1 2.21±0.61 2±0.12 1.92±0.21 3.77±0.32 3.28±0.12 2.57±0.00

2 8.72±0.11 8.15±0.21 8.15±0.21 11.57±0.23 10.67±0.33 9.15±0.44

3 15.36±0.21 14.4±0.42 14.55±0.32 19.74±0.22 18.43±0.33 16.2±0.54

4 22.59±0.33 20.91±0.20 21.13±0.21 27.15±0.24 25.89±0.43 23.87±0.32

5 28.85±0.3 27.53±0.24 27.59±0.68 35.37±0.38 33.53±0.44 31.07±0.53

6 35.37±0.55 34.47±0.21 33.37±0.53 42.97±0.75 41.44±0.98 37.88±0.54

7 42.78±0.95 40.73±0.11 40.48±0.72 50.93±0.61 49.01±1.19 45.42±0.22

8 49.59±0.42 47.68±0.33 47.92±0.84 59.28±0.55 57.14±0.58 53.13±0.45

9 56.49±0.22 54.66±0.43 54.39±0.84 67.14±0.48 64.81±0.82 60.27±0.55

10 63.37±0.19 60.44±0.30 61.8±0.77 76.01±0.25 73.14±0.87 67.53±0.38

11 77.86±0.19 70.02±0.26 71.5±1.17 85.29±0.74 84.31±1.07 79.96±0.26

12 85.14±0.22 82.97±0.33 81.1±0.56 99.24±0.33 95.66±0.35 90.05±0.12

*All values are expressed as Mean ±SD, n = 3

Figure No.7: Dissolution profiles of Ramipril loaded calcium alginate floating microbeads (F1 to F6)

Stability study of selected batch of microbeads

Table No. 12: Effect of temperature and humidity on selected batch of microbeads

Formulation Initial 40ºC ± 2 ºC at 75% ± 5% RH CompatibilityF4 F4 1 Month 2Month 3 MonthColour Light

YellowLight Yellow Light

YellowLight Yellow

Complies

Odour No No No No Complies% Entrapment efficiency

66.78±0.20 66.80±0.12 66.85±0.22 66.85±0.56 Complies

% cumulative drug release

99.24±0.33 99.29±0.24 99.35±0.12 99.39±0.11 Complies

% Swelling index

2.55±0.19 2.51±0.15 2.50±0.56 2.51±0.45 Complies

Here no any significance difference was found to be in initials and after three month in stability data, so it can be concluded that microbeads showed good stability after three months.

DISCUSSION

The identity of Ramipril was confirmed by carrying out pH study in distilled water &

solubility at room temperature.(Table no.3 &4) Analytical study such as λ max values of

Ramipril in 0.1N HCl and Phosphate buffer (pH 7.4) was also carried out.(Table no.5, Fig.1&2

respectively) Calibration curve of Ramipril in phosphate buffer pH 7.4 followed Beer-Lamberts

law.(Fig.3) FTIR spectrum of pure drug was also carried out.(Fig.4)

Differential scanning colorimetry of Ramipril also showed satisfied results.(Fig.5)

Drug-polymer interactions were studied by FTIR spectrophotometer and no interaction was

found.(Fig.6) Ramipril loaded microbeads were formulated by using ionotropic gelation

technique for 6 batches(Table no.1).The formulated microbeads were evaluated for

microbeads size & sphericity, (Table no. 6), Floating lag time and floating duration (Table

no.7), and percent entrapment efficiency (Table no.8), percent swelling index, (Table no.9)

and in-vitro percent cumulative drug release(Table no.10). The concentration of sodium

alginate had significant impact on drug particle size was observed. 3.5 % w/v sodium alginate

polymeric solution was too viscous to pass through needle therefore microbeads could not

form. Hence sodium alginate concentration from 0.5 to 3 % w/v which forms desired

viscosity to obtain desired microbeads. HPMCK100M was selected in combination with

sodium alginate to decreases the burst release and to sustain the release. Also good floating

properties i.e. increased floating duration more than 12 hrs as seen in formulation F4. F5 and

F6 respectively.(Table no.11 & Fig. no.7) CaCO3:polymer ratio, fulfilled maximum

requisites because of better drug entrapment efficiency and % cumulative sustained release of

the drug therefore-F4formulation was selected as best batch. Here no any significance

difference was found to be in initials and after three month in stability data, (Table no.12) so

microbeads showed good stability after three month results of selected batch & proved the

porous nature of microbeads.

CONCLUSION:

Ionotropic gelation can be promising method to formulate floating microbeads of

calcium alginate containing Ramipril. Percent entrapment efficiency of selected batch found

66.78 % also the percent cumulative drug release was found to be 99.24 % and good floating

>12 hrs. In-vitro drug release study of F4 indicated that Ramipril was released in controlled

and sustained manner up to 12 hours. No significance difference was found to be in initials

and after one two and three months in stability data, so it can be concluded that microbeads

showed good stability after three months.

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