Formulation and In-vitro characterization of Gelatin Micro spheres loaded with Lisinopril dihydrate

Sudha Talasila*1, Yamini Pendyala2, Deepthi Madhu3, Dr.K.L.Senthil kumar.
1,2 Department of Pharmaceutics,
Padmavathi College of Pharmacy and Reserch institute,
periyanahalli, Tamilnadu, India.
3 Department of Pharmaceutics, Gitams university,
Visakhapatnam, India.

Aim of our present study was to formulate the Gelatin Microspheres Loaded with Lisinopril Dihydrate by using Co-acervation phase separation technique for control prolonged release of drug. Micro-particulate drug delivery of Lisinopril Dihydrate was prepared by using a blend of gelatin-carbopol 934P NF and Gelatin-Sodium alginate as release retardant.Lisinopril Dihydrate Microspheres were formulated by using different drug, gelatin-carbopol and gelatin-sodiumalginate in 6 batches was F1, F2, F3, F4, F5 & F6. All the formulations were investigated for various evaluation parameters like particle size, Bulk density, flow behavior, Entrapment efficiency, percentage yield and in vitro drug release etc. All the formulations showed good flow behavior. SEM study revealed that the spheres were almost spherical in shape with smooth surface. In-vitro drug release study showed that by increasing the polymer concentration the drug release of all the formulations were gradually decreased and the optimized formulation (F6) was able to sustain the drug release for 24 hours. So, it was concluded that gelatin microspheres loaded with Lisinopril Dihydrate can be prepared by Coacervation phase seperation  technique and used for sustaining the drug release for prolonged period of time.

Reference Id: PHARMATUTOR-ART-1262

Micro sphere technology has been studied extensively for the sustained delivery of therapeutic agents1-3. Control release system includes any drug delivery system that releases the drug over an extended period of time. If it is unsuccessful in this, it is only extents the duration of actions. Advancement in drug delivery could come from innovative improvement to existing drug delivery system. This is because of reducing frequency of administration, sustained release dosage form and ambulatory patient compliance. If the system is successful in maintaining a constant drug level in the blood or target tissue4-5.The objectives in designing a controlled release system are to deliver the drug at a rate necessary to achieve and maintain a constant drug blood level. This rate should be analogous to that achieved by continuous intravenous infusion where a drug is provided to the patient at a rate just equal to its rate of elimination. This complies that the rate of delivery must be independent of the amount of drug remaining in the dosage form and constant over time. That is release from the dosage form should follow zero-order kinetics6.

Lisinopril is a potent, competitive inhibitor of angiotensin-converting enzyme (ACE) and is used to treat high blood pressure (hypertension), heart failure and to improve survival after heart attack. Lisinopril, one of the few ACE inhibitors that is not a pro-drug, compete with ATI for binding to ACE and inhibits and enzymatic proteolysis of AT-I to AT-II. Lisinopril, a synthetic peptide derivative, is chemically described as (S)-1-[N2-(1-carboxy-3- phenylpropyl)-L-lysyl]-L-proline dihydrate7.Lisinopril inhibits angiotensin-converting enzyme (ACE) in human subjects and animals. ACE is a peptidyl dipeptidase that catalyzes the conversion of angiotensin I to the vasoconstrictor substance, angiotensin II. Angiotensin II also stimulates aldosterone secretion by the adrenal cortex.
The beneficial effects of Lisinopril in hypertension and heart failure appear to result primarily from suppression of the renin-angiotensin-aldosterone system. Inhibition of ACE results in decreased plasma angiotensin II which leads to decreased vasopressor activity and to decrease aldosterone secretion. The latter decrease may result in a small increase of serum potassium. In hypertensive patients with normal renal function treated with Lisinopril alone for up to 24 weeks, the mean increase in serum potassium was approximately 0.1mEq/L however, approximately 15% of patients had increases greater than 0.5mEq/L and approximately 6% had a decrease greater than 0.5mEq/L. In the same study, patients treated with Lisinopril and hydrochlorothiazide for up to 24 weeks had a mean decrease in serum potassium of 0.1mEq/L; approximately 4% of patients had increases greater than 0.5 mEq/L and approximately 12% had a decrease greater than 0.5mEq/L.

Materials and Methods

Materials Lisinopril Dihydrate (Watson Pharma Pvt. Ltd, Thane.), Gelatin B IP grade (Supra pharmaceuticals Pvt. Ltd, India), Carbopol 934P NF grade (Mclareu Bio-tech (P) Ltd, Hosur), Sodium alginate, formaldehyde, Isopropyl alcohol (S.D.Fine chemicals ltd, India).

Method of Preparation
Gelatin, gelatin-carbopol and gelatin-sodium alginate mixture containing Lisinopril dihydrate Microspheres were prepared by “coacervation phase separation technique” utilizing temperature change. In this method weighed amount of Gelatin was dissolved in 10ml of water which was previously heated to 50?C, to this Lisinopril was added and stirred approximately at 300 rpm with the help of magnetic stirrer for 15 mins to get a stable dispersion.  The dispersion was poured drop wise into the 100ml of seasome oil which was also previously heated to 50? C on a water bath. The mixture was stirred with a help of magnetic stirrer for 2 hrs at 300rpm at room temperature. At the end of 2 hrs cross linking agent formaldehyde 0.5ml was added to the dispersion medium and stirring was continued for next 30 mins8. Finally it was kept in refrigerator for 24 hrs to ensure the rigidization of microspheres. Then the microspheres were washed with propylene glycol to wash off excess oil. This Procedure was followed to prepare 6 batches of Lisinopril microspheres with different ratios of gelatin and gelatin-carbopol, gelatin-sodium alginate mixtures.

Evaluation of Microspheres
Particle size analysis

The Particle size analysis was carried out by using optical microscopy. About 200 Micro spheres were selected randomly and their size was determined by using optical microscope fitted with standard micrometer scale.

Micromeritic properties of microspheres
Angle of Repose

A glass funnel is held in place with a clamp on a ring support over a glass plate. The glass plate is placed on a stand. Approximately 100 mg of a particle is transferred into funnel keeping the orifice of the funnel blocked by the lower thumb. As the thumb is removed the particles are emptied from funnel, and the angle of repose is determined.

Bulk Density
Bulk density is defined as the weight per unit volume of material.Interparticulate interaction is one of the most important parameter that affects the bulk and flow characteristics ofpowder9.The test can provide a gross measure of particle size and dispersion which can affect material flow consistency and reflect packaging quantity.

Morphological Examination
The texture of surface and shape of the microspheres were investigated by using Scanning electron microscopy (JSM 5610 LV SEM, JEOL, Datum Ltd, Tokyo, Japan). The sample (optimized formulation F6) was spread on stub and coated for 120 s with a layer of gold using a sputter coater. Afterwards, the stub containing the sample was placed in the scanning electron microscope chamber. The scanning electron photomicrograph image was taken at the acceleration voltage of 10 kV, chamber pressure of 0.6 mm Hg.

Production yield (% w/w)
The percentage yield of each batch was calculated on weight basis with respect to the weight of starting material. Thoroughly dried microspheres were collected and weighed accurately. The percentage yield was then calculated using formula

              % Yield = Mass of microspheres obtained  ×100
                           Total weight of drug and polymer

Entrapment efficiency
The entrapment efficiency of the prepared formulations was determined by the method of extraction of the drug present in the microsphere. The dried microspheres (20mg) were taken and extracted in 100 mL of phosphatebuffer (pH 6.8) for 4 hours. Then the dispersion of microspheres was sonicated for 30 min (Imeco Sonifier, Imeco Ultrasonics, India) and the solution was filteredthrough a 0.45 μm filter. Finally, the polymeric debris was washed twice with fresh solvent (phosphate buffer) to extract any adhering drug. The drug content of filtrateand washings was determined spectrophotometrically at 258 nm (UV-2450, Shimadzu, Japan).

Encapsulation efficiency was calculated using the following formula

Theoretical drug content =   Amount of drug in bead  ×100
                                         Amount of bead taken

Encapsulation efficiency =   Practical drug content    x 100
                                     Theoretical drug content

Drug Polymer Interaction (FTIR) Study
FTIR spectra of pure drug, pure polymers and formulations containing both drug and polymers were performed to study the drug polymer interaction. FTIR study was performed by using Fourier transformed infrared spectrophotometer (Prestige-21, Shimadzu, Japan).

In vitro drug release study
In-vitro release profile of Lisinopril Dihydrate from the microspheres was examined in phosphate buffer (pH 7.4) using USP (XXI) six stage dissolution rate test apparatus I (Thermolab®, Mumbai, India). Microspheres equivalent to 20 mg of drug was suspended in dissolution medium at 50 rpm and 37 ± 0.5°C. An aliquot of 5 ml was withdrawn periodically at intervals of one hour and same volume of fresh medium was replaced. The samples were filtered through Whatman filter paper and analyzed spectrophotometrically at 258 nm for amount of drug released10, 11.

The formulations were stored in a oven at 37 ± 2?C and 65% RH for a period of six weeks. The samples were analyzed for drug content every week by spectrophotometer at 258nm.



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