INTRODUCTION:
Background
Pulsatile
drug delivery system is
defined as the
rapid and transient
release of certain
amount of molecules
within a short
time period immediately after
predetermined off-release periods i.e. lag time1. Ramipril is a prodrug belonging to the
angiotensin-converting enzyme (ACE) inhibitor class of medications. It is
metabolized to ramiprilat in liver and, to a lesser
extent in kidneys. Ramiprilat is a potent,
competitive inhibitor of ACE, the enzyme responsible for the conversion of
angiotensin I (ATI) to angiotensin II (ATII). ATII regulates blood pressure and
is a key component of the renin-angiotensin-aldosterone
system (RAAS).
Ramipril may be used in the treatment
of hypertension, congestive
heart failure, nephropathy,
and to reduce
the rate of
death, myocardial infarction and
stroke in individuals at high risk of cardiovascular events2.
In present study Ramipril
granules and Ramipril albumin microspheres were
formulated as pulsincap for better patient
compliance, to reduce dose frequency, reduced first pass metabolism for
sustained release, for better absorption, and release of drug over
predetermined period.
METHODS AND MATERIALS:
Ramipril was obtained as gift sample from vee laboratories, kailer, Dist. Solan (H.P). Egg albumin was procured from Fisher
Scientific. Size 1 capsules were procured from Gowtham
distributors; HPMCK4M was obtained as gift sample from MMC
Healthcare Chennai. All other chemicals used were of analytical grade.
Formulation
of Pulsatile Device of Ramipril2-10
Preparation of Immediate release granules2,3,13:
Granules of Ramipril were made by wet granulation method. Ramipril, Sodium starch glycolate,
Microcrystalline cellulose were weighed accurately and
blended homogeneously. Polyvinyl pyrollidone was
dissolved in isopropyl alcohol and mixed with the powder blend to get a
coherent mass. The mass was passed through sieve no 22. The
formulation of immediate release granules in Table 1.
Table:
1 Formulation of Immediate Release Granules
|
S. No |
Ingredient |
G1 (mg) |
G2 (mg) |
G3 (mg) |
G4 (mg) |
|
1 |
Ramipril |
500 |
500 |
500 |
500 |
|
2 |
Sodium starch glycolate |
86 |
178 |
272 |
386 |
|
3 |
Microcrystalline cellulose |
180 |
180 |
180 |
180 |
|
4 |
PVP K30 |
3.6 |
3.6 |
3.6 |
3.6 |
Preparation of Ramipril Albumin
Microspheres4
Ramipril albumin microspheres were prepared by
single emulsion polymerization technique. 100ml of liquid paraffin was mixed
with 0.4% w/v span 60, Stirred and heated to 70ºC. Drug and polymer were
dissolved in methanol and phosphate buffer respectively and mixed together.
This mixture was then added drop wise to liquid paraffin using hypodermic
syringe with continuous stirring at 600 rpm. 0.25ml of glutaraldehyde
was added and stirred for 3 hours. Microspheres were separated by decantation,
washed 6 times with petroleum ether and dried at room temperature. The
microspheres were stored in a dessicator. The
formulation of microspheres is given in Table 2.
Table: 2 Formulations of
Microspheres
|
S. No. |
Drug (mg) |
Polymer (mg) |
|
F1 |
500 |
500 |
|
F2 |
500 |
1000 |
|
F3 |
500 |
1250 |
|
F4 |
500 |
1500 |
|
F5 |
500 |
2000 |
Preparation
of Cross- Linked Gelatin Capsules5, 6
The “1” sized hard gelatin capsules about
100 in number were taken. The body of the capsules was placed on a wire mesh.
25ml of 15%v/v formaldehyde was taken into a desiccator
and potassium permanganate was added to it to generate formalin vapours. The wire mesh along with the body was kept in the dessicator. The reaction was carried out for 12 hours,
after which the body were removed and dried at 50ºC for 30 minutes to ensure
completion of reaction between gelatin and formaldehyde vapour.
They were dried at room temperature to facilitate removal of residual
formaldehyde.
Preparation
of Hydrogel Plug6
Plug for sealing the capsule body was
prepared by compressing HPMCK4M granules using 9mm punches on rotary
tablet press.
Designing
of Pulsincap6
The pulsincap is
similar in appearance to a hard gelatin capsules, but the body is water
insoluble. Microspheres equivalent to 2.5mg of Ramipril
were accurately weighed and filled into the formaldehyde treated body. The
capsule body containing the microspheres was plugged with hydrogel
plug and the capsule cap was filled with Ramipril
granules equivalent to 2.5mg and sealed over the body.
Evaluation
of Ramipril Immediate Release Granules
Angle
of repose 9, 10
Angle of repose was determined using funnel
method. The frictional forces can be measured by Angle of repose. θ = tan-1(h
/ r) where, θ is the angle of repose, h is the height in cm and r is the
radius in cm.
Compressibility
index
It is an important measure that can be obtained
from the bulk and tapped densities. The percentage compressibility of the bulk
drug was determined using the following formula.
I = DT – Db / DT x 100
Where,
I is the
Compressibility index, DT is
the tapped density
of the powder
and DB is the bulk density of the
powder.
Hausner’s ratio
It indicates the flow properties of the
powder and is measured by the ratio of tapped density to the bulk density. H =
DT / Db Where, H is the Hausner’s ratio DT is the
tapped density of the powder and Db is the bulk density of the powder.
Drug
content2
Granules were dissolved in a small quantity
of methanol and the volume was made up to 100ml with phosphate buffer pH 7.4.
It was stirred for 12hrs. After stirring the solution was filtered through whatman filter paper, the absorbance was measured
spectrophotometrically at 210nm after suitable dilution and the drug content
was calculated.
Evaluation of Microspheres5,6,10
Particle
Size Analysis
The size
was measured using an optical microscope and the mean particle size was
calculated by measuring 100 particles with the help of a calibrated ocular
micrometer.
Angle
of repose
Angle of repose was determined using funnel
method. The frictional forces can be measured by Angle of repose. θ = tan-1(h
/ r) where, θ is the angle of repose, h is the height in cm and r is the
radius in cm.
Percentage yield
The
prepared microspheres were collected and weighed. The yield was calculated by
dividing the measured weight by the total weight of all non-volatile
components. The percentage yield of microspheres was calculated as follows.
% Yield = Weight of microsphere
×100
Theoretical weight of drug and
polymer
Drug content2
Drug loaded
microspheres were dissolved in a small quantity of methanol and the volume was
made up to 100ml with phosphate buffer pH 7.4. It was stirred for 12hrs. After
stirring the solution was filtered through whatman
filter paper and the absorbance was measured spectrophotometrically at 210nm
after suitable dilution and the drug content was calculated.
Drug Loading
capacity7,8
Drug loading capacity
was calculated by formula
Drug
loading (%) = M actual / weighed quantity of powder microspheres × 100
Where M
actual is the actual drug content in weighed quantity of powder of microspheres
Physicochemical Characterization Of Hydrogel Plug5
Hydrogel Plugs were studied for hardness,
friability, weight variation, lag time and Swelling Index table 6.
Determination
of Swelling Index of Hydrogel Plug
Hydrogel plugs were kept immersed in three
different pH conditions. Plugs were taken out
carefully at 2,4,6,8,10,12
hours and their weights were determined
accurately table 7.
% Swelling = Wet weight − dry weight X 100
Wet weight
Evaluation Of Cross Linked Empty Capsules5
Various physical and chemical tests were
carried out for formaldehyde treated and untreated capsules.
Physical
tests
Identification, Solubility test for
formaldehyde treated capsules, dimension measurement was performed.
Chemical
Test
Qualitative
Chemical Test for Free Formaldehyde
Formaldehyde solution (0.0002%w/v) was used
as a standard solution. A sample solution was prepared by cutting 25
formaldehyde treated body of the capsules into small pieces and placed in
distilled water. This was stirred for 1hr with a magnetic stirrer, to solubilize the free formaldehyde. The solution was then
filtered into a 50ml volumetric flask, washed with distilled water and the
volume made up to 50 ml with the washings. To 1ml of sample solution, 9ml of
water was added. 1ml of the resulting solution was mixed with 4ml of water and
5ml of acetone. The solution was warmed in a water bath at 40ºC and allowed to
stand for 4 minutes.
Invitro Dissolution Studies2,5,6,13
For Ramipril
Immediate release granules
The in
vitro dissolution was carried out using USP Type I (Basket) dissolution
apparatus under sink condition. The dissolution medium was 900 ml of a 0.1N HCl solution (pH=1.2), at 370C±0.20C
and the stirring speed was 50 rpm. The in
vitro release studies were carried out for 2 hours.10ml samples were taken
at 10 minutes intervals for 2 hours and were replaced with fresh dissolution
medium. The absorbance of the solution was recorded at 210 nm using UV
spectrophotometer.
For Ramipril
Microspheres
The in
vitro dissolution was carried out using USP Type I (Basket) dissolution
apparatus under sink condition. The dissolution medium was 900 ml of a
phosphate buffer pH 6.8 at 370C±0.20C
and the rotating speed was 50 rpm. 10ml samples were taken at 1hour intervals
and were replaced with fresh dissolution medium. The absorbance of the solution
was recorded at 210 nm using UV spectrophotometer.
a.
In
Vitro Release of Pulsatile
Capsule
Dissolution studies were carried out using
USP XXIII dissolution test apparatus (paddle method). A Capsule was tied to paddle with a cotton
thread so that the capsule was immersed completely in dissolution media but not
float. In order to simulate the pH changes along the GI tract, three
dissolution media with pH 1.2, 7.4 and 6.8 were sequentially used, (sequential
pH change method). The pH 1.2 was
first used for
2 hrs then removed
and the fresh phosphate buffer pH 7.4was added. After 3 hrs the medium was
removed and colonic
fluid phosphate buffer pH 6.8 was
added for subsequent study. Nine hundred milliliters of the dissolution
medium was used at each time. Rotation
speed was 100 rpm and temperature was maintained at 37±0.5ºC. 10ml samples were
withdrawn at predetermined time intervals and fresh dissolution media was
replaced. The withdrawn samples were
analyzed at 210 nm, by UV absorption spectroscopy and the cumulative percentage
release was calculated.
Kinetic
Analysis of Ramipril In Vitro Release Data10
To analyze the mechanism for the drug
release and release kinetics, the data obtained from the in vitro drug release studies was fitted to various kinetics models
shown in figure 6.
RESULT
AND DISCUSSION:
Physical
Compatibility Study:
The drug
and excipients mixtures were kept at room temperature at 40 ± 2oC /
75 ± 5% RH. The mixtures did not show any physical changes. They were
compatible.
Chemical
Compatibility Study (FTIR)
FTIR
spectroscopy was carried out to study the compatibility of pure drug Ramipril with the polymer albumin, and other excipients
like microcrystalline cellulose, sodium starch glycolate,
polyvinylpyrrolidone, hydroxyl propyl
methylcellulose. There is no appearance or disappearance of any characteristic
peaks. This shows that there is no chemical interaction between the drug and
excipients showed in figure 1.