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6. FORMULATIONS, EVALUATION AND IN-VIVO STUDY OF MUCOADHESIVE DRUG DELIVERY SYSTEM USING A 23 FULL

FACTORIAL DESIGN

Numerous buccal mucoadhesive drug delivery systems have been

developed to prolong the drug release. The biopharmaceutical classification

system (BCS) 153,154 is used to group the API depending upon the solubility

and permeability characteristics of the drug.

BCS Class II compounds are poorly water soluble and highly

permeable. Nifedipine was chosen as a model drug to formulate and

optimize the sustained release mucoadhesive drug delivery system using 23

factorial designs. Nifedipine belongs to BCS Class II compound for treatment

of angina pectoris.

BCS Class III compounds are highly soluble and poorly permeable.

Hydralazine HCl was chosen as a model drug to formulate and optimize the

Sustained release mucoadhesive drug delivery system using 23 factorial

designs. Hydralazine HCl belongs to BCS Class III compound for treatment

of hypertension and congestive heart failure.

6.1 Formulation of nifedipine and Hydralazine HCl mucoadhesive buccal drug delivery system using a factorial design by two level-three factor

A factorial design was applied in this experiment where the effects of

different conditions or factors, on experimental results are to be elucidated.

A factor is an assigned variable such as concentration, temperature,

lubricating agent, drug treatment or diet. The choice of factors to be

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included in an experiment depends on experimental objectives and is

predetermined by the experimenter.

In this present investigational research work the mucoadhesive buccal

tablets of nifedipine and hydralazine HCl was prepared separately employing

23 randomized full factorial design by using xanthan gum or Pectin,

carbopol-974P, HPMC-K4M, In this experimental model, our target is to

determine how the t90% of drug release and mucoadhesive characters can be

affected by adjusting three parameters, concentration of polymers xanthan

gum or Pectin, HPMC-K4M, carbopol-974P, of the mucoadhesive buccal

tablets. For each of these factors, the levels will explain for use in this 2-

level experiment.

23 full factorial studies were designed to determine the interaction of

three independent variables at two levels (low and high level concentration).

The factorial design, simplifying the method and highlighting the

relationships between variables, it also allows the effects of manipulating a

single variable to be isolated and analyzed singly.

6.1.1 Experimental design (23 full factorial)

A 23 full factorial design was created to determine and optimize the

effect of the three formulation factors using t90% as response factor.

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Table.6.1 23factorial design represents three variables at two levels

Level Factor A Factor B Factor C High + + + Low - - -

Table.6.2 Concentration of polymers variables

Level Factor A Factor B Factor C

High 60 40 40

Low 35 25 20

Table.6.3 23 factorial design represents three variables Factors

Std Factor A Factor B Factor C

A -1 -1 -1 A +1 -1 -1 B -1 +1 -1

AB +1 +1 -1 C -1 -1 +1

AC +1 -1 +1 BC -1 +1 +1

ABC +1 +1 +1

+1 denotes High Level, -1 denotes Low Level

Factor A, B, C are Variables

6.1.2 Statistical optimization technique

In this present research work nifedipine and hydralazine HCl

mucoadhesive buccal tablets optimized has been done by statistically using

23 full factorial designs. In this study, three variables factors were evaluated

each at two levels, and investigational were performed at all eight possible

combinations. In which three variables namely such as xanthan gum or

pectin, HPMC-K4M, carbopol-974P, was kept at two levels, one is low level

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and another one is high level. Except the optimization phase whose purpose

was validated by extra design check point and main interactive influences

were tested using statistical method. The eight formulation of optimization

phase were categorized in to four groups for ease of investigation and

similarity as follows.

Group 1: All Three changeable factors at Low Level

Group 2: Any one of the changeable factors at High Level

Group 3: Any two of the changeable factors at high level

Group 4: All three changeable factors at high level

6.1.3 Formulation of mucoadhesive sustained release buccal tablets

containing nifedipine

Mucoadhesive sustained release buccal tablets155 of nifedipine were

prepared by a direct compression technique by using various proportions of

polymers such as xanthan gum or pectin, HPMC-K4M, carbopol-974P, and

with ethyl cellulose & magnesium sterate were used as an impermeable &

backing membrane respectively. The tablets were prepared by involving two

consecutive steps. In the first step, the mucoadhesive layer was prepared by

weighing accurately all the ingredients as shown in Tables 6.4, 6.5, 6.6 &

6.7 tablets incorporated with drug and mixed thoroughly in a glass mortar

for 15 min. Then this mixture was compressed using an 10-mm-diameter

die in the 10-station Rotary tablet punching machine (Chamunda Pharma

Pvt Ltd, Ahmadabad), followed by second step, after compression of the

adhesive core layer the upper punch was raised and ethyl cellulose and

magnesium stearate were added on the above compact and then again

compressed for the backing layer.

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The same procedure was adopted for the preparation of mucoadhesive

sustained release buccal tablets of Hydralazine HCl.

Table.6.4 Composition variables of nifedipine buccal tablets using xanthan gum

DRUG RESERVOIR(mg) DRUG FREE

BACKING LAYER(mg)

Formula code Drug Xanthan

gum HPMC K4M

CP-974P EC Mg.

Stearate FNX1 30 35 25 20 20 10

FNX2 30 60 25 20 20 10 FNX3 30 35 40 20 20 10 FNX4 30 60 40 20 20 10 FNX5 30 35 25 40 20 10 FNX6 30 60 25 40 20 10 FNX7 30 35 40 40 20 10 FNX8 30 60 40 40 20 10

Table.6.5 Composition variables of nifedipine buccal tablets using pectin

DRUG RESERVOIR(mg) DRUG FREE

BACKING LAYER(mg)

Formula code Drug Pectin HPMC

K4M CP-974P EC Mg. Stearate

FNP1 30 35 25 20 20 10 FNP2 30 60 25 20 20 10 FNP3 30 35 40 20 20 10 FNP4 30 60 40 20 20 10 FNP5 30 35 25 40 20 10 FNP6 30 60 25 40 20 10 FNP7 30 35 40 40 20 10 FNP8 30 60 40 40 20 10

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Table.6.6 Composition variables of hydralazine HCl buccal tablets using xanthan gum

Table.6.7 Composition variables of hydralazine HCl buccal tablets using Pectin

DRUG RESERVOIR DRUG FREE

BACKING LAYER(mg)

Formula code Drug Pectin HPMC

K4M CP-

974P EC Mg. Stearate

FHP1 25 35 25 20 20 10 FHP2 25 60 25 20 20 10 FHP3 25 35 40 20 20 10 FHP4 25 60 40 20 20 10 FHP5 25 35 25 40 20 10 FHP6 25 60 25 40 20 10 FHP7 25 35 40 40 20 10 FHP8 25 60 40 40 20 10

HPMCK4M- Hydroxyl propyl methyl cellulose, CP-974P – Carbopol-

974P, EC – Ethyl cellulose, Mg.Sterate-Magnesium sterate

DRUG RESERVOIR DRUG FREE

BACKING LAYER(mg)

Formula code Drug Xanthan

gum HPMC K4M

CP-974P EC Mg.

Stearate FHX1 25 35 25 20 20 10 FHX2 25 60 25 20 20 10 FHX3 25 35 40 20 20 10 FHX4 25 60 40 20 20 10 FHX5 25 35 25 40 20 10 FHX6 25 60 25 40 20 10 FHX7 25 35 40 40 20 10 FHX8 25 60 40 40 20 10

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6.2. Evaluation of Post compression parameters of sustained release mucoadhesive buccal tablets of nifedipine

The prepared mucoadhesive buccal tablets were estimated for post

compression factors156 such as thickness, friability, drug content and

hardness.

The same parameters evaluations were carried out for hydralazine HCl

buccal tablets

6.2.1 Thickness

The thickness of randomly selected average of the five buccal tablets

was used from each formulation, resoluted by using digital vernier calipers

and results were articulated in millimeter.

6.2.2 Hardness

Tablets have need of a definite sum of resistance and hardness or

strength to resist involuntary shocks of managing in packaging, shipping

and manufacture. The rigidity of five tablets randomly selected from each

formulation, calculated by means of monsanto hardness tester apparatus

and results were and expressed in Kg/cm2.

6.2.3 Friability

Friability test is assessing the strength of the granules; friability test

was done by using Roche friability test apparatus was used to conclude the

friability of the prepared buccal tablets. Twenty pre-weighed buccal tablets

was located in the friabilator apparatus and activated for 100 revolutions

(25 rpm) in four minutes and the buccal tablets freed from dust and

reweighed. The prescribed limit for loss on friability is not more than 1%

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w/w. The percentage friability was evaluated according to the following

formula.

% Friability = Pre weight-Final Weight Pre weight

6.2.4 Drug content

Ten prepared buccal tablets were selected randomly from each

formulation were delicately powdered and powder comparable to 30 mg of

nifedipine was exactly weighed and place in to 100 ml volumetric flasks

having 50 ml of phosphate buffer pH 6.8. The volumetric flasks were shaken

to mix the stuffings carefully. The amount was made up to the mark with

phosphate buffer pH 6.8 and filtered. 1 ml of the filtrate with appropriate

dilution was calculated for nifedipine content at 238 nm by means of a

double beam UV-visible spectrophotometer.

The same procedure was adopted for the evaluation of drug content

studies of Hydralazine HCl buccal tablets (25mg) at 260 nm.

6.2.5 Surface pH study

The surface pH study157 was conducted on nifedipine and hydralazine

HCl mucoadhesive buccal tablets, carried out to predict the comfort of the

buccal formulation into the possibility of any side effects in buccal mucosa

environment. The prepared buccal tablet were permissible to distend by

maintaining it make contact with 5ml of phosphate buffer containing 2%

w/v agar medium (pH 6.8 ± 0.01) at room temperature for 2 hrs. The surface

pH was deliberate by keeping the electrode the in make contact with the

surface of the buccal tablet and permiting it to equilibrate for 1 minute. The

mean of three reading was recorded.

X 100

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6.3 Swelling index characteristics for buccal tablets

The swelling index performance study was carried out on nifedipine

and hydralazine HCl buccal tablets. The degree of swelling index was on

purpose in terms of % weight gain158 by the mucoadhesive buccal tablets.

The swelling index velocity of the bioadhesive buccal tablet was estimated by

means of 1% agar gel plate. The initial weight of the buccal tablet was

deliberate (W1). The buccal tablet from each formulation was located on gel

surface in a petridish incubator at 37 ± 5°C. The buccal tablets were

detached at dissimilar time intervals (1, 2, 3, 4, 5 and 6 h) and wiped with

filter paper and weighed again (W2). The swelling index performance was

estimated by the formula.

S.I = [(W2-W1)/ W1] X 100

Where S.I= Swelling Index

W1- Initial weight of buccal tablet

W2- weight of swollen buccal tablet at time (t)

6.4 Measurement of ex -vivo buccoadhesive strength

Measurement of mucoadhesive strength159 required breaking the

adhesive bond between a buccal membrane and buccal tablets was carried

by modifying balance method. Fresh Sheep buccal mucosa was employed as

model membrane. Fresh sheep buccal mucosa were acquired from a local

slaughter-house and utilized for the study within 2 h of slaughter.

The mucosal membrane was detached by eliminating underlying loose

tissues and fat. The membrane was washed with distilled water and followed

with phosphate buffer solution (pH 6.8). The both sides of the balance were

made identical prior to the experiment, by keeping a 5 g weight. A piece of

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buccal mucosa was fixed to the glass vial, which was packed with phosphate

buffer. The mucoadhesive buccal tablet was trapped to the inferior side of a

rubber stopper with cyanoacrylate paste and adds load on the right-hand

pan. A weight of 5 g was isolated from the right hand pan, which lowered the

pan along with the tablet over the mucosa. The balance was kept in this

position for 5 minutes contact time. The weights were slowly added to the

right hand pan until the tablet detached from the mucosal surface.

The same procedure was adopted for the evaluation of ex-vivo buccoadhesive

strength on Hydralazine HCl buccal tablets.

Fig.6.1. Sheep buccal mucosa

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Fig.6.2. Modified Physical balance for measurement of Ex -vivo buccoadhesive strength

6.5 In-vitro drug release studies

The in-vitro dissolution study of nifedipine buccal tablets was carried

out for all formulation by means of Tablets Dissolution Tester (USP-II) 160.

The buccal tablet is positioned such that core faced to the 900 ml of

dissolution medium (pH 6.8 Phosphate buffer) maintained temperature at

37 ± 5°C and stirred at 50 rpm. The backing layer of mucoadhesive buccal

tablet was fixed in to the glass disk by instantaneous adhesive

(cyanoacrylate adhesive). The glass disk was owed to the bottom of the

dissolution container. The samples were withdrawn at regular time intervals

and substituted with same quantity of fresh buffer medium in order to

retain sink conditions. The samples were filtered; apposite dilutions were

made with phosphate buffer pH 6.8 and the samples were analyzed using

UV Visible spectrophotometrically at 238 nm.

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The same procedure was adopted for the evaluation of in-vitro drug release

studies of Hydralazine HCl buccal tablets at 260 nm.

6.6 Ex-vivo Drug Permeation through sheep buccal mucosa

An ex-vivo buccal permeation study161 of nifedipine buccal tablet was

carried through the sheep buccal mucosal membrane. The buccal tablet was

positioned in such a way that it fixed on the mucous membrane and the

compartments clamped together. The receptor compartment was packed

with isotonic phosphate buffer pH 6.8. The assembly was sustained

temperature at 37 ± 5°C and stirred with a magnetic bead at 50 rpm.

Samples were withdrawn and filtered through whatman filter paper; at

regular time intervals analyzed by means of UV Spectrophotometer at 238

nm.

The same process was adopted for the evaluation of ex-vivo drug

permeation through sheep buccal mucosa studies of Hydralazine HCl buccal

tablets at 260 nm.

Fig.6.3. Franz diffusion assembly used for drug Permeation studies

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6.7 In-vitro – Ex-vivo Correlation

The record obtained from the in-vitro drug release was correlated with

in-vivo drug permeation across buccal membrane of sheep for the

formulation FNX6. The correlation162 was carried out by plotting graph, in-

vitro cumulative percentage of drug release on x-axis and in-vivo cumulative

percentage of drug release on y-axis for the similar phase of time and

calculated the regression r2.

6.8 In-vitro kinetics studies

The in-vitro liberate data was fit into dissimilar equations and kinetic

models to explain the release kinetics163 of nifedipine and hydralazine HCl

from the buccal tablets. The kinetic models used were a zero-order equation,

First order kinetics, higuchi’s and Korsemeyer- Peppa’s models. The attained

results in these buccal tablets formulations to find out the method of drug

release from nifedipine and hydralazine HCl matrices, the in-vitro dissolution

data of each formulation with dissimilar kinetic drug release equations.

Namely zero order: Q=K0t; first order: Log C =Log C0 –k1t/2.303 higuchi’s

square rate at time: Q=KHt1/2 and peppas: F=KKtn, where Q is quantity of

drug release at time t, F is portion of drug release at time t, K0 is zero order

kinetic drug release constant, KH is higuchi’s square root of time kinetic drug

release constant, Km is constant incorporating geometric and structural

characteristic of the tablets and n is the diffusion exponent suggestive of the

release mechanism. The correlation coefficient values (r) from higuchi’s

model specify the diffusion exponent values (n) and kinetic of drug release

from peppa’s model signify the mechanism of drug release.

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6.9 In-vivo drug absorption studies on rabbits

The pharmacokinetic parameters164,165 of nifedipine were determined

by using two groups (six for each group) of male New Zealand white rabbits

(1-1.5 kg) were selected for the in-vivo drug absorption study, which was

previously verified for lack of any diseases. All animals were fasted for 12h

but drinking water was allowed ad libitum. On next morning nifedipine 8mg

per kg body weight in hard gelatin capsules was administered orally for each

of a group of six rabbits and an another group of rabbits were

administreated optimized buccal tablet (formulation FNX6) by located in the

buccal membrane with the help of a clip. Then 1 ml of blood sample was

taken every 60 minutes time intervals using 22 gauge needle, which

previously enclosed 1 ml of heparin solution to avert blood clotting. These

blood samples were subjected for centrifuging at 2,500 rpm for about 30

minutes. 1 ml of supernatant samples was taken, and after appropriate

dilution, analyzed at 238 nm by means of HPLC. The study was accepted by

the Institutional Ethical Committee (IAEC) Reg. No.

CPCSEA/PCP/IAEC/PhD/125/12 (Padmavathi College of Pharmacy).

According to BCS Class III Categories drugs when formulated as

sustained release preparation no IVIVC is expected, Hence Hydralazine HCl

also comes this categories.Therefore IVIVC study was not carried out for this

drug.

6.9.1 In-vivo drug absorption pharmacokinetics of nifedipine

The pharmacokinetic factors such as peak plasma concentration

(Cmax), time to attain highest plasma concentration (Tmax) and total area

under the plasma concentration time curve (AUC0-t,) were expected from the

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plasma concentration-time profiles of absorption records followed by buccal

administration of nifedipine buccal tablet formulation. The obtained data’s

are run through the PK solver model for nifedipine formulations.

6.9.2 Construction of calibration curve by HPLC

A calibration curve was created by plotting the nifedipine peak-height

ratio against the corresponding added plasma concentration. The standard

nifedipine concentration (0-12 µg/ml) was prepared by using mobile phase

ratio of methanol: acetate buffer (75:25).The calibration curve to calculate

regression lines and to evaluate the linearity.

6.9.3 Collection of blood and preparation of analysis

0.5 ml of blood was collected in heparinised eppendorf tube and the

plasma removed by centrifugation. Extraction was carried out with

methanol by shaking for 5 min at 110 rpm. The phases were detached by

centrifugation (10 min at 1500 rpm) and the aqueous layer was isolated by

vacuum aspiration. The organic layer was transfer into culture tubes and

evaporated. The residue was reconstituted in mobile phase and injected in

to the chromatograph.

6.9.4 Quantification for Nifedipine

The HPLC system composed of a rheodyne sample injector fitted with

a 20 µl sample loop and an Agilent compact LC 1120 pump.

A ODS (Octadecyl silane) C18 column (10µm, 250x4.6 mm) fitted with

a guard column was used for partition. Stock solution (1 mg/ ml) of

nifedipine was prepared in methanol. The solutions were kept secluded from

light with an aluminum foil and they were stored at 4°C.

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The mobile phase was composed166 of methanol: acetate buffer pH 4.0

(75:25) prepared freshly prepared with deionizer water. The mobile phase

filtered through a 0.45 µm membrane filter (Sartorius USA) and was then

degassed by ultrasonication. The flow rate was set at 1.0 ml/min and

ambient temperature retained for column. The detector was activated using

a wavelength of 238 nm.

6.10 Correlation studies

A simple in-vitro dissolution test on the drug product will be

inadequate to calculate its therapeutic efficiency. Therefore in order to

ensure batch to batch consistency in the physiological performance of a

drug product by use of such in-vitro values, the correlation167, of drug

release was carried out for the remedial effectiveness of a pharmaceutical

formulation and is managed by the features related to in-vivo and in-vitro

characteristics of the drug. The collective percentage of drug releases both

in-vitro and in-vivo was plotted.

6.11 Stability Studies

Short term stability studies168 were carried out the optimized

nifedipine and hydralazine HCl buccal tablets. Adequate number of buccal

tablets were filled in amber colored rubber Stoppard bottles and reserve in

stability compartment maintained at temperature at 40 ± 2oC / 75 ± 5%

RH for three months were analysed regularly, for their Swelling index,

physical appearance, buccoadhesive strength, drug content, and in-vitro

drug release.