World Journal of Pharmaceutical Research - WJPR · Reshma et al. World Journal of Pharmaceutical...

www.wjpr.net Vol 5, Issue 7, 2016.

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Reshma et al. World Journal of Pharmaceutical Research

DEVELOPMENT OF CONTROLLED RELEASE FORMULATION OF

AN ANTIEMETIC DRUG USING NOVEL PARENTERAL DRUG

DELIVERY SYSTEMS

Mirajkar Reshma*1, Madgulkar Ashwini

1 and Kulkarni Shirish

2

1Department of Pharmaceutics, AISSMS College of Pharmacy, Near RTO, Kennedy Road,

Pune 411001, Maharashtra, India.

2Formulation Development, Sun Pharmaceutical Industries Ltd, Vadodara, Gujarat,

Maharashtra.

ABSTRACT

The overall aim of this work was to develop Controlled release

parenteral drug delivery system using in situ gel forming mechanism

for an anti-emetic drug typically co-administered with

chemotherapeutic agents. The study involved formulation of the drug

Ondansetron Hydrochloride in an in situ gel forming system involving

thermoreversible polymer Pluronic F127 (PF127), copolymer

Hydroxypropylmethyl cellulose (HPMC) K100M and temperature

modifier Polyethylene glycol 400 (PEG 400) to form an optimized

solution intended to be administered subcutaneously. Ondansetron

Hydrochloride is parenteraly administered twice a day in

chemotherapy induced nausea and vomiting over a period of 5 days.

The formulation was optimised and evaluated for all prerequisites of parenterals and other

parameters like gel formation at 37°c, viscosity, rheology, gel stability, gel strength, invitro

drug release, in vivo pharmacokinetics and stability studies. The formulation showed rapid in

situ gelation at 37°c forming stable and viscous gel showing thixotropic behavior, following

all prerequisites of a parenteral product. The in vitro drug release was found to be over 95%

in Simulated Body fluid pH 7.4 at 37°C over a prolong period of 5days and the invivo

pharmacokinetic evaluation showed increase in tmax, Cmax and AUC as compared to

immediate release formulation which may be attributed to the slow diffusion of the drug from

the polymeric matrix. The formation invivo of a stiffer gel confirmed the in situ gelation of

the formulation and it was found to be biocompatibile by the histopathological studies. The

*Corresponding Author

Mirajkar Reshma

Department of

Pharmaceutics, AISSMS

College of Pharmacy, Near

RTO, Kennedy Road, Pune

411001, Maharashtra, India.

Article Received on

29 April 2016,

Revised on 19 May 2016,

Accepted on 09 June 2016

DOI: 10.20959/wjpr20167-6489

World Journal of Pharmaceutical Research SJIF Impact Factor 6.805

Volume 5, Issue 7, 931-949. Research Article ISSN 2277– 7105


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formulation was physically and chemically stable at ambient temperature, accelerated

conditions and at refrigerator conditions with most suitable storage condition at the

refrigerator temperature.

KEYWORDS: anti-emetic, Pluronic F127, in situ gelling system, thermosensitive.

INTRODUCTION[1-5]

Pain and Patient incompliance are the major drawbacks of parenteral formulation that are

worsened in case of frequent administration. Therefore there is the need to formulate and

develop controlled release parenteral drug delivery systems which are designed to provide a

prolonged effect (from few hours to few years) mainly in chronic diseases. In situ gel forming

injectables offer an important alternative to achieve controlled release. These are in sol form

prior to administration in the body and undergo gelation in situ, due to various stimuli like pH

change, temperature modulation and thereby control the drug release.

Ondansetron hydrochloride is a selective 5-HT3 inhibitor exhibiting anti-emetic activity for

the management of nausea and vomiting induced by cytotoxic chemotherapy and

radiotherapy. To prevent delayed or prolonged emesis, the treatment needs to be continued

for up to five days at a rate of 8 mg twice daily. The therapeutic use of the drug is also limited

by its considerable hepatic fist pass metabolism requiring daily administration. Therefore it

would be rationale to develop a controlled drug delivery system without this limitation of

frequent administration.

MATERIALS AND METHODS

MATERIALS

Ondansetron Hydrochloride was obtained as a generous gift from HiKAL Ltd. (Bangalore,

India). Pluronic F127 was purchased from Sigma Aldrich (Mumbai, India). Polyethylene

glycol 400 was purchased from LobaChemie (Mumbai, India). HPMC K100M was

purchased from Colorcon Asia (Goa, India). All other chemicals and reagents were of the

analytical grade.

Methods

Optimization

A response surface statistical experimental design was used to optimize the effect of different

independent factors on response. The responses were investigated using a Box–Behnken

statistical experimental design using Design-expert software® 9.0.4 (Stat-Ease, Inc., USA)


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This design was based on a 33 factorial design, three replicates of the central run, leading to

13 sets of experiments, enabling each experimental response to be optimized.

Different batches were prepared with different independent variables at different levels and

responses. The criterion for selection of optimum formulations was based on the gelation at

body temperature and highest gel strength to remain stable for prolonged period of time i.e.

for 5 days.

Preparation of in situ gel[5]

In situ gels were prepared by cold method. The method involved initial solubilisation of drug

(80mg) in water using Citric acid (4%) as a solubilising agent with continuous stirring on

magnetic stirrer followed by addition of Pluronic F127, stirred and kept in refrigerator at 40c

for 24 hrs to dissolve completely. The other excipients like HPMC K100M, PEG 400 and

NaCl were added to this solution and stirred continuously to give complete clear viscous

solution which was filled aseptically in amber glass ampoules 2ml and sterilized by

autoclaving at 121°C at 15 psi for 20 min and stored at room temperature.

Table: 1-Box Behnken experimental design

Batch no Factors

PF 127 HPMC K100M PEG400

F1 18 2.25 1

F2 22 2.25 1

F3 18 2.75 1

F4 22 2.75 1

F5 18 2.5 0.5

F6 22 2.5 0.5

F7 18 2.5 1.5

F8 22 2.5 1.5

F9 20 2.25 0.5

F10 20 2.75 0.5

F11 20 2.25 1.5

F12 20 2.75 1.5

F13 20 2.5 1

The quality of the fitted model was expressed by the coefficient of determination R2, and its

statistical significance was checked by an F-test (analysis of variance) at the 5% significance

level. The statistical significance of the regression coefficients was determined by using the t-

test (only significant coefficients with p-value < 0.05 are included). The optimum processing


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conditions were obtained by using graphical and numerical analysis based on the criteria of

the desirability function and the response surface.

Evaluation of formulation

Physical appearance & pH

The optimized formulation was visually checked for its appearance and pH. The pH of the sol

form was measured using standardized digital pH meter (Deluxe pH meter 101/EI) at room

temperature by taking adequate volume in a 10 ml beaker.

Gelation temperature[8]

Gelation temperature was determined by modification of Miller and Doravan technique. A 2

ml aliquot of gel was transferred to test tubes immersed in a water bath at 40°C and sealed

with aluminum foil. The temperature of water circulation bath was increased with increments

of 10°C and left to equilibrate for 5 min. at each new setting. The samples were examined for

gelation which was said to have occurred when the meniscus would no longer move upon

tilting through 90°C. The gel melting temperature, a critical temperature when the gel starts

flowing upon tilting through 90°C was recorded.

Gel strength[9-10]

Gel strength was determined by Surimi test using Texture Analyzer (Texture Pro CT V1.4).

Formulation (20 ml) was placed in a 25 ml beaker and the gelation was induced using heating

plate at 37˚C. The probe (TA 3/100) was allowed to traverse the gel up to 1 cm at a speed of

1mm/s. The load reading was taken in g/cm for gel strength.

Rheology

The rheological property of the polymeric formulation was determined with Brookfield

rheometer. Rheogram was plotted (shear rate vs shear stress).

Viscosity

Viscosity was measured on Brookfield viscometer using RV (spindle no. 21) and T-type

helipath spindle (spindle no. S21) at 37˚C under variable shear stress.

Osmolality

The samples were subjected to osmolality test using an Osmometer (Model 3250, Version

2.4).


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Syringeability[11]

Syringeability is the ability or ease by which a formulation can be dispensed out of a syringe.

Syringeability of the formulations at room temperature was determined using 21 to 26 G

needles. All prepared formulations were withdrawn into identical 5 ml plastic syringes placed

with 21 to 26 gauge needles to a constant volume (1 ml). The solutions which were easily

passed from a particular syringe were termed as pass and the ones which were difficult to

pass were termed as fail.

Drug content estimation[12]

0.25 ml of the test formulation was diluted with excess of methanol and evaporated to

dryness. The residue was diluted up to 100 ml with mobile phase to get a stock solution of

100μg/ml. From this stock solution, a solution of 40μg/ml was prepared and analysed by

HPLC. The concentration of the drug present in formulation was computed from the

calibration curve using the equation y=mx+c.

Sterility Testing[13-14]

Sterility testing was carried out as per the IP 1996. The formulation was incubated for not

less than 14 days at 30°-35°C in the alternate fluid thioglycolate medium to find the growth

of bacteria & at 20°-25°c in Soya bean casein digest medium to find the growth of fungi. The

test was performed using positive and negative controls. The bacterial strain used for positive

control was Clostridium sporogenes whereas the fungal strain was Candida albicans.

In-vitro Drug Release Studies[15]

In vitro release studies of OndansetronHCl from the formulation were performed at 37˚C

using membrane less dissolution model and simulated body fluid (SBF, pH 7.2) was used as

the dissolution medium. Orbital shaking Incubator was used to maintain the desired

temperature (37˚C) and agitation (30rpm).1ml of dissolution medium was withdrawn at

specific time intervals (1, 24, 48, 72, 96, 120 hrs) and replenished with fresh medium to

maintain sink conditions. Aliquots withdrawn were suitably diluted and analyzed using UV

spectrophotometer at 248nm.

Pharmacokinetic analysis[5-9]

In vivo examinations were accomplished using 8 male Wistar rats. Animals were housed and

handled according to the CPCSEA guidelines. The process of killing the animals was

performed using anaesthetic overdose method. In vivo experimental protocol was approved


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by the IAEC. A HPLC method for quantitative estimation of drug in plasma was developed

and validated.

Eight male rats, weighing 210–250 g, were divided in four study groups (n=2) and

administered with the injection as shown in Table: 2 as follows. The blood samples were

collected from retro-bulbar vein at 1,5,24,48,72,96 and 120 hr after injections for controlled

release and at 1, 3, 5, 7, 8hrs for immediate release. The blood serum was separated and

assayed for Ondansetron content by HPLC.AUC from time zero to the last day of sampling

(AUC), maximum blood concentration (Cmax), time to the maximum concentration (Tmax)

and plasma drug concentration were evaluated and graphs were plotted.

Table: 2- Animal groups for in vivo studies

Group Administration

Group A (Control) 0.5 ml of SWFI given

subcutaneously.

Group B (Test) 0.5 ml of controlled release test

formulation given subcutaneously.

Group C (Placebo) 0.5 ml of placebo formulation given

subcutaneously.

Group D (IR) 0.5ml of IR formulation given

subcutaneously.

In vivo gelation[5]

Formulation treatment rat was given subcutaneous injection in the abdomen region. The

formulation was allowed for gelation in vivo. After 1 hour, the skin surrounding the site of

injection was carefully cut open to reveal in vivo gelation.

Histopathological evaluation of subcutaneous tissue[9]

Formulation treatment, placebo treatment and control rats were killed at a scheduled time,

carefully cut open the skin at the injection site, together with the surrounding tissue. To

evaluate the biocompatibility of the in situ-forming gel, the injection site was observed and

the surrounding tissue was harvested for histopathological analysis. The harvested tissues

were preserved in 10% formalin followed by paraffin embedding. Sections were cut to 4μ

thickness and stained with Standard hematoxylin eosin solution. The stained sections were

studied for biocompatibility.


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RESULTS AND DISCUSSIONS

Optimization

Design of experiments (DOE) is an efficient procedure for planning experiments so that the

data obtained can be analyzed to yield valid and objective conclusions. The Box- Behnken

Design is an independent quadratic design which does not contain an embedded factorial or

fractional factorial design. 3-D response surface plot were used to plot relationship between

variables and responses.

Table: 3- Box Behnken experimental design with response

Batch no

Response

Gelation

temp(0C)

Gel strength

(gm/cm)

F1 29 14.21

F2 31 13.93

F3 30 13.19

F4 32 14.21

F5 35 14.24

F6 36 15.43

F7 32 16.33

F8 35 16.94

F9 32 15.49

F10 33 14.98

F11 32 14.1

F12 35 16.07

F13 37 15.80

Fig. 1-Response surface plot for gelation temperature

Gelation Temperature=+1.63*A-0.37*B-0.25*C+0.25*AB-16*AC-0.50*BC-3.38*A2-

0.87*B2- 1.62*C2.


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Fig.2- Response surface for gel strength

GelStrength=+16.47*A+003*B-3*C-003*AB-016*AC-003*BC+0.30*A2-0.021*B2-

0.014*C2.

Table: 4- Desirability function of optimized formulation

Formulation

Code PF127(% w/v)

HPMCK100M

(% w/v) Desirability

F13 20 2.5 0.974

Table: 5- ANOVA TEST RESULT

Response Model R2 F value P value

Gelation

Temperature (°C) Quadratic 0.9831 63.92

0.0008

(significant)

Gel strength

(gm/cm) Quadratic 0.9734 479.51

0.0001

(significant)

Where, A is concentration of Pluronic F127, B is the concentration of HPMCK100M and C is

the concentration of PEG400. Pluronic F127 is a triblock copolymer containing POE-PPO-

POE. As the temperature increases dehydration of PPO block causes formation of micelle and

hydration of PEO block leads to form gel. But as the concentration of Pluronic F127 increase,

gelation temperature decreases and viscosity of sol also increases. Addition of PEG 400

causes modification in process of micelle formation due to similarity in PE moieties. So

gelation temperature of formulation was achieved by addition of PEG 400 which ultimately

resulted in increased gelation temperature i.e. 37°C with optimized viscosity of formulation.

The formulation F13was chosen as optimized batch with gelation temperature at 37°c.


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In order to obtain a controlled release over a 5 day period it was necessary for the insitu gel

formed to have a sufficient gel strength and stability. Plain Pluronic F127 could not impart

these parameters so HPMCK100M was added as a copolymer to achieve sufficient gel

strength, stability and controlled release. As the concentration of HPMCK100M increases,

gel strength increases along with increase in viscosity of sol. Although HPMCK100M and PF

127 differ in chemical structure, both have hydrophobic regions in their chains. It is reported

that solvation of hydrophilic chains (POE moiety) of PF 127 at high temperature in water

promotes interaction between hydrophobic moieties of HPMCK100M and Pluronic F 127.

Thus combination of PF 127 and HPMC imparts the characteristic to the formulation to

obtain controlled release over the time period. The formulation F13 was chosen as optimized

batch with gel strength as 15.8g/cm at 37°c and optimized viscosity.

Appearance and pH

All the formulations were clear and colorless. Terminal sterilization with autoclaving had no

effect on physical and chemical properties of the formulation. The pH of the all the

formulations was found to be between 6.2-6.5. The pH values were found to be in the range

tolerated by the subcutaneous tissue. Moreover, the drug was found to be most stable in this

pH range. Thus the formulation follows the prerequisites of parenteral dosage form.

Gelation temperature

The gelation temperature for the optimized formulation was found to be 37°C. This

temperature was sufficient to trigger in situ gel formation which is the body temperature. As

the temperature increases, dehydration of the PPO leads to formation of a micelle core while

hydration of the PEO causes it to expand and form a hydrogel. So as the concentration of

Pluronic F127 increases it dehydrate the micelle structure which leads to lowering of gelation

temperature. So to increase gelation temperature, PEG400 were added, which possess same

hydrophobic portion which interfere with micelle association of Pluronic F127 and causes

entanglement, which ultimately leads to increase in gelation temperature.

Gel strength

Gel strength of the formulation was found to be 15.8 g/cm. The gel strength was adequate to

withstand shear forces in the body and remain stable upto a prolong period of 5 days and

maintain controlled release.


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Viscosity[18]

The viscosity of the sample was found to be 2100 cPs. High viscosity of the sample indicated

higher strength of the resultant gel formation. The high viscosity of the sample could be

attributed to the high viscosity grade of HPMC. The increase in viscosity may also be

attributed to thermoreversible property of PF127. As concentration increases the solution

viscosity was found to be increased and at or beyond gelling temperature the extent of

micelle formation and hydrogen bonding of PF127 with water also increased which resulted

in increase in viscosity.

Rheology

The rheogram showed the thixotropic behavior of the sol. Thus the formulation is in sol form

during administration and forms gel in situ.

Fig. 3- Rheogram

Osmolality

The osmolality of the sample was found to be 310mOsmol/L. The formulation was found to

be isotonic with the physiological fluids. The normal physiologic osmolarity range is

approximately 280-320 mOsmol/L.

Syringeability

The formulation easily passed through the needle gauge 21. This needle size was suitable for

subcutaneous injection.


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Table 6-Syringeability of optimized formulation

Needle gauge no. Outcome

21 Pass

22 Fail

24 Fail

26 Fail

Drug content estimation

The percentage drug content in the formulation was calculated and found to be 98.54%,

indicating insignificant loss of drug during the formulation.

Sterility testing

Parenteral formulations are sterile preparations. The formulation showed no turbidity after 14

days and thus was found to be free from bacteria and fungi. Hence, it passes the test for

sterility as per I.P. The sterility of the formulation may be attributed to aseptic process of

preparation and filling the container and autoclaving at 121°C at 15 psi for 20 min.

Table 7-Observations for bacterial growth

Sample Observation

Positive control Growth

Negative control No growth

Test sample No growth

Table 8-Observations for fungal growth

Sample Observation

Positive control Growth

Negative control No growth

Test sample No growth

In-vitro Drug Release Studies[21]

In vitro drug release study serve as a comparative tool in formulation and development. Drug

release from Pluronic F127 gel into release medium was regulated by dissolution or diffusion

of drug, depending upon experimental conditions. In vitro drug release of gel formulation

was regulated by diffusion mechanism. As the concentration of HPMC K100M increases

drug release was retarded. When the formulation got exposed to the preheated phosphate

buffer pH 7.4 at 370C it undergoes phase transition from sol-gel and form barrier for drug

release. As the time passes the outer layer become more hydrated and the polymer chains

become fully relaxed and can no longer maintain its integrity, leading to disentanglement and

erosion from the surface. Thus drug release was regulated by diffusion and erosion


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mechanism. About 97.43% of drug release was observed on the last day which indicated

sustenance of the release for 5 days. Thus the combination of thermosensitive polymer

Pluronic 127 and Release retardant HPMC K 100M serve to form a controlled release

formulation over the period of 5 days reducing the dosing to once a day for a 5 day period.

Fig.4 - In vitro release profile for optimized formulation.

It is evident from above data that Korsemeyes-Peppas model was the best fit model for

optimized batch. The value of diffusion exponent (n) of the optimized formulation was 0.679.

The obtained n-value for the optimized formulation was 0.679, indicating that the optimized

formulation followed non-Fickian diffusion mechanism for drug release.

Pharmacokinetic analysis

The pharmacokinetic parameters for the Controlled release test formulation and immediate

release injection were studied and compared.


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A) B)

Fig. 5 - Plasma drug conc. Vs Time profile in rat for A) Controlled release B) Immediate

release.

Table 9-Pharmacokinetic data for controlled release and immediate release formulation

in rats

Parameters Controlled release

formulation

Immediate Release

injection

Cmax 8402ug/ml 38.61ug/ml

Tmax 5hr 15min

t1/2 6 hr 10min

AUC0-

120(Controlled R)

AUC0-30(IR)

3044 342.79

It was seen from the above figures and table that the plasma concentration profile of the drug

after administration of in situ gel showed controlled drug release in the systemic circulation

for 120 hours. The increase in peak plasma concentration (Cmax) for OND controlled release

formulation in plasma after subcutaneous administration, time (tmax) to reach Cmax and

AUC as compared to immediate release injection may be attributed to the slow dffusion of

the drug from the polymeric matrix.

In vivo gelation

A stiffer gel clot was observed after 1 hour which confirmed the in situ gelation of the

formulation.


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Fig. 6 - In vivo gel forming evaluation by subcutaneous injection of formulation in

animal

The in vivo gelation occurred due to the meeting of physiological conditions required to form

gel within the body. The body temperature of rat at 37°C was adequate to trigger in vivo

gelation. Thus this justifies the use of Pluronic F127 as a thermoreversible polymer and its

combination with HPMC K100M to form a stable viscous gel showing thixotropic behavior.

Histopathological evaluation of subcutaneous tissue[20]

Histopathological reports revealed the following outcomes.

Fig.7 - Optical micrographs of subcutaneous tissues a) Control b) Test c) Placebo

Table 10-Observations for histopathology studies

Sample Observations

Control Mild increase in collagen fibres, reduced number

of hair follicles, No signs inflammation.

Test No abnormality detected, no inflammation

Placebo

Deeper dermis and subcutaneous fat and muscle

tissue showed Mononuclear cells (macrophages

and monocytes) infiltration and few polymorphs

indicating inflammation.


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From the above figures and table the formulation was found to be biocompatible with no

abnormality detected. The biocompatibility could be attributed to the use of biocompatible

polymers and other excipients within their approved limits. Moreover, the pH and osmolality

of the formulation within the desired ranges did not trigger any immunological response.

Mild increase in collagen fibers in control group indicated initiation of healing of the

disrupted tissue at the site of injection. Absence of collagen fibers in test formulation

indicated complete healing of the damaged tissue at the injection site.

Accelerated stability study

Stability studies indicates that formulation F13 was physically and chemically stable at

ambient temperature i.e at 25°C ± 2°C/60% RH ± 5% RH and at accelerated conditions 40°C

± 2°C/75% RH ± 5% RH and at refrigerator conditions i.e at 5°C ± 3°C for period of 6

months with most suitable storage condition at the refrigerator temperature. It shows no

change in appearance, clarity, pH, gelation temperature with negligible changes in gel

strength and in vitro drug release profile. Thus the formulation packed in Amber colored

ampoule sterilsed by autoclaving proves to be stable.

Table 11-Stability studies at Room temperature (25°C ± 2°C/60% RH ± 5%)

Appearance

/clarity pH

Gel

temperature °C

Gel strength

g/cm

In vitro

Drug (%)

1month Transperant, clear 6.2 37 15.8 96

3 month Transperant, clear 6.2 37 15.0 92

6 month Transperant, clear 6.0 36.8 14.8 87

Table 12-Stability studies at 40°C ± 2°C/75% RH ± 5% RH)

Appearance/

clarity pH

Gel

temperature °C Gel strength

g/cm In vitro

drug (%)


3 month Transperant, clear 6.0 36.5 15.0 92


Table 13-Stability studies at Refrigerator (5°C ± 3°C)

Appearance/

clarity pH

Gel temperature °C

Gel strength

g/cm

In vitro

drug (%)





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Fig. 8-Drug release after 1month

Fig. 9-Drug release after 3month


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Fig. 10-Drug release after 6 month

CONCLUSION

Thus formulation of Ondansetron hydrochloride as an insitu gelling parenteral drug delivery

system is an alternative to the conventional formulations owing to its ability to Control drug

release over the length of treatment with reduced dosing frequency and increased patient

compliance experiencing long term treatment. The formulation involves less complicated

fabrication and easy administration.

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