SOLUBILITY ENHANCEMENT OF CLOMIPHENE CITRATE USING

www.wjpps.com Vol 6, Issue 6, 2017.

530

Mansuri et al. World Journal of Pharmacy and Pharmaceutical Sciences

SOLUBILITY ENHANCEMENT OF CLOMIPHENE CITRATE USING

NANOTECHNOLOGY

Mansuri Mohammadfarhan Mohammadharun*1, Dr. Anil S. Solanki

2, Dr. Vishnu M.

Patel3 and Dr. Anand K. Patel

4

1A.P.M.C. College of Pharmaceutical Education and Research, Himatnagar-383001.

2Professor and Principal, A.P.M.C. College of Pharmaceutical Education and Research,

Himatnagar-383001.

3Asso. Professor, A.P.M.C. College of Pharmaceutical Education and Research, Himatnagar-

383001.

4Assi. Professor, Pharmaceutics Department, A.P.M.C. College of Pharmaceutical Education

and Research, Himatnagar-383001.

ABSTRACT

The ambition of the present investigation is to improve solubility and

dissolution rate of clomiphene citrate by nanotechnology (solvent

diffusion metod) using mixture of pvp k – 30 and tween – 80. To

prepare Nano suspension of optimized batch (FF6).The prepared

Clomiphene citrate Nano suspension were characterized in term of

Fourier transform infrared spectroscopy (FTIR), gas chromatography,

particle size distribution, poly dispersity index (Zeta sizer),

microscopic (SEM) studies, solubility, dissolution studies. The FTIR

indicates no interaction between drug and polymer and no change in

chemical nature. The aqueous solubility and dissolution rate of

clomiphene citrate Nano suspension was significantly increased. The

optimized Nano suspension after stability showed no significant

change in formulation that was same as before stability. The prepared Nano suspension of

clomiphene citrate showed more solubility and in vitro drug release compared to marketed

formulation.

KEYWORDS: Clomiphene citrate, chitosan, PVP k - 30, PLGA, solvent diffusion method,

Nano suspension, Nano technology, solubility enhancement.

*Corresponding Author

Mansuri

Mohammadfarhan

Mohammadharun

A.P.M.C. College of

Pharmaceutical Education

and Research, Himatnagar-

383001.

Article Received on

19 April 2017,

Revised on 10 May 2017,

Accepted on 31 May 2017

DOI: 10.20959/wjpps20176-9278

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

S SJIF Impact Factor 6.647

Volume 6, Issue 6, 530-562 Review Article ISSN 2278 – 4357


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INTRODUCTION

Solubility: ―Solubility is defined as maximum amount of solute that can be dissolved in a

given amount of solvent to form a homogenous system at specified temperature.‖

Need of Solubility Enhancement

Recently, more than 40% drugs are lipophilic and having a problem of poor water solubility.

Dissolution of such drugs should be improved for better drug profile. Oral bioavailability of a

drug depends on its solubility or dissolution rate, so that to increase dissolution of drugs with

limited water solubility is often needed.

Importance of Solubilty

Therapeutic effectiveness of a drug depends upon the bioavaibility and ultimately upon the

solubility of drug molecules.Solubilty is one of the important parameter to achieve desired

concentration of drug in system circulation for pharmacological response. Now only 8% of

new drug candidates have both high solubility and permeability. A new discovery in

chemistry comes out with novel entities of poorly water soluble drugs.

More than one third of the drugs listed in U.S. pharmacopeia fall into the poorly water

soluble or water insoluble categories. Low water solubility is the major drawback

encountered with formulation development of new chemical molecules.

An Overview of Disorders

Ovulatory disorders is a period that describes a group of disorders in which ovulation fails to

occur, or occurs on an infrequent or irregular basis. Ovulatory disorders are one of the

primary causes of infertility. ―Anovulation (no ovulation) is a disorder wherein eggs do not

develop properly, or are not released from the follicles of the overies.‖ Women who have this

disorder may not menstruate for several months. Others may menstruate still though they are

not ovulating. Anovulation may result of hormonal discrepancy, eating disorders, and other

medical disorders, the cause is often unfamiliar. Women who exercise extremely can also

stop ovulating.

Oligoovulation is a disorder wherein ovulation does not occur on regular basses and your

menstrual cycle may be longer than the normal cycle of 21 to 35 days. Around 1/4th of

infertile women have problems with ovulation. These comprise the lack of ability to produce

fully matured eggs or failure to ovulate an egg. Fertility specialists use agroup of


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medications, called ―fertility drugs‖ for the moment correct ovulatory problems and enhance

a woman’s chance for pregnancy.

Drugs use for ovulation

Clomiphene citrate, Metformin, Follicle Stimulating Hormone (FSH), Human Chorionic

Gonadotropin (HCG), Luteinizing Hormone (LH), Dopamine agonists, Human Menopausal

Gonadotropin (HMG), Gonadotropin releasing Hormone (GnRH). The most commonly

approved ovulation drug is clomiphene citrate. This drug is most frequently used to stimulate

ovulation in women who have irregular or absent ovulation.

But Clomiphene citrate is slightly soluble in water, so that solubility enhancement of

clomiphene citrate is important part to enhance its oral bioavailability.

Nanotechnology

In the pharmacy, important application of size reduction is unit operation. It improves the

solubility and also improves bioavailability increasing the release rate and producing best

formulation for drugs. In case of, Micro size rang is limited of size reduction.eg, Drug having

nanosize range, it enhance the performance in verious dosage forms. Main advantages of

nanosizing dosage forms:

Increase surface area.

Increase solubility.

Increase rate of dissolution.

More rapid onset of therapeutics.

Less amount of dose required.

Reduce fasted/fed variability.

Nanotechnology which is sometimes shortened to "Nanotech‖ refers to a field of applied

science and technology whose theme is to control the matter on an atomic and molecular

scale. Generally nanotechnology deals with structures of the size 1-100 nanometers or

smaller, and involves developing materials or devices within that size.

Nanotechnology is on its way to make a big impact in Biotech, Pharmaceutical and Medical

diagnostics sciences. A dynamic collaboration is observed within the Researchers,

Government, Pharmaceutical - Biomedical companies and educational institutions all over

the world in developing the nanotechnology.


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Water is the solvent of choice for liquid pharmaceutical formulation. Hence various

techniques are used for the improvement of the solubility of poorly water soluble drugs.

Various Shapes of Nanoparticles.

Nanosuspension

Nanosuspensions are submicron colloidal dispersions of pure drug particles in an outer liquid

phase. Nanoparticle engineering allows poorly soluble drugs to be formulated as

nanosuspensions either alone or with a combination of pharmaceutical excipients. The

nanosuspension engineering methods recently used are precipitation, high-pressure

homogenization and pearl milling, either in water or in mixtures of water and water-miscible

liquids or in nonaqueous media.

Nanosuspension is a well - organized and intelligent drug delivery system for water insoluble

drugs, as the saturation solubility and the surface area available for dissolution increased.

Generally, the biopharmaceutical benefits of water insoluble drugs formulated as

nanosuspensions with improvement in formulation performance, such as reproducibility of

oral absorption, improved dose-bioavailability proportionality, minimized toxicity and side

effects and increased patient compliance by reduction of frequency of oral units to be taken.

Most commonly used stabilizers to stabilize nanosuspension are either polymer like (e.g.,

polyvinyl pyrrolidone (PVP), crystalline cellulose, amphiphilic amino acid, hydroxypropyl

cellulose (HPC) and hydroxypropyl methyl cellulose (HPMC). Whereas, surfactant such as

ionic are (e.g., sodium lauryl sulphate (SLS), poly (ethyleneimine) (PEI), chitosan and non-


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ionic surfactant (e.g., polysorbate (tween 80), block copolymer like pluronic) and some food

protein are also used as stabilizers.

Nanosuspension are prepared by two methods first is Bottom-up and second is Top-down

method. In the present work nanosuspension is prepared by bottom up method in which drug

is dissolved in a solvent, which is then added to non-solvent that cause precipitation of the

fine drug particle and the system is stabilize by polymer and or surfactant to prevent them

from aggregation or agglomeration.

Organoleptic Characteristics

The color, odour and taste of the drug were characterized and recorded using descriptive

terminology.

Determination of Melting Point

Melting point of Clomiphene citrate was determined by capillary method. Clomiphene citrate

was filled in capillary and tied with a thermometer. The thermometer with capillary was

placed in Paraffin bath. The bath was subjected to external heat. The point at which

Clomiphene citrate melts is recorded from thermometer.

Standard Calibration Curve

Accurately weighed 50 mg Clomiphene citrate was dissolved in small amount of 0.1N Hcl

and dilute with Distilled water upto 50 ml to get the stock solution having concentration 1000

mcg/ml. From the stock solution take 5ml and dilute with distilled water up to 50 ml to get

the stock solution having concentration 100 mcg/ml. From the stock solution aliquots of 2.5,

5, 7.5, 10 & 12.5 ml were withdrawn and further dilute to 50 ml with distilled water to obtain

a concentration range of 5 to 25 mcg/ml. The absorbance of the solutions was measured at

232 nm by UV- Visible spectrophotometer.

Solubility of Drug

Excess (usually more than1mg/ml concentration) of solid drug were added to 20ml distilled

water and different dissolution media taken in stopper conical flasks and mixture were shaken

for 24hrs in rotary flask shaker. After shaking to achieve equilibrium, 1ml aliquots were

withdrawn at 1hr intervals and filtered through Whatmann filter paper. The filtrate was dilute

with water up to 10ml to get 100mcg/ml solution. From that withdrawn 1ml and dilute with


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water up to 10ml to get 10mcg/ml solution and analyzed by UV‐ spectrophotometer at 232

nm. Shaking was continued until three consecutive readings were same.

Compatibility Study

Drug: Polymer compatibility study was carried out for any interference of drug and polymer.

The interference study was carried out using FTIR. The infrared absorption spectra of pure

drug and physical mixture of drug and polymer were performed for polymer drug interaction

studies.

Fourier transform infrared spectroscopy (FTIR) has also been used to evaluate the interaction

between polymer and drug molecules in the solid state. The chemical interaction between the

drug and the polymer frequently leads to certain changes in the infrared (IR) profile of

mixture.

Experimental Work

Preparation of Nano Particles by Solvent Diffusion Method

Trial batches with Chitosan

Drug was dissolve in small quantity of methanol to get clear solution. Chitosan polymer was

dissolve in glacial acetic acid where quantity of glacial acetic acid is 1.5 times greater than

concentration of chitosan. Prepare aqueous solution of Tween 20 (0.1 %) used as stabilizer.

Add solution of drug and aqueous solution of stabilizer to polymeric solution respectively

with continueous stirring at 6000 RPM using high speed homogenizer for 3 hours. Add

glutaraldehyde solution 25 % as a hardening agent dropwise during process. Due to addition

of glutaraldehyde (25 % solution) as a Hardening agent, prepared solution becomes highly

viscous or gel like structure. So that deletion of Gluteraldehyde from next formulation

method.

Trial batches with PLGA

Drug was dissolve in small quantity of methanol to get clear solution. PLGA polymer was

dissolve in DCM. Prepare aqueous solution of Tween-80 (0.1 %) used as stabilizer. Add

solution of drug and polymeric solution to solution of stabilizer respectively with continueous

stirring at 8000 RPM using high speed homogenizer for 3 hours.


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Preliminary Trials of Nanosuspension

Nanosuspensions were prepared according to solvent diffusion method. Clomiphene citrate

was dissolved in (0.5ml) methanol to get clear solution. The prepared organic solution was

then added slowly dropwise with the help of a syringe into an aqueous phase (25 ml)

containing polymer (PVP k - 30) and stabilizer (Tween - 80) using magnetic stirrer at 400C

temperature for 2 hrs. to evaporate organic solvent. Complete evaporation of methanol was

determined by gas chromatographic method. The volume was then adjusted with the addition

of distilled water to recover loss in keeping other parameters constant.

Formulation and Development of Nanosuspension of Chlomiphene Citrate Using 32 Full

Factorial Design

It is desirable to develop an acceptable pharmaceutical formulation in shortest possible time

using minimum number of man, hours and raw materials. Traditionally pharmaceutical

formulations after developed by changing one variable at a time approach. The method is

time consuming and it may be difficult to develop an ideal formulation using this classical

technique since the joint effects of independent variables are not considered. It is therefore

very essential to understand the complexity of pharmaceutical formulations by using

established statistical tools such as factorial design. In addition to the art of formulation, the

technique of factorial design is an effective method of indicating the relative significance of a

number of variables and their interactions.

A statistical model incorporating interactive and polynomial terms was used to evaluate the

responses. The number of experiments required for these studies is dependent on the number

of independent variables selected. The response (Y) is measured for each trial.

Y b0 b1 X1 b2 X2 b12 X1 X2 b11 X11 b22 X22

Where, Y is the dependent variable,

b0 is the arithmetic mean response of the nine runs and

bi is the estimated coefficient for the factor Xi.

The main effects (X1 and X2) represent the average result of changing one factor at a time

from its low to high value. The interaction terms (X1X2) show how the response changes

when two factors are simultaneously changed. A 32 randomized full factorial design was

utilized in the present study. In this design two factors were evaluated, each at three levels,

and experimental trials were carried out at all nine possible combinations.


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Selection of dependent and independent variables

On the basis of preliminary trial results, 2 independent variables at 3 levels were selected. For

optimization 32 designs was employed to study the effect of independent variables (i)

concentration of PVP k - 30 (X1) and (ii) Concentration of Tween - 80 (X2) on dependent

variables solubility after 30 min. (Q30) (Y1), solubility after 24 hours. (QEq.) (Y2) and % Drug

Release (Y3). All the batches were prepared according to the experimental design.

Characterization of Formulation

Particle size determination

The mean diameter of solid lipid nanoparticle in the dispersion was determined by a

ZetaSizer (Malvern Instruments ltd, Malvern UK) at a fixed angle of 90 degrees. The particle

size analysis data were reported by using the volume distribution as calculated automatically

by the device.

Determination of polydispersity index (PDI)

PDI was determined by ZetaSizer (Malvern Instruments ltd., Malvern UK).

Scanning electron microscopy

Scanning electron microscopy (SEM) is behavior to characterize the surface morphology of

the NPs. The samples are mounting on alumina stubs using double adhesive tap. Then the

sample is observed in JEOL JSM-5610LV SEM at an acceleration voltage of magnification

of 270X and 20kV.

Solubility study (Q30)

Excess (usually more than 1mg/ml concentration) of Nanosuspension was added to 20ml

distilled water taken in stopper conical flasks and mixture was shaken for 30 min. in rotary

flask shaker. 5ml aliquots were withdrawn filtered through Whatmann filter paper. The

filtrate was diluted if necessary and analyzed by UV‐ spectrophotometer at 232 nm. Shaking

was continued until three consecutive readings were same.

Solubility study (QEq.)

Excess (usually more than 1mg/ml concentration) of Nanosuspension was added to 20ml

distilled water taken in stopper conical flasks and mixture was shaken for 24hrs in rotary

flask shaker. After shaking to achieve equilibrium, 5ml aliquots were withdrawn at 1hr

intervals and filtered through Whatmann filter paper. The filtrate was diluted if necessary and


538


analyzed by UV‐ spectrophotometer at 232 nm. Shaking was continued until three

consecutive readings were same.

In vitro drug release study

The experiments were conducted according to the following procedure:

900 ml of water, maintained at 37±0.5°C was used as a dissolution medium. The stirring

speed of the paddle was at 100rpm. After the required amount of each sample had been

placed into the dissolution medium, an aliquot portion of the solution was withdrawn at

appropriate time intervals and diluted with 0.1N HCl then analyzed by UV spectrophotometer

at 232 nm. Each point on the dissolution profiles represented the average of three

determinations.

Accelerated stability study

Stability studies were carried out as per ICH guidelines (40°C ±2 °C and 75 % RH ± 5 %) for

a period of 30 days for optimized formulation by storing the sample in stability chamber. The

samples were placed in vials with bromo butyl rubber plugs and sealed with aluminium caps.

The samples were evaluated for solubility study and in vitro drug release.

Gas chromatography for estimation of organic solvent residuals

Gas chromatography was carried out using the Thermo Scientific Flame Ionized Detector

(Trace 0001). Data were reported as calculated automatically by the device.

Table and Figure

Trial batches of Chitosan with glutaraldehyde

Name of method Formulation

code

Drug: polymer

(mg)

Hardening agent

concentration (ml)

Solvent Diffusion

Method

CGF1 1:5 1 2 3

CGF2 1:10 1 2 3

CGF3 1:15 1 2 3

Trial batches of Chitosan

Name of method Formulation code Drug: polymer (mg)

Solvent Diffusion Method

CF1 1:5

CF2 1:10

CF3 1:15


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Trial batches of PLGA

Name of method Formulation code Drug: polymer (mg)

Solvent Diffusion Method

PLF1 1:1

PLF2 1:2

PLF3 1:3

The batches were prepared according to the formulation design in below table

Trial Batches of PVP k-30 & Tween-80

Formulation of Preliminary Batches

Batches Code PVF1 PVF2 PVF3 TF4 TF5 TF6 PVTF7 PVTF8 PVTF9

Clomiphene

citrate (mg) 25 25 25 25 25 25 25 25 25

PVP k30 (mg) 25 37.5 50 ---- ---- ---- 25 37.5 50

Tween 80 (mg) ---- ---- ---- 25 37.5 50 50 37.5 25

Methanol (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Water (ml) 25 25 25 25 25 25 25 25 25

Variables of 32 Full Factorial Design

Independent and Dependent Variable

Independent variable

(concentration) Dependent variable

X1 X2 Y1 Y2 Y3

PVP k – 30

(polymer)

Tween – 80

(stabilizer)

Solubility after

30 min. (Q30)

Solubility after

24 hours (QEq.)

% Drug

Release

32 Full Factorial Design

Levels of Independent Variables

Coded value Actual value

X1 (PVP k – 30 in mg) X2 (Tween – 80 in mg)

-1 25 50

0 37.5 37.5

1 50 25

32 Full Factorial Design Layout

Factorial Design Layout

Formulation Code X1 (PVP k - 30) X2 (Tween - 80) Coded value

FF1 -1 -1 (-1,-1)

FF2 -1 0 (-1,0)

FF3 -1 1 (-1,1)

FF4 0 -1 (0,-1)

FF5 0 0 (0,0)

FF6 0 1 (0,1)

FF7 1 -1 (1,-1)

FF8 1 0 (1,0)

FF9 1 1 (1,1)


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Trial Batches of 32 Full Factorial Design

Composition of The Factorial Design Batches

Ingredients FF1 FF2 FF3 FF4 FF5 FF6 FF7 FF8 FF9

Clomiphene citrate

(mg) 50 50 50 50 50 50 50 50 50

PVP k - 30 (mg) 25 25 25 37.5 37.5 37.5 50 50 50

Tween - 80 (mg) 50 37.5 25 50 37.5 25 50 37.5 25

Methanol (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Water (ml) 50 50 50 50 50 50 50 50 50

Result of Preformulation Study

Organoleptic charecteristics of Drug

Sr. No. Organoleptic Properties Result

1 State Solid Crystalline powder.

2 Color White to pale yellow

3 Taste Tasteless

4 Odor Odorless

Melting point of Drug

Reported value Actual value

Mean ± SD

116.5◦c - 118

◦c 117.5

◦c ± 0.5

◦c

Figure 2: Standard calibration curve of Clomiphene citrate.


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Standard linear calibration curve of clomiphene citrate.

Abs. – Conc. Data of Clomiphene citrate

Concentration

(μg/ml)

Absorbence Average value

(λ max = 232 nm) Mean ± SD I II III

0 0 0 0 0

5 0.1362 0.1361 0.1363 0.1362 ± 0.0001

10 0.3270 0.3274 0.3272 0.3272 ± 0.0002

15 0.4969 0.4968 0.4967 0.4968 ± 0.0001

20 0.6456 0.6453 0.6450 0.6453 ± 0.0003

25 0.8549 0.8545 0.8553 0.8549 ± 0.0004

Standard calibration curve of clomiphene citrate (Graphycally)

Absorbence (y) = 0.034 * Concentration (x) – 0.016

Correlation coefficients (R2) = 0.997 Slope = 0.034 and Intercept = 0.016


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Solubility of Drug in different Dissolution media

Solvent or Dissolution Media Solubility (mcg/ml) Mean ± SD

Water 179.40 ± 0.06

pH 1.2 Acid buffer 212.45 ± 0.05

pH 4.6 Phosphate buffer 198.41 ± 0.02

pH 6.8 Phosphate buffer 183.32 ± 0.04

pH 7.4 buffer 174.46 ± 0.03

Fourier Transform Infrared spectroscopic (FTIR) studies

FTIR of pure drug clomiphene citrate.

FTIR of Clomiphene citrate + Chitosan.


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FTIR of Clomiphene Citrate + PLGA.

FTIR of Clomiphene Citrate + PVP k – 30.

FTIR interpretation

Sr.no. Functional

group

Drug Frequency

(cm-1

)

Mixture

Frequency

(PVP k-30)

(cm-1

)

Mixture

Frequency

(Chitosan)

(cm-1

)

Mixture

Frequency

(PLGA)

(cm-1

)

1 C=O 1730.14 (1900-1500) 1728 .95 1727.82 1729.60

2 N-H 2714.81 (3800-1500) 2935 .90 2996.60 2992.12

3 O-H 1214.88 (1200-1250) 1213 .56 1210.63 1173.68


544


GC of standard methanol

GC of Clomiphene citrate Nanosuspension


545


Solubility after 30 min

Result of Preliminary Trials

Result of preliminary trials Solubility study after 30 min.

Formulation

Code

Solubility

(mg/ml)

Mean ± SD

Drug 0.056 ± 0.06

CF1 0.098 ± 0.02

CF2 0.140 ± 0.03

CF3 0.053 ± 0.06

PLF1 0.080 ± 0.04

PLF2 0.103 ± 0.02

PLF3 0.091 ± 0.08

PVF1 0.371 ± 0.04

PVF2 0.385 ± 0.04

PVF3 0.300 ± 0.02

TF1 0.215 ± 0.03

TF2 0.424 ± 0.08

TF3 0.338 ± 0.04

PTF1 0.626 ± 0.06

PTF2 0.544 ± 0.02

PTF3 0.503 ± 0.03

Solubility study of preliminary trial batches


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Result of Factorial Batches

Solubility study of factorial batches

solubility profile of factorial batches

Formulation

Code

Solubility after 30 min. (Q30)

(mg/ml) Mean ± SD

Solubility after 24 hrs.

(QEq.) (mg/ml) Mean ± SD

Drug 0.056 ± 0.06 0.179 ± 0.03

FF1 0.296 ± 0.04 0.373 ± 0.06

FF2 0.249 ± 0.02 0.339 ± 0.02

FF3 0.243 ± 0.03 0.302 ± 0.04

FF4 0.447 ± 0.02 0.539 ± 0.03

FF5 0.495 ± 0.06 0.521 ± 0.06

FF6 0.698 ± 0.03 0.713 ± 0.04

FF7 0.314 ± 0.04 0.326 ± 0.03

FF8 0.302 ± 0.02 0.318 ± 0.03

FF9 0.568 ± 0.03 0.602 ± 0.04


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Zeta sizer of Chitosan optimized batch

Zeta sizer of Factorial optimized batch (FF6)


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Zeta sizer of optimized PLGA batch

Result of SEM Study

Chitosan optimized batch

Full Factorial Optimized Batch (FF6)


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PLGA optimized batch

Result of In-vitro Drug Release

% drug release of factorial batches

% Drug Release

Formulation

Code

Time (min).

5 15 30 45 60

Drug 17.36 ± 0.14 32.66 ± 0.18 41.60 ± 0.12 42.85 ± 0.11 40.95 ± 0.25

FF1 36.91 ± 0.22 56.71 ± 0.16 72.18 ± 0.23 81.35 ± 0.25 80.05 ± 0.09

FF2 34.86 ± 0.25 53.84 ± 0.31 71.54 ± 0.26 80.12 ± 0.18 79.75 ± 0.25

FF3 38.09 ± 0.11 56.86 ± 0.25 69.22 ± 0.20 77.95 ± 0.15 76.80 ± 0.22

FF4 38.71 ± 0.09 59.86 ± 0.14 74.45 ± 0.15 87.16 ± 0.18 85.12 ± 0.08

FF5 39.49 ± 0.25 60.31 ± 0.20 75.12 ± 0.09 86.37 ± 0.11 84.45 ± 0.15

FF6 43.17 ± 0.15 62.66 ± 0.18 77.89 ± 0.06 90.55 ± 0.25 88.15 ± 0.13

FF7 37.78 ± 0.36 58.52 ± 0.22 73.18 ± 0.25 78.45 ± 0.20 77.60 ± 0.14

FF8 37.05 ± 0.30 55.64 ± 0.11 68.66 ± 0.09 78.05 ± 0.28 77.36 ± 0.18

FF9 40.12 ± 0.25 60.15 ± 0.17 74.20 ± 0.12 88.62 ± 0.23 86.17 ± 0.27

In vitro drug release study of factorial batches


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Effect of Formulation Variable on solubility after 30 min. (Q30) (Y1)

Design-Expert® Software

Factor Coding: ActualSolubility (30 min) (mg/ml)

Design Points

0.698

0.243

X1 = A: PVP K30

X2 = B: TWEEN 80

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1Solubility (30 min) (mg/ml)

A: PVP K30 (mg)

B: T

WE

EN

80

(mg)

0.3

0.3

0.4

0.4

0.5

0.6

Counter plot of Solubility (Q30)


Factor Coding: ActualSolubility (30 min) (mg/ml)

Design points above predicted value

Design points below predicted value

0.698

0.243

X1 = A: PVP K30

X2 = B: TWEEN 80

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

10.2

0.3

0.4

0.5

0.6

0.7

Sol

ubili

ty (

30 m

in)

(mg/

ml)

A: PVP K30 (mg)B: TWEEN 80 (mg)

Surface plot of Solubility (Q30)

ANOVA for (Q30)

ANOVA for solubility after 30 min. (Q30)

Source Sum of Squares Df Mean square F value p– value

Prob > F

Significant

Model 0.19 5 0.038 9.38 0.0474

A 0.026 1 0.026 6.41 0.0853

B 0.034 1 0.034 8.35 0.0630

AB 0.024 1 0.024 5.78 0.0955

A2 0.095 1 0.095 23.31 0.0169

B2 0.012 1 0.012 3.06 0.1785

Residual 0.012 3 4.078


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Regression analysis for (Q30)

Summary output of regression analysis for effect of X1 & X2 on Y1

Regression statistics

Prediction R Square 0.2672

R Square 0.9399

Adjusted R square 0.8397

Observations 9

F Value 9.38

Coefficient value

Coefficient value Coefficient value

Intercept 0.49

A 0.066

B 0.075

AB 0.077

A2

-0.22

B2

0.079

Equation:

Y2=+0.49+0.066A+0.075B+0.077AB-0.22(A)2+0.079(B)

2

Effect of Formulation Variable on solubility after 24 hrs. (QEq.) (Y2)


Factor Coding: ActualSolubility (24hrs) (mg/ml)

Design Points

0.713

0.302

X1 = A: PVP K30

X2 = B: TWEEN 80

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1Solubility (24hrs) (mg/ml)

A: PVP K30 (mg)

B: T

WE

EN

80

(mg)

0.4

0.4

0.50.5

0.6

Countor plot of Solubility (QEq.)


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Factor Coding: ActualSolubility (24hrs) (mg/ml)



0.713

0.302

X1 = A: PVP K30

X2 = B: TWEEN 80

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

10.2

0.3

0.4

0.5

0.6

0.7

0.8

Sol

ubili

ty (

24hr

s) (

mg/

ml)


Surface plot of Solubility (QEq.)

ANOVA for (QEq.)

ANOVA for solubility after 24 hrs. (QEq.)

Source Sum of Squares Df Mean square F value p– value Prob >F

Significant

Model 0.17 5 0.034 10.73 0.0394

A 8.971 1 8.971 2.85 0.1898

B 0.024 1 0.024 7.61 0.0702

AB 0.030 1 0.030 9.57 0.0535

A2 0.092 1 0.092 29.22 0.0124

B2 0.014 1 0.014 4.40 0.1269

Residual 9.433 5 0.034

Regression Analysis for (QEq.)




R Square 0.9471


Observations 9

F Value 10.73

Coefficient value


Intercept 0.54

A 0.039

B 0.063

AB 0.087

A2

-0.21

B2

0.083

Equation:

Y2=+0.54+0.039A+0.063B+0.087AB-0.21(A)2+0.083(B)

2


553


Effect of Formulation Variable on % drug release (Y3)


Factor Coding: Actual% Drug release (%)

Design Points

90.55

77.95

X1 = A: PVP K30

X2 = B: TWEEN 80

-1 -0.5 0 0.5 1

-1

-0.5

0

0.5

1% Drug release (%)

A: PVP K30 (mg)

B: T

WE

EN

80

(m

g)

80

80

8585

90

Countor plot of % Drug release


Factor Coding: Actual% Drug release (%)



90.55

77.95

X1 = A: PVP K30

X2 = B: TWEEN 80

-1

-0.5

0

0.5

1

-1

-0.5

0

0.5

175

80

85

90

95

% D

rug r

ele

ase

(%

)


Surface plot of % Drug release


554


ANOVA for % drug release

ANOVA for % Drug release

Source Sum of Squares Df Mean square F value p– value

Prob > F

Significant

Model 186.86 5 37.37 9.48 0.0467

A 5.41 1 5.41 1.37 0.3257

B 17.20 1 17.20 4.37 0.1278

AB 46.04 1 46.04 11.68 0.0419

A2 105.71 1 105.71 26.83 0.0140

B2 12.50 1 12.50 3.17 0.1729

Residual 11.82 3 3.94

Regression analysis for % Drug release




R Square 0.9405


Observations 9

F Value 9.48

Coefficient value


Intercept 86.36

A 0.95

B 1.69

AB 3.39

A2

-7.27

B2

2.50

Equation:

Y2=+86.36+0.95A+1.69B+3.39AB-7.27(A)2+2.50(B)

2

Comparison study with marketed formulation

Comparison study with marketed formulation

Time

(min).

% Drug Release

Marketed Formulation

(CLOFERT 50 Tab.)

Clomiphene citrate

Nanosuspension

0 0 0

5 18.36 ± 0.14 43.17 ± 0.15

15 35.91 ± 0.22 62.66 ± 0.18

30 51.86 ± 0.25 77.89 ± 0.06

45 65.09 ± 0.11 90.55 ± 0.25

60 74.71 ± 0.09 88.15 ± 0.13


555


STABILITY STUDY

Solubility study before and after stability study

Solubility data of Stability study

Batch

Solubility after

30 min. (Q30)

At 0 month

Solubility after

24 hrs. (QEq.)

At 0 month

Solubility after

30 min. (Q30)

At 1 month

Solubility after

24 hrs. (QEq.)

At 1 month

FF6 0.698 ± 0.03 0.713 ± 0.04 0.542 ± 0.18 0.508 ± 0.06

% Drug release profile before and after stability study

% Drug release data of stability study

Time

(min).

% Drug Release

At 0 Month After 1 Month

0 0 0

5 43.17 ± 0.15 42.07 ± 0.06

15 62.66 ± 0.18 60.05 ± 0.10

30 77.89 ± 0.06 76.19 ± 0.18

45 90.55 ± 0.25 88.15 ± 0.20

60 88.15 ± 0.13 86.55 ± 0.15

SUMMARY AND COCLUSION

Improving the solubility characteristics of slightly water soluble drug Clomiphene citrate is

important to improve its systemic availability, its dissolution. so that it could reach the

systemic circulation and have its enhanced effect. The most commonly prescribed ovulation

drug is clomiphene citrate. This drug is most often used to stimulate ovulation in women who

have infrequent or absent ovulation. But Clomiphene citrate is slightly soluble in water, so

that solubility enhancement of clomiphene citrate is important part to enhance its oral

bioavailability.

Clomiphene citrate is slightly water soluble drug, their water solubility is increased by Nano

technology approach. The highest improvement in solubility and in vitro drug release were

observed in Nanosuspension (FF6) prepared by solvent diffusion method with PVP k – 30

(polymer) and Tween – 80 (stabilizer) mixtures.

IR studies of drug and polymer mixture showed the principle peak of API and polymers and

no additional peak were found, which indicates that there was no interaction between drug

and polymer.

The Nano technology is an important tool in this direction. The biodegradable polymers

(Chitosan and PLGA) had been tried to improve the solubility and dissolution profile of


556


Clomiphene citrate. But, there was no significant results obtained. Then, PVP k-30 and

Tween-80 were used to make Nano suspensions were prepared by solvent diffusion technique

with different drug to polymer ratio and evaluated for the solubility and dissolution. Then,

optimization of Nano suspension was carried out. The prepared Nano suspension of

optimized batch analyzed for particle size determination (Zeta Sizer), SEM studies to

conform particle size. The highest improvement in solubility and dissolution were observed

in Nano suspension with ratio of 1.5:1 prepared by solvent diffusion method with PVP k - 30

polymer and Tween - 80. The prepared Nano suspension of optimized batch also studied for

gas chromatography to calculate residual amount of organic solvent (methanol). The prepared

Nano suspension of optimized batch showed more solubility and in vitro drug release

compared to marketed tablet. The formulation was subjected to stability study. Formulation

was found to be stable as there was no change in the in-vitro drug release.

Hence, it was concluded that a Nanosuspension system of Clomiphene citrate with polymer

chitosan provide a method of enhancing solubility and in vitro drug release.

CONCLUSION

Clomiphene citrate is slightly water soluble drug; their water solubility is increased by Nano

technology approach. The FTIR indicates no interaction between drug and polymer and no

change in chemical nature.The highest improvement in solubility and in vitro drug release

were observed in batch FF6 prepared by solvent diffusion method with PVP k - 30 (polymer)

and Tween - 80 (stabilizer) mixtures. The prepared Nano suspension of optimized batch

analyzed for particle size determination (Zeta Sizer), SEM studies conforms particle size. The

prepared Nano suspension showed more solubility and in vitro drug release compared to

marketed tablet. Fomulation was found to be stable as there was no change in the in-vitro

drug release.

Hence, it was concluded that a Nanosuspension (Nanotechnology) of Clomiphene citrate with

polymer PVP k 30 and Tween 80 in appropriate combination provide a method of enhancing

solubility and in vitro drug release.

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Documents

SOLUBILITY ENHANCEMENT OF CLOMIPHENE CITRATE USING