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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
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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
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GC of standard methanol
GC of Clomiphene citrate Nanosuspension
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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)
Design-Expert® Software
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)
Design-Expert® Software
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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Design-Expert® Software
Factor Coding: ActualSolubility (24hrs) (mg/ml)
Design points above predicted value
Design points below predicted value
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)
A: PVP K30 (mg)B: TWEEN 80 (mg)
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.)
Summary output of regression analysis for effect of X1 & X2 on Y2
Regression statistics
Prediction R Square 0.3672
R Square 0.9471
Adjusted R square 0.8588
Observations 9
F Value 10.73
Coefficient value
Coefficient value 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
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Effect of Formulation Variable on % drug release (Y3)
Design-Expert® Software
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
Design-Expert® Software
Factor Coding: Actual% Drug release (%)
Design points above predicted value
Design points below predicted value
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
(%
)
A: PVP K30 (mg)B: TWEEN 80 (mg)
Surface plot of % Drug release
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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
Summary output of regression analysis for effect of X1 & X2 on Y2
Regression statistics
Prediction R Square 0.2747
R Square 0.9405
Adjusted R square 0.8413
Observations 9
F Value 9.48
Coefficient value
Coefficient value 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
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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
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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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