Doctor of Philosophy - Pakistan Research Repository

Application of various strategies to enhance the

solubility and bioavailability of Rosuvastatin calcium

A dissertation submitted in partial

fulfillment of the requirements for the degree of

Doctor of Philosophy (Pharmaceutics)

by

Rai Muhammad Sarfraz Pharm. D., M.Phil.

Department of Pharmacy

Faculty of Pharmacy and Alternative Medicine,

The Islamia University of Bahawalpur,

Pakistan

2013-2016

i

In the name of Allah, Most Gracious, Most Merciful

ii

Dedicated to,

My Dearest Parents

For their endless support, appreciation, encouragement and ever sincere prayers

My Dearest Brothers, Sisters & Wife

For their love, care, confidence and giving me strength to chase my dreams

IV

I, Rai Muhammad Sarfraz, Ph.D. Scholar of Department of Pharmacy, The Islamia

University of Bahawalpur, hereby declare that the research work entitled “Application

of Various Strategies to Enhance the Solubility and Bioavailability of Rosuvastatin

Calcium” is done by me. I also certify that nothing has been incorporated in this

research work without acknowledgement and that to the best of my knowledge and

belief it does not contain any material previously published or written by any other

person or any material previously submitted for a degree in any university where due

reference is not made in the text.

Rai Muhammad Sarfraz

V

It is hereby certified that this dissertation entitled “Application of Various Strategies to

Enhance the Solubility and Bioavailability of Rosuvastatin Calcium” is based upon

the results of experiments carried out by Mr. Rai Muhammad Sarfraz under my

supervision. No portion of this work has previously been presented for higher degree in

this university or any other institute of learning and to the best of the knowledge, no

material has been used in this dissertation which is not her own work except where due

acknowledgement has been made. He has fulfilled all the requirements and is qualified to

submit this dissertation for the Degree of Doctor of Philosophy in Pharmaceutics.

Prof. Dr. Mahmood Ahmad

Supervisor,

Dean Faculty of Pharmacy and Alternative medicine,

The Islamia University of Bahawalpur

V

All praises to Allah Almighty who created the universe and knows whatever is in it,

hidden or evident, and who bestowed upon me the intellectual ability and wisdom to

search for its secrets.

All respects, blessings and love to the last Holy Prophet MUHAMMAD, (Sal-

Allaho-alaihe-wa-aalehee-wasallam), who enabled me to recognize Creator and His

creations and changed me from man to Muslim by teaching, “Seek knowledge from

cradle to grave.”

I feel ample pleasure in expressing my heartiest gratitude to my ever affectionate

supervisor Prof. Dr. Mahmood Ahmad for his supervision and constant support. His

precious assistance of constructive comments and suggestions throughout the

experimental and thesis works have contributed to the success of this research.

I would like to encompass my deep felt gratitude and appreciations to Dr. Naveed Akhtar,

Chairman Department of Pharmacy for his support to the completion of this milestone.

I record my sincere thanks and appreciation for my seniors, particularly Assistant

Professor Dr. Muhammad Usman Minhas for his valuable guidance and support.

I would like to thanks Asif Mahmood, who as a good friend was always willing to help

and give his best suggestions. It would have been a lonely lab without his. It would like

to acknowledge cooperation and support of my seniors Mrs. Ayesha Yaqoob and Mr.

Muhammad Rouf Akram.

Also I would like to acknowledge Hafiz Muhammad Saleem, peon Dean Office and

Muhammad Binyamin, Lab attendant, M.Phil/Ph.D. Research Lab. 25 for their services

and cooperation during my research work.

I express my heartfelt gratitude to my mother, father, sisters, brothers and all other

family members. Their unlimited love and affection served me a beacon of light. It is

through their benevolent help and wholehearted prayers that enabled me to complete my

studies. I am also indebted to all those people who prayed for my success.

At the last, but not the least, I am grateful to Higher Education Commission of

Pakistan for financial support in the form of Indigenous Scholarship.

RAI MUHAMMAD SARFRAZ

VI

VII

RST Rosuvastatin Calcium

HMG-CoA 3-hydroxy-3-methyl glutaryl CoA

CoA Co enzyme A

BCS Biopharmaceutics classification system

β-CD β- cyclodextrin

MAA Methacrylic acid

HPMC Hydroxypropyl methylcellulose

NCEs New Chemical Entities

PVP Polyvinyl pyrrolidone

PEG Polyethylene glycols

Log P Partition coefficient

SCF Supercritical fluid

Tc Critical temperature

Tp Critical pressure

CMC Critical micelle concentration

USP United State Pharmacopoeia

BP British Pharmacopoeia

FDA Food and Drug Agency

CEFIC European Chemical Industry Council

ApoB Apolipoprotein B

CK Creatine kinase

VIII

APS Ammonium per sulphate

MBA N, N/ methylene-bis-acrylamide

TEM Transmission electron microscopy

FTIR Fourier transforms infrared spectroscopy

PXRD Powder X-ray diffraction

SEM Scanning electron microscopy

TGA Thermal gravimetric analysis

DSC Differential scanning calorimetry

FDT’s Fast disintegrating tablets

ODT’s Oro dispersible tablets

LDL Low-density lipoprotein

HDL High-density lipoprotein

Hrs Hours

UV Ultraviolet

AUC0-∞ Area Under the Curve from zero to infinity

AUMC0-∞ Area Under the First Moment Curve from

zero to infinity

Cmax Maximum Plasma Concentration

HPLC High Performance Liquid Chromatography

GIT Gastro-intestinal Tract

MRT Mean Residence Time

Tmax Time for Maximum Plasma Concentration

t1/2 Half life

IX

b.w Body weight

ALT Alanine transaminase

AST Aspartate transaminase

MCH Mean corpuscular hemoglobin

MCV Mean corpuscular volume

MCHC Mean corpuscular hemoglobin concentration

Hb Hemoglobin

ANOVA Analysis of variance

X

S. No Contents Page

No

1 Bismillah I

2 Dedication II

3 Declaration III

4 Certificate IV

5 Acknowledgment V

6 Abbreviations VI

7 Table of Contents IX

8 List of Figures XIV

9 List of Tables XIX

10 Abstract XXI

CHAPTER # 1

1 INTRODUCTION 1

1.1 Physical Modifications 3

1.2 Chemical Modifications 3

1.3 Miscellaneous Methods 3

CHAPTER # 2

2 LITERATURE REVIEW 7

2.1 Solubility 7

2.2 Importance of solubility 10

2.3 Techniques for solubility enhancement 12

2.3.1 Physical Modifications 12

2.3.2 Chemical Modifications 12

2.3.3 Miscellaneous Methods 12

2.3.4 Particle size reduction 12

2.3.5 Solid dispersion 13

2.3.5.1 Hot-Melt Method (Fusion Method) 13

2.3.5.2 Solvent Evaporation Method 14

2.3.5.3 Hot-Melt Extraction 14

2.3.6 Nanosuspensions 15

2.3.6.1 Precipitation Technique 15

2.3.6.2 Media Milling 15

2.3.6.3 High Pressure Homogenization 16

2.3.6.4 Combined Precipitation and Homogenization 16

2.3.7 Supercritical fluid (SCF) process 16

2.3.8 Cryogenic techniques 17

2.3.8.1 Spray Freezing onto Cryogenic Fluids 17

2.3.8.2 Spray Freezing onto Cryogenic Liquids (SFL) 17

2.3.8.3 Spray Freezing into Vapor over Liquid (SFV/L) 18

XI

2.3.8.4 Ultra-Rapid Freezing (URF) 18

2.3.9 Inclusion complex formation-based techniques 18

2.3.9.1 Kneading Method 20

2.3.9.2 Lyophilization/Freeze-Drying Technique 20

2.3.9.3 Microwave Irradiation Method 20

2.3.10 Micellar solubilization 21

2.3.11 Hydrotrophy 21

2.3.12 Crystal engineering 22

2.3.13 Hydrogel microparticles 24

2.3.13.1 Drug Loading in Hydrogel Microparticles 25

2.3.13.2 Drug Release Mechanisms 26

2.3.14 Fast disintegrating tablets (FDT’s) 26

2.3.15 Other techniques 34

2.4 Cyclodextrins 35

2.5 Methacrylic acid 36

2.6 Acrylic acid 37

2.7 2-Acrylamido-2-methylpropane sulfonic acid (AMPS) 37

2.8 Statins 38

2.9 Rosuvastatin Calcium 39

2.9.1 Properties of Rosuvastatin 40

2.9.2 Pharmacokinetics 40

2.9.3 Dosing information 41

2.9.4 Adverse effects 42

2.9.4.1 Myopathy 42

2.9.4.2 Liver toxicity 42

2.9.4.3 Pharyngitis 43

2.9.5 Drug interactions 43

2.9.5.1 Cytochrome P450 43

2.9.5.2 Cyclosporine 43

2.9.5.3 Gemfibrozil 43

2.9.5.4 Antacid 43

2.9.5.5 Oral contraceptives 43

CHAPTER # 3

3 MATERIAL AND METHODS

3.1 Materials 44

3.2 Instrumentation and apparatus 44

3.3 Methods 46

3.3.1 Preparation of solid dispersions 46

3.3.2 Preparation of inclusion complexes 46

3.3.3 Preparation of RDT’s 46

3.3.4 Synthesis of Hydrogel Microparticles 48

3.3.4.1 Drug loading 48

3.4 Characterization of microparticles 50

XII

3.4.1 Product yield and entrapment efficiency 50

3.4.2 Micromeritics properties 50

3.4.2.1 Angle of repose 50

3.4.2.2 Bulk density 51

3.4.2.3 Tapped density 51

3.4.2.4 Carr’s compressibility index 51

3.4.2.5 Hausner ratio 51

3.4.3 Particle size analysis 52

3.4.4 FTIR spectroscopy 52

3.4.5 Thermal analysis 52

3.4.6 Scanning electron microscopy (SEM) 52

3.4.7 Transitions electron microscopy (TEM) 52

3.4.8 Zeta size measurement 53

3.4.9 Dissolution studies 53

3.4.10 Solubility studies 54

3.4.11 X-Ray diffraction studies 54

3.4.12 In vitro dispersion time 54

3.4.13 Water absorption ratio 54

3.4.14 Friability 55

3.4.15 Tablet disintegration 55

3.4.16 Wetting time 55

3.4.17 Swellability of hydrogels microparticles 55

3.5 In vivo studies 55

3.5.1 Study design 56

3.5.2 Instrumentation and analytical conditions 56

3.5.3 Preparation of standard solutions 56

3.5.4 Sample preparation 56

3.5.5 Chromatographic conditions 57

3.5.6 Method validation 57

3.5.7 Linearity 57

3.5.8 Precision 57

3.5.9 Specificity/selectivity 58

3.5.10 Accuracy 58

3.5.11 LLOD and LLOQ 58

3.5.12 Stability 59

3.5.13 Robustness 59

3.5.14 Pharmacokinetic analysis 59

3.6 Acute oral toxicity studies of prepared formulatios 59

3.7 Statistical analysis 59

CHAPTER # 4

4 RESULTS AND DISCUSSION

4.1 Fast Disintegrating Tablets 61

XIII

4.1.1 MICROMERITICS PROPERTIES 61

4.1.2 FTIR studies 63


4.1.4 Post compression studies 70

4.1.4.1 Weight variation and Hardness 70

4.1.4.2 Friability 72

4.1.4.3 PXRD studies 72

4.1.4.4 Scanning electron microscopy 73

4.1.4.5 Wetting time 74

4.1.4.6 Disintegration time 74

4.1.4.7 IN VITRO DISPERSION TIME 74

4.1.4.8 Water absorption ratio 76

4.1.4.9 Dissolution studies 76

4.1.4.10 Solubility studies 81

4.1.4.11 Stability studies 82

4.2 Inclusion complexes and solid dispersions 83

4.2.1 Product yield and entrapment efficiency 83

4.2.2 Micromeritics properties 84

4.2.3 Optical microscopy 85


4.2.5 Dissolution studies 87



4.2.8 Zeta sizer and zeta potential 100

4.2.9 PXRD studies 101

4.2.10 Scanning electron microscopy 102

4.2.11 Transmission electron microscopy 104

4.2.12 Stability studies 106

4.3 Hydrogel microparticles 107

4.3.1 SWELLING BEHAVIOUR 110



4.3.4 Powder X-ray diffraction studies 127

4.3.5 Scanning electron microscopy 129

4.3.6 In vitro drug release studies 131


4.3.8 Zeta size and zeta potential studies 140

4.3.9 Transmission electron microscopy 141

4.3.10 STABILITY STUDIES 143

4.4 In vivo Pharmacokinetic studies 146

XIV

4.4.1 Standard curve 146

4.4.2 HPLC method development 146

4.4.3 Pharmacokinetic evaluation of Rosuvastatin calcium 151

4.5 Acute oral toxicity study of prepared formulations 167

4.5.1 Biochemical blood analysis 167

4.5.2 Histopathological study 169

4.5.3 Acute oral toxicity study of prepared formulations 170

4.5.4 Clinical Observations 170

4.5.5 Biochemical blood analysis 171

4.5.6 Histopathological Study 171

CHAPTER # 5

5 CONCLUSION 175

CHAPTER # 6

6 REFERENCES 177

XV

Fig. No. Contents Page No.

2.1 Biopharmaceutics Classification System 9

2.2 Representations of hydrophobic cavity and hydrophilic outer

surface of cyclodextrin 19

2.3 1:1 drug cyclodextrin complexes 19

2.4 1:2 drug cyclodextrin complexes 19

2.5 Structure of β-Cyclodextrin 36

2.6 Structure of Methacrylic acid 37

2.7 Structure of acrylic acid 37

2.8 Structure of 2-Acrylamido-2-methylpropane sulfonic acid

(AMPS) 38

2.9 Mechanism of action of statins 39

2.10 Structure of Rosuvastatin calcium 40

4.1(a) FTIR spectra of rosuvastatin calcium 63

4.1(b) FTIR spectra of β-cyclodextrin 64

4.1(c) FTIR spectra of Kyron T134 64

4.1(d) FTIR spectra of sodium starch glycolate 65

4.1(e) FTIR spectra of FDT’s 65

4.2(a) TGA curves of Rosuvastatin calcium 66

4.2(b) DSC curves of Rosuvastatin calcium 67

4.2(c) TGA curves of β-cyclodextrin 67

4.2(d) DSC curves of β-cyclodextrin 68

4.2(e) TGA curves of FDT’s 69

4.2(f) DSC curves of FDT’s 69

4.3 SEM image of FDT 70

4.4 X-Ray diffraction patterns of (A) Rosuvastatin calcium, (B) β-

cyclodextrin and (C) FDT’s 73

4.5(b) Standard Curve of Rosuvastatin calcium at pH 1.2 78

4.5(c) Cumulative % drug release from different formulations of FDT’s

and RST tablets at 6.8 pH 78

4.6 Solubility studies of pure drug and FDT’s 82

4.7 Optical microscope images of microparticles 85

4.8 Solubility studies of pure drug and microparticles 87

4.9(a) Cumulative % drug release from different formulations and RST

tablet at pH 6.8 88

4.9(b) Cumulative % drug release from different formulations and RST

tablet at pH 1.2 89

4.10(a) FTIR spectra of rosuvastatin calcium 91


4.10(c) FTIR spectra of Physical mixture of rosuvastatin and β-

cyclodextrin 92

XVI

4.10(d) FTIR spectra of solid dispersions 93

4.10(e) FTIR spectra of inclusion complexes 94

4.11(a) DSC curves of Rosuvastatin calcium 95

4.11(b) TGA curves of Rosuvastatin calcium 95

4.11(c) TGA curves of β-cyclodextrin 96

4.11(d) DSC curves of β-cyclodextrin 96

4.11(e) DSC curves of inclusion complexes 98

4.11(f) TGA curves of inclusion complexes 98

4.11(g) TGA curves of solid dispersions 99

4.11(h) DSC curves of solid dispersions 99

4.12(a) Zeta size measurement of microparticles (µm) 100

4.12(b) Zeta potential measurement of microparticles 101

4.13 X-Ray diffraction patterns of (A) Rosuvastatin calcium, (B) β-

cyclodextrin and (C) solid dispersions (D) inclusion complexes 102

4.14 SEM images of lyophilized microparticles at various

magnifications 103

4.15 TEM images of lyophilized microparticles at various

magnifications 105

4.16(a) FTIR spectra of Rosuvastatin calcium 112


4.16(c) FTIR spectra of methacrylic acid 113

4.16(d) FTIR spectra of MBA 113

4.16(e) FTIR spectra of Acrylamido-2-methyl propane sulphonic acid 114

4.16(f) FTIR spectra of Ammonium per sulphate 114

4.16(g) FTIR spectra of Physical mixture of cyclodextrin and

Acrylamido-2-methyl propane sulphonic acid 115

4.16(h) FTIR spectra of Physical mixture of β-cyclodextrin and

Rosuvastatin calcium 116

4.16(i)

FTIR spectra of Physical mixture of rosuvastatin, ammonium

persulfate, Acrylamido-2-methyl propane sulphonic acid and

methylene bisacrylamide

116

4.16(j) FTIR spectra of Hydrogel microparticles containing methacrylic

acid 118

4.16(k) FTIR spectra of Hydrogel microparticles containing Acrylamido-

2-methyl propane sulphonic acid 118

4.17(a) DSC curve of rosuvastatin calcium 120

4.17(b) TGA curve of rosuvastatin calcium 120

4.17(c) TGA curve of β-cyclodextrin 121

4.17(d) DSC curve of β-cyclodextrin 121

4.17(e) TGA curve of AMPS 122

4.17(f) DSC curve of AMPS 122

4.17(g) TGA curve of MBA 123

4.17(h) DSC curve of MBA 123

4.17(i) TGA of hydrogel microparticles containing methacrylic acid 125

4.17(j) TGA of hydrogel microparticles containing AMPS 126

XVII

4.17(k) DSC of hydrogel microparticles containing methacrylic acid 126

4.17(l) DSC of hydrogel microparticles containing AMPS 127

4.18

Powder X-Ray diffraction pattern of (A) Rosuvastatin calcium,

(B) β-cyclodextrin, (C) hydrogel microparticles of methacrylic

acid, (D) hydrogel microparticles of AMPS

128

4.19 SEM images of lyophilized hydrogels microparticles at various

Magnifications 130

4.20(a) Cumulative % drug release from hydrogel microparticles

containing MAA and from RST tablets at pH 6.8 132

4.20(b) Cumulative % drug release from hydrogel microparticles

containing MAA and from RST tablets at pH 1.2 133

4.20(c) Cumulative % drug release from hydrogel microparticles

containing AMPS and from RST tablets at pH 6.8 134

4.20(d) Cumulative % drug release from hydrogel microparticles

containing AMPS and from RST tablets at pH 1.2 135

4.21(a) Solubility studies of pure drug and hydrogel microparticles

containing MAA 138

4.21(b) Solubility studies of pure drug and hydrogel microparticles

containing AMPS 139

4.22(a) Zeta size measurement of hydrogel microparticles (µm) 140

4.22(b) Zeta potential measurement of hydrogel microparticles 141

4.23 TEM images of lyophilized hydrogel microparticles at various

magnifications 142

4.24 Proposed reaction of β-cyclodextrin-g-MMA hydrogel

microparticles 144

4.25 Proposed reaction of β-cyclodextrin-g-AMPS hydrogel

microparticles 145

4.26 Standard curve of Rosuvastatin calcium in rabbit plasma 146

4.27 HPLC chromatogram of blanked Plasma 148

4.28 HPLC chromatogram of Rosuvastatin calcium in mobile phase 149

4.29 HPLC chromatogram of spiked Plasma 149

4.30 HPLC chromatogram of rabbit’s samples taken after

administration of drug 150

4.31 Plasma profile of Rosuvastatin calcium after administration of

FDT’s formulation 153


hydrogel microparticles formulation 154


commercially available tablets 155

4.34

Combined plasma profile of Rosuvastatin calcium after

administration of oral drug of FDT’s, hydrogel microparticles and

commercially available RST tablets

159


FDT’s formulation 160

4.37 Plasma profile of Rosuvastatin calcium after administration of 162

XVIII

commercially available tablets

4.38

Combined plasma profile of Rosuvastatin calcium after



166

4.39

Histological examination of heart of rabbit of control group I (A),

after FDT’s treatment group II (B), and hydrogel microparticles

group III (C)

172

4.40

Histological examination of liver of rabbit of control group I (A),


group III (C

173

4.41

Histological examination of lung of rabbit of control group I (A),


group III (C)

173

4.42

Histological examination of kidney of rabbit of control group I

(A), after FDT’s treatment group II (B), and hydrogel

microparticles group III (C)

174

4.43

Histological examination of small intestine of rabbit of control

group I (A), after FDT’s treatment group II (B), and hydrogel

microparticles group III (C)

174

XIX

Table

No. Page

No.

2.1 USP and BP solubility criteria (USP: United States

Pharmacopoeia, BP: British Pharmacopoeia) 9

2.2 Commonly used cross linking agents in hydrogel microparticles 28

2.3 Various Monomers used in the formulation of Hydrogel

Microparticles 29

2.4 Polymers used in fabrication of Hydrogel Microparticles 32

3.1 Composition of fast disintegrating tablets F1 –F15 47

3.2 Composition of (β-CD-g-MAA) hydrogel microparticles 49

3.3 Composition of (β-CD-g-AMPS) hydrogel microparticles 49

4.1 Results of bulk density, tapped density, angle of Repose,

Hausner’s ratio and Carr’s index 62

4.2 Results of weight variation, hardness, thickness, friability,

disintegration time and wetting time 71

4.3 Results of wetting volume, dispersion time, pH of tablet solution

and water absorption ratio 75

4.4 In vitro drug release kinetics of FDT’s 80

4.5 Entrapment efficiency and product yield of inclusion complexes

and solid dispersions 84

4.6 Micromeritic properties of microparticles of inclusion complexes

and solid dispersions 85

4.7 In vitro drug release kinetics of solid dispersions and inclusion

complexes 90

4.8 Entrapment efficiency and product yield of HA1 to HA 9 108

4.9 Entrapment efficiency and product yield of HS1 to HS 9 108


Hausner’s ratio and Carr’s index of HA1 to HA 9 109


Hausner’s ratio and Carr’s index of HS1 to HS9 110

4.12 In vitro drug release kinetics of hydrogel microparticles 136

4.13 Intra-day and inter-day precision data (n = 3) 150

4.14 Mean ± SEM plasma concentration of Rosuvastatin calcium after

administration of FDT’s in rabbits (n=6) 153


administration of hydrogel microparticles in rabbits (n=6) 154

4.16

Mean ± SEM plasma concentration of Rosuvastatin calcium after

administration of RST commercially available tablets in rabbits

(n=6)

155

4.17 Mean values of pharmacokinetic parameters of Rosuvastatin 156

XX

calcium following administration of FDT’s (F14)

4.18

Mean values of pharmacokinetic parameters of Rosuvastatin

calcium following administration of hydrogel microparticles

(HS2)

157

4.19


calcium following administration of commercially available RST

tablets

158


administration of FDT’s in rabbits (n=6) 160

4.21

Mean ± SEM plasma concentration of Rosuvastatin

calcium after administration of hydrogel microparticles in

rabbits (n=6)

161

4.22

Mean ± SEM plasma concentration of Rosuvastatin calcium after


(n=6)

162

4.23 Mean values of pharmacokinetic parameters of Rosuvastatin

calcium following administration of FDT’s (F14) 163

4.24


calcium following administration of hydrogel microparticles

(HS2)

164

4.25


calcium following administration of commercially available RST

tablets

165

4.26 ANOVA table for pharmacokinetic parameters of prepared

formulations (HS2 & F14) and RST tablets 166

4.27 Clinical observations of acute oral toxicity test for different

formulations 167

4.28 Biochemical blood analysis of rabbits 168

4.29 Liver, kidney and lipid profile of rabbits treated with prepared

formulations and control 169

4.30 Effect of oral administration of prepared formulations on the

organ weight (gm) of rabbits 169

XXI

ABSTRACT

The term ‘solubility’ is defined as an excess amount of solute that can be

incorporated in a given amount of solvent. Solubility of poorly water-soluble drugs is

one of the most emerging issue associated with these drugs to form a suitable dosage

form that will provide desired pharmacological response. Their low solubility causes

elimination of most of the drug from body as such, and desired therapeutic levels are not

achieved. In recent years, a large number of drugs have been developed, but nearly 70%

of new drugs have poor water solubility. Major part of the human body is made up of

water. Therefore, drugs must be having a certain aqueous solubility. The solubility of

drugs ultimately has strong impact on their bioavailability. Rosuvastatin calcium (RST)

belongs to the Biopharmaceutics Classification System class II having low solubility and

high permeability. It is a poorly water-soluble 3-hydroxy-3-methyl glutaryl CoA (HMG-

CoA) reductase inhibitor.

Efforts have been made to enhance solubility of these drugs. Different techniques

have been used to enhance solubility of these poorly water soluble drugs such as

reduction in particle size to increase surface area, thus increasing the dissolution rate of

drug, solubilization in surfactant systems, formation of water-soluble complexes, drug

derivatization such as strong electrolyte salt forms that usually have higher dissolution

rate, producing liquisolid formulations, manipulation of the solid state of a drug

substance to enhance drug dissolution i.e. by decreasing crystallinity of the drug

substance through formation of solid solutions, solid dispersion formulations.

Polymers are major players in these formulations to enhance solubility e.g.,

chitosan, polyvinyl pyrolidone, polyvinyl alcohol, β-cyclodextrin, etc. β-Cyclodextrin is

one of the most efficient polymer among all of these to work as a carrier for these drugs

to enhance solubility.

In present work, fast disintegrating tablets (FDT’s) of rosuvastatin calcium were

prepared by using β-cyclodextrin as polymer along with different super disintegrants such

as kyron T134 and sodium starch glycolate and microparticles were prepared by using β-

cyclodextrin as polymer to enhance solubility. Microparticles were prepared by using

solvent evaporation (solid dispersions), kneeding technique (inclusion complexes) and

XXII

free radical polymerization to prepare hydrogel microparticles. Prepared formulations

were evaluated by Fourier Transform Infrared Spectroscopy (FTIR), Differential

Scanning Calorimetry (DSC), Thermo Gravimetric Analysis (TGA), dissolution studies,

powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), zeta size and

zeta potential, transmission electron microscopy (TEM), and stability studies to confirm

enhancement in solubility. FDT’s were further characterized by wetting time, wetting

volume, disintegration time, dispersion time etc. Different in vitro kinetic models such as

zero order, first order, Higuchi, and Korsmeyer–Peppas were applied to determine the

release behavior of drug from prepared formulations. Results were also statistically

analyzed by mean, one-way analysis of variance (ANOVA), and p value was determined

to check significant results.

Results of FTIR and DSC of prepared formulations had revealed that stable

complex was formed between drug and polymer. SEM study of formulations had shown

that small openings were present on their surfaces. These openings facilitated the

penetration of water and rapid release of drug. From PXRD study it was observed that

drug had changed from crystalline to amorphous form. Internal morphology of TEM

images had shown that drug was present inside of FDT’s and microparticles. Zeta size

and zeta potential studies confirmed that microparticles had micron size and net charge

was neutral. Wetting time, wetting volume, disintegration time, dispersion time, water

absorption ratio of FDT’s were 43±1.15-96±1.5 seconds, 80±0.5-22±1.50 seconds,

3±1.50-77±1.50 seconds, 29±0.58-57±0.58 seconds and 1.10±0.01-2.00±0.02,

respectively.

FDT’s and microparticles dissolution studies had shown that FDT’s released 91-

97% (p=0.025) of drug while inclusion complexes and solid dispersions released 71-92%

(p=0.15) of drug and hydrogel microparticles released upto 92% of drug (p=0.02). In

contrast to prepared formulations, drug released from commercially available tablets of

Rosuvastatin calcium was very less (43%). Due to acidic nature of Rosuvastatin calcium

drug was more soluble at higher pH value i.e., at 6.8 as compared to 1.2 pH. Hydrogel

microparticles containing Acrylamido-2-methyl propane sulphonic acid (AMPS) as

monomer had shown pH independent swelling and shown better release than methacrylic

XXIII

acid (MAA) containing hydrogel microparticles. AMPS containing hydrogel

microparticles released drug at both pH values but it was better at 6.8 than 1.2.

Solubility studies revealed that prepared formulations had greater solubility at 6.8

pH phosphate buffer, 1.2 pH HCl buffer and in pure water than alone drug. All three

types of formulations had enhanced solubility of Rosuvastatin calcium but it was highest

at 6.8 pH phosphate buffer. FDT’s enhanced solubility of Rosuvastatin calcium 7.42

folds in HCl buffer of 1.2 pH, while in phosphate buffer of 6.8 pH 11.71 folds and in pure

water 9.05 folds solubility was enhanced. Microparticles prepared by solvent evaporation

had enhanced solubility 3.32 folds, 8.54 folds and 5.86 folds at 1.2 pH, 6.8 pH and in

pure water, respectively. Hydrogel microparticles prepared by AMPS had enhanced

solubility 7.53 folds, 10.66 folds and 7.30 folds at 1.2 pH, 6.8 pH and in pure water,

respectively. In case of hydrogel microparticles containing MAA had no greater impact

on solubility of Rosuvastatin calcium (RST) at 1.2 pH, while these enhanced solubility

upto 9.59 folds and 6.9 folds at pH 6.8 and in pure water. From findings it was observed

that solubility of Rosuvastatin calcium was enhanced by using these techniques.

Pharmacokinetic data had also depicted that Cmax and AUC0-24 were also greater

for prepared formulations in contrast to RST commercially available tablets. Elimination

half-life of drug was reduced upto 4 hours in our formulations. Toxicology data also

shown that no toxic effects were observed from hematological, biochemical and

histological studies.

From findings of this study it was concluded that solubility of Rosuvastatin

calcium was successfully enhanced by using techniques. Prepared formulations were

found stable during stability studies of 6 month period. Thus, we can conclude that

solubility of BCS class drugs can be enhanced by using these techniques with improved

bioavailability.

1

1. INTRODUCTION

A number of new active compounds show a very low solubility in

biological media due to lipophilic nature. Most of the pharmaceutical industries are

facing a major challenge to increase solubility of drugs by using different

formulation techniques to reach an acceptable bioavailability. More than one-third

of available drugs are poorly water soluble which are listed in USP and 41% of failures

in new drug development have been attributed due to poor biopharmaceutical properties

including poor solubility. This limits their dissolution in gastrointestinal tract and leads

to low levels of oral absorption and bioavailability [1].

Present era belongs to the development of those chemical entities that have low

dose to produce desired effects within body [2]. For this purpose main problem

originated with these chemicals is that of their low water solubility and ultimately low

bioavailability [3]. Poorly water soluble drugs are eliminated from gastrointestinal tract

before their dissolution that results in low bioavailability and reduced clinical effects

[4]. Most part of human body is composed of water and drug should have to be water

soluble to produce desired effects at low dose [3]. For this purpose work is going to be

done to improve the solubility of poorly water soluble drugs and ultimately

bioavailability [5].

The group of medications referred to as statins, 3-hydroxy-3-methylglutaryl-

coenzyme A (HMG-CoA) reductase inhibitors, has been the most popular and widely

prescribed for treating hypercholesteremia and atherosclerosis. Statins are used to lower

cholesterol levels by inhibiting the enzyme HMG-CoA reductase that catalyzes the

conversion of HMG-CoA to mevolanate, which plays a major role in the production of

cholesterol in liver [6–7].

Rosuvastatin calcium (RST) is one of the newest member of drugs that belongs

to statin group, and was approved in the United States in 2003 for the treatment of

dyslipidemia. RST also reduces the level of low-density lipoprotein (LDL) in plasma,

modestly increases level of HDL cholesterol in blood and is satisfactorily tolerated by

patients [8].

Rosuvastatin calcium, a widely prescribed antihyperlipidemic HMG-CoA

reductase inhibitor drug belongs to Class II under biopharmaceutics classification

2

system (BCS) and exhibit low and variable oral bioavailability due to its poor aqueous

solubility. It is crystalline in nature having only 20% bioavailability. Peak plasma level

is achieved within 3-5 hours and plasma protein binding is 90%. Its oral absorption is

dissolution rate limited and it requires enhancement in solubility and dissolution rate

for increasing its oral bioavailability. Several techniques such as micronization,

cyclodextrin-complexation, use of surfactants and solubilizers, solid dispersion in water

soluble and dispersible carriers, use of salts, prodrugs and polymorphs which exhibit

high solubility, microemulsions and self-emulsifying micro and nano disperse systems

have been used to enhance the solubility, dissolution rate and bioavailability of poorly

soluble drugs [9].

Different technologies can be used to enhance dissolution rate of highly

lipophilic drugs there by improving their bioavailability [10–13]. Usually, two-

component systems consisting of a hydrophilic carrier in which drug is incorporated.

The drug incorporated in hydrophilic carrier may be molecularly dispersed or may

occur as nanocrystals or amorphous nanoparticles. The improved dissolution rate of

drug can be ascribed to (i) an increased solubility of drug because of its amorphous

state or small particle size (Kelvin’s law) [14–17] (ii) an increasing surface area

available for drug dissolution because of small size of drug particles [18, 19] and (iii)

an improved wetting of drug caused by the hydrophilic carrier [20, 21].

There are number of methods for enhancing dissolution rate of poorly water-

soluble drugs including reducing particle size to increase surface area, thus increasing

the dissolution rate of drug [22], solubilization in surfactant systems [23,24], formation

of water-soluble complexes [25], drug derivatization such as strong electrolyte salt

forms that usually have higher dissolution rate [26], producing liquisolid formulations

[27], manipulation of the solid state of a drug substance to enhance drug dissolution i.e.

by decreasing crystallinity of drug substance through formation of solid solutions and

solid dispersion formulations [28].

Variety of carriers have been used in previously discussed technologies to

promote solubility enhancement of poorly water soluble drugs [29]. These includes

polyethylene glycols (PEG), polyvinyl pyrrolidone (PVP), lactose, Chitosan, β-

cyclodextrin (β-CD), and hydroxypropyl methylcellulose (HPMC) are most commonly

used enhancers [30, 31]. Now a day’s poloxamers, a group of block copolymer

3

nonionic surfactants have also been used for this purpose in various techniques [32].

These carriers have strong effect on major parameters of drugs to enhance solubility,

dissolution and bioavailability of many hydropobic drugs making them suitable

chemical moieties with improved solubility [33].

Hydrotropes have also been employed to increase aqueous solubility of poorly

soluble drugs. In many cases, the aqueous solubility of poorly soluble drugs has been

increased by 2-4 folds of magnitude, simply by mixing hydrotropes in water [34].

Despite this advantage, application of low molecular weight hydrotropes in drug

delivery has not been practical, because it may result in absorption of a significant

amount of hydrotropes themselves into the body along with drug. One approach to

prevent absorption of hydrotropes along with drug from the gastrointestinal tract after

oral administration, is to make polymeric hydrotropic agents (hydrotropic polymers).

Hydrotropic polymers are expected to provide an alternative approach for increasing

aqueous solubility of poorly soluble drugs [35, 36].

Different solubility enhancement techniques are used that can be categorized

into physical modifications, chemical modifications of the drug substance and other

miscellaneous techniques.

1.1 Physical Modifications

Particle size reduction like micronization and nanosuspension, drug dispersion

in carriers like eutectic mixtures, solid dispersions, solid solutions and cryogenic

techniques.

1.2 Chemical Modifications

Change of pH, use of buffer, derivatization, complexation and salt formation.

1.3 Miscellaneous Methods

Supercritical fluid process, use of adjuvant like surfactant, solubilizes,

cosolvency and hydrotrophy.

In kneading method, drug and polymers are triturated in pestle and mortar by

the addition of liquid drop wise which may be water or hydro alcoholic mixture

resulting in formation of slurry and reduction of particle size resulting in enhanced

4

bioavailability due to kneading. Then kneaded mixture is dried and passed through

mesh if required to bring uniformity in contents [37].

In solvent evaporation method drug and carrier are dissolved in separate

miscible solvents and then evaporation is done under vacuum to yield a solid solution.

Many researchers have studied solid dispersion of meloxicam, naproxen and

nimesulide using solvent evaporation technique. Dissolution study revealed that the

modified solvent evaporation is the most convenient and effective method for solubility

enhancement of poorly water soluble drugs, among various methods of preparation of

solid dispersions [38].

Hydrogels are three dimensional particles having capability of absorbing large

amount of water maintaining insolubility behavior due to crosslinking agents. Cross-

linked hydrophilic polymer gels, or “hydrogels,” have become an important class of

formulations in nanotechnology, biotechnology, and in pharmaceuticals due to their

distinctive material properties. When hydrogels are in the form of macroscopic links or

confined to smaller dimensions, they are termed as microgels or hydrogel

microparticles, also called as cross-linked polymeric particles. When the size of

microgels is reduced up to the range of submicrmeters, they are known as nanogels.

Microparticles are suitable for delivery of drugs because of their large surface area and

ability to modify their size and hydrophobicity. Hydrogel microparticles are employed

in solid dosage forms, i.e. tablets as superdisintegrants, e.g., polyacrylic acid (AA)

super porous hydrogel microparticles. Microgels/nanogels/hydrogel microparticles are

used in diagnostic imaging and as semiconductor nanocrystals. Hydrogel microparticles

and hydrophilic polymers are used in patterning surfaces, immobilizing cells, and

proteins within the microenvironment of hydrogels.

Cell-laden hydrogels are used as scaffolding materials in tissue engineering and

as immune isolation barriers in microencapsulation technology. Hydrogel

microparticles have been used in fabrication of tunable micro lenses that respond to pH

and temperature changes, for example, N-isopropyl acrylamide hydrogel microparticles

prepared by aqueous polymerization. Hydrogel microparticles are employed in

enhancement of hydrophobic and poorly water-soluble drugs like acyclovir whose

solubility has been enhanced by the formation of chitosan hydrogel microparticles.

5

Efforts are being continuously made to utilizing these emerging tools in solubility

enhancement of hydrophobic drugs [39].

Another interesting method to improve the dissolution of poorly water soluble

drugs is fast disintegrating tablets with a high drug load might be the incorporation of

superdisintegrants. Superdisintegrants do not irritate the gastrointestinal tract and can

be used at low amounts in the formulations. We speculate that by the incorporation of

superdisintegrants, the tablets will rapidly disintegrate which prevents crystallization of

the drug. Superdisintegrants added in the formulation increase the drug release, thus

increasing the bioavailability of drug. Mouth disintegrating tablets when placed in the

mouth, disintegrate instantaneously, releasing the drug, which dissolves or disperses in

the saliva [40].

6

Schematic representation of work plan to enhance solubility of Rosuvastatin

calcium

Methods of Preparation

Preparation of

Inclusion

Complexes

Preparation of

Hydrogel

Microparticles

Solvent

Evaporation Kneading

Technique

Free Radical

Polymerization

In-vitro Characterization

Particle Size Analysis

Pre- and Post-

compression studies

for Fast Dissolving

Tablets

Determination of

loading efficiency

Zeta potential

measurement

Swelling Studies

Drug content

estimation

Dissolution studies

DSC Studies

FTIR Studies

XRD Studies

TGA Studies

Scanning electron

microscopy

Stability Studies

Characterization

In-vivo Studies

HPLC method

development and

analysis

DD Solver/Kinetica

Statistical Analysis

One way ANOVA

Microsoft Excel

Preparation of

Solid

Dispersions

Preparation of

Fast Dissolving

Tablets

Direct

Compression

Presentation of results

7

2. LITERATURE REVIEW

2.1 Solubility

The term solubility is defined as an excess amount of solute that can be

dissolved in a certain volume of solvent. It can also be defined both quantitatively and

qualitatively. Quantitatively it is solute concentration in a saturated solution at a certain

temperature. In qualitative terms, solubility is spontaneous interaction of two or more

substances to form a homogeneous molecular dispersion. Solubility has a number of

expressions of concentration as parts, percentage, molarity, molality, volume fraction

and mole fraction [41-43].

Solubility is the property of a solid, liquid, or gaseous chemical material

called solute to dissolve in a solid, liquid, or gaseous solvent to form a uniform solution

of solute in solvent. The solubility of a substance mostly determined by solvent used as

well as on temperature and pressure. The degree of solubility of a substance in a

particular solvent is calculated as saturation concentration where adding more solute

does not increase its concentration in the solution [44].

The degree of solubility varies extensively, from infinitely soluble (fully

miscible) such as ethanol in water, to poorly soluble, such as silver chloride in water.

The term insoluble is frequently related with poorly or very poorly soluble compounds

[45]. Solubility occurs under dynamic equilibrium, which means that solubility results

from the parallel and disparate methods of dissolution and phase joining (e.g.,

precipitation of solids). Solubility equilibrium occurs when the two processes proceed

at a constant rate. Under certain conditions equilibrium in solubility may be exceeded

to give a so-called supersaturated solution, which is metastable [46].

Solubility does not to be mixed up with the capability to dissolve or liquefy a

substance, since these procedures may occur not only because of dissolution but also

because of a chemical reaction. For example, zinc is not soluble in hydrochloric acid,

but it can be desolved by chemically reacting zinc chloride and hydrogen, where zinc

chloride is soluble in hydrochloric acid. Solubility does also be governed by particle

size or other kinetic factors; given enough time, even large particles will eventually

dissolve [47].

8

Saturated solutions of ionic complexes of comparatively less solubility are

sometimes defined by solubility constants. It is a case of equilibrium procedure. It

defines balance between dissolved ions from salt and undissolved salt. Similar to other

equilibrium constants, temperature would affect the numerical value of solubility

constant. The value of this constant is usually independent of the existence of additional

species in solvent.

The Flory-Huggins solution theory is a theoretical model elaborating solubility

of polymers. The Hansen solubility factors and the Hildebrand solubility factors are

empirical approaches for the forecast of solubility. It is also probable to guess solubility

from other physical constants such as enthalpy of fusion. The partition coefficient (Log

P) is an amount of differential solubility of a compound in a hydrophobic solvent and

a hydrophilic solvent. The logarithm of these two values allows compounds to be

classified in terms of hydrophilicity (or hydrophobicity).

The Biopharmaceutics Classification System (BCS) is a guide for forecasting

the intestinal drug absorption provided by the U.S. Food and Drug Administration. This

system confines the forecasting using solubility and intestinal permeability factors.

Solubility is constructed on the highest-dose strength of fast release product. A drug is

judged extremely soluble when the maximum dose strength is soluble in 250 mL or less

of aqueous media at pH range of 1 to 7.5. The volume assessment of 250 mL is

derivative from typical bioequivalence study procedures that recommend

administration of a drug product to fasting human volunteers with a glass of water [48].

The intestinal permeability classification is based on a comparison to

intravenous injection. All those factors are extremely vital, since 85% of mostly sold

drugs in USA and Europe are orally administered. According to Biopharmaceutics

Classification System (BCS), all the drugs have been divided into four classes: class I

- high soluble and high permeable, class II - low soluble and high permeable, class III

- high soluble and low permeable and class IV - low soluble and low permeable. Drugs

belonging to Class II under BCS have low and variable oral bioavailability due to their

poor aqueous solubility as shown in Figure 2.1 [49]. United State Pharmacopoeia (USP)

and British Pharmacopoeia (BP) has also established solubility criteria as shown in

Table 2.1.

9

Figure 2.1: Biopharmaceutics Classification System.

Table 2.1: USP and BP solubility criteria (USP: United States Pharmacopoeia, BP:

British Pharmacopoeia).

Descriptive term Part of solvent required

per part of solute

Very soluble Less than 1

Freely soluble From 1 to 10

Soluble From 10 to 30

Sparingly soluble From 30 to 100

Slightly soluble From 100 to 1000

Very slightly soluble From 1000 to 10,000

Practically insoluble 10,000 and over

10

The formulation of poorly water-soluble drugs have always been a serious

problem faced by pharmaceutical scientists, and it is expected to increase, because 40%

or more of the New Chemical Entities (NCEs) created by drug discovery programs are

poorly soluble in water. Such drugs often have an uneven absorption profile and

variable bioavailability, because their performance is dissolution rate limited and is

affected by the fed and fasted state of the patient [50, 51].

If a medication is administered per-orally in solid pharmaceutical dosage forms,

such as tablets, capsules or suspension it must be released from the dosage form and

dissolved in the gastrointestinal fluids before it can be absorbed. The bioavailability of

many poorly water-soluble drugs is limited by their dissolution rates, which in turn

controlled by the surface area that is exposed to dissolution media. There are

consecutive two processes which can be identified to describe oral absorption of drugs

from solid dosage forms:

Dissolution of the drug in vivo to produce a solution and Transportation of

dissolved drug through gastrointestinal membrane. These processes are defined upon

the basis of rate constant. Slowest step is rate limiting step that control release of drug.

If rate of dissolution of drug is significantly slower than rate of absorption, then

dissolution of drug becomes rate-limiting step in absorption process [52].

Subsequently, several efforts have been made to change dissolution features of various

drugs to obtain more prompt and more complete absorption. Particle size of drug is also

very vital in transport from gastrointestinal (GI) tract to site of action by increasing

dissolution rate in gastrointestinal tract [53].

2.2 Importance of solubility

Oral ingestion is the utmost suitable and frequently used route of drug delivery

due to its ease of administration, high patient compliance, cost efficacy, minimum

sterility controls, and flexibility in the design of dosage form. As a result, several of the

generic drug companies are prone more to develop bioequivalent oral drug products

[54]. However, the main problem associated with the design of oral dosage forms is

their poor bioavailability. The oral bioavailability based on numerous aspects including

aqueous solubility, drug permeability, dissolution rate, first-pass metabolism,

presystemic metabolism, and vulnerability to efflux mechanisms. The most frequent

causes of low oral bioavailability are attributed to poor solubility and low permeability.

11

Solubility also plays a vital role for other dosage forms like parenteral

formulations [55]. Solubility is one of the significant factor to obtain desired

concentration of drug in systemic circulation for attaining necessary pharmacological

response [56]. Poorly water soluble drugs frequently require high doses in order to

reach therapeutic plasma concentrations after oral administration. Low aqueous

solubility is the main problem with formulation development of new chemical entities

as well as generic development. Any drug to be absorbed must be present in the form

of an aqueous solution at site of absorption. Water is the solvent of choice for liquid

pharmaceutical formulations. Most of the drugs are either weakly acidic or weakly basic

having poor aqueous solubility.

More than 40% NCEs (new chemical entities) developed in pharmaceutical

industry are practically insoluble in water. These poorly water soluble drugs having

slow drug absorption leads to insufficient and variable bioavailability and

gastrointestinal mucosal toxicity. For orally administered drugs solubility is the most

important rate limiting parameter to achieve their desired concentration in systemic

circulation for pharmacological response. Problem of solubility is a major challenge for

formulation scientists [57].

The improvement of drug solubility and thereby its oral bio-availability remains

one of the most challenging aspects of drug development process especially for oral-

drug delivery system. There are numerous approaches available and reported in

literature to enhance solubility of poorly water-soluble drugs. These techniques are

chosen on the basis of certain aspects such as properties of drug under consideration,

nature of excipients to be selected, and nature of intended dosage form.

The poor solubility and low dissolution rate of poorly water soluble drugs in the

aqueous gastrointestinal fluids often cause insufficient bioavailability. Especially for

class II (low solubility and high permeability) substances according to BCS, the

bioavailability may be enhanced by increasing solubility and dissolution rate of drug in

the gastro-intestinal fluids. As for BCS class II drugs, rate limiting step is drug release

from dosage form and solubility in gastric fluid and not absorption, so increasing the

solubility in turn increases bioavailability for BCS class II drugs [54, 57, 58].

The negative effect of compounds with low solubility include poor absorption

and bioavailability, insufficient solubility for intravenous (IV) dosing, development

12

challenges leading to increasing the development cost and time, burden shifted to

patient (frequent high-dose administration) [55].

2.3 Techniques for solubility enhancement

Solubility enhancement methods can be classified into physical modification,

chemical modifications of drug substance, and other techniques.

2.3.1 Physical Modifications

Decrease in particle size e.g., micronization and nanosuspension, change in

crystal form like polymorphs, amorphous form and cocrystallization, distribution of

drug in carrier system like eutectic mixtures, solid dispersions, solid solutions and

cryogenic techniques.

2.3.2 Chemical Modifications

This method involve alteration in pH, use of buffer derivatization, complexation, and

salt formation.

2.3.3 Miscellaneous Methods

Supercritical fluid method, use of adjuvants like surfactants, solubilizing agents,

cosolvency, hydrotrophy, and unique excipients.

2.3.4 Particle size reduction

The solubility of drug is frequently associated with particle size of drug. When

size of particles is reduced then surface area of particles has been increased as a result

there is increase in surface to volume ratio. This increase in surface area causes more

contact of particles with solvent that ultimately enhances its solubility.

Normally several methods are used to reduce particle size. These include

comminution and spray drying that based upon the mechanical stress to separate

aggregates of active compound. This reduction in particle size enhances solubility. This

method of solubility enhancement is very economic, cost effective and efficient.

Grinding and milling are two basic mechanical forces that are associated with

comminution. These forces apply desired level of physical stress on drug product to

separate aggregates of drug particles. In comminution and spray drying, thermal stress

is also generated that must be kept in mind while dealing with thermosensitive drugs.

13

Using conventional methodologies for practically insoluble drugs may not be able to

improve the solubility up to anticipated level.

Micronization is an alternative traditional practice for reduction in particle size.

Micronization enhances dissolution rate of drugs by increasing surface area, it does not

improves equilibrium solubility. Reduction in particle size of these drugs causes

increase in surface area, as a result rate of dissolution has increased. Micronization of

drugs is carried out by milling methods by means of jet mill, rotor stator colloid mills.

This technique cannot be used for drugs which have high dose [59].

These methods were applied to griseofulvin, progesterone, spironolactone and

fenofibrate. For these drugs, micronization enhances digestive absorption, and

subsequently their bioavailability and clinical effectiveness. Micronized fenofibrate

shown greater than 10-fold (1.3% to 20%) rise in dissolution within 30 minutes in bio

relevant media [60, 61].

2.3.5 Solid dispersion

The theory of solid dispersions was initially suggested by Sekiguchi and Obi,

who examined generation and dissolution enactment of eutectic melts of a sulfonamide

drug and a water-soluble carrier in the start of 1960s [62]. Solid dispersions exemplify

a beneficial pharmaceutical system for improving dissolution, absorption, and

therapeutic effectiveness of drugs in dosage forms. The term solid dispersion represent

to a group of solid products involving at least two dissimilar constituents, usually a

hydrophilic medium and a hydrophobic drug. The most frequently applied hydrophilic

carriers for solid dispersions comprise of polyvinylpyrrolidone (Povidone, PVP),

polyethylene glycols (PEGs), Plasdone-S630. Surfactants like Tween-80, Docusate

sodium, Pluronic-F68, and Sodium lauryl sulphate (SLS) also find a place in the

formulation of solid dispersion.

The solubility of celecoxib, halofantrine, and ritonavir has been enhanced by

solid dispersion using appropriate hydrophilic carriers like celecoxib with povidone

(PVP) and ritonavir with gelucire. Numerous methods to formulate solid dispersion of

poorly water soluble drugs with an objective to increase their aqueous solubility are

listed below [63-65].

2.3.5.1 Hot-Melt Method (Fusion Method)

14

The major benefits of direct melting process is its easiness and inexpensive. The

melting or fusion technique was first suggested by Sekiguchi and Obi to formulate rapid

release solid dispersion dosage forms. In this technique, physical blend of a drug and a

water-soluble carrier is heated directly till both of these will melts. The melted blend is

then cooled and frozen quickly in an ice bath with continuous stirring. Obtained solid

mass is then crumpled, ground, and passed through sieve which can be compacted into

tablets by using other ingredients required for tablets. The melting point of this two

phase system is reliant on its composition, i.e. the nature of chosen carrier system and

quantity of drug used [66].

The most important requirement for the development of solid dispersion by hot-

melt process is miscibility of drug and carrier in melted state. Drug and carrier should

be thermostable when using this technique for preparation of solid disperssions.

2.3.5.2 Solvent Evaporation Method

Tachibana and Nakamura [67] first time prepare solid disperssions by

dissolving carrier and drug in same solvent and then evaporated solvent under vacuum

to yield a solid solution. This allowed them to prepare a solid solution of extremely

lipophilic β-carotene in hydrophilic carrier povidone. Various researchers studied solid

dispersion of meloxicam, naproxen, and nimesulide by means of solvent evaporation

method. These outcomes recommend that said approaches can be used effectively for

enhancement of solubility and stability of solid dispersions of hydrophobic drugs [68,

69].

The major benefit of solvent evaporation technique is that thermal breakdown

of drugs or carriers can be prohibited because less temperature is needed for evaporation

of organic solvents. The basic disadvantage associated with this technique is much

greater cost required for preparation and it’s very difficult to remove organic solvents.

The probable adverse effect of apparently insignificant quantity of solvent on chemical

stability of drug, the choice of a common volatile solvent, and problem in reproducing

crystal forms [70, 71].

2.3.5.3 Hot-Melt Extrusion

Hot-melt extrusion is basically same as fusion method excluding powerful

blending of different ingredients carried out by extruder. Similar to conventional fusion

method, miscibility of drug and matrix could be difficult [72, 73]. High-shear forces

15

increases temperature of extruder that is problematic for thermosensitive compounds.

This method has advantage over conventional fusion method because it can be used for

large scale production due to continuity of this approach. Moreover, handling of

product is very convenient because at the end of extruder, shape of final product can be

adjusted for further processing without grinding [74].

2.3.6 Nanosuspension

Nanosuspension is also a favorable approach that has been developed for

effective delivery of poorly water soluble drugs. This technique is equally helpful for

both water insoluble drugs as well as oil insoluble [75-77]. A pharmaceutical

nanosuspension is a two phase system comprising of nano sized drug units stabilized

by surfactants for oral and topical use or parenteral and pulmonary application. In

nanosuspensions, generally the size of solid ingredients is kept below than one micron

with an average size of 200-600 nm [78- 80].

Several approaches applied for preparation of nanosuspensions comprise of

precipitation method, media milling, high-pressure homogenization in water, high

pressure homogenization in non-aqueous media and combination of precipitation and

high-pressure homogenization [81, 82].

2.3.6.1 Precipitation Technique

In precipitation method drug is dissolved in a solvent, which is then added to

antisolvent to precipitate crystals. This technique is advantageous because it involve

cost effective equipment’s but there is challenge of preventing microparticles formation

during growth of crystals. The selection of solvents is also critical because drug have

to be soluble at least in one solvent and this solvent should have been miscible with

antisolvent. This approach of solubility enhancement cannot be applied for those drugs

that are insoluble in aqueous and non-aqueous media [83]. Nanosuspension of Danazol

and Naproxen have been formulated by precipitation method to enhance their

dissolution rate and oral bioavailability. Decrease in particle size of naproxen was also

linked with increase in the rate of absorption nearly 4-times [84, 85].

2.3.6.2 Media Milling

The nanosuspensions can also be formulated by the application of high-shear

media mills. The milling chamber filled with milling media, water, drug, and stabilizer

16

is rotated at a very high-shear rate under controlled temperatures for some days (at least

2–7 days). The milling medium is composed of glass, Zirconium oxide, or highly cross-

linked polystyrene resin. High energy shear forces are produced as a consequence of

the impaction of milling media with drug that convert microparticles of drug into nano

sized particles [82].

2.3.6.3 High Pressure Homogenization

Nanosuspension of many hydrophobic drugs can be prepared by using High-

pressure homogenization. In this technique, suspension of drug and surfactant has been

passed through a high pressure homogenizer having nano size aperture. The principle

of this process is constructed on cavitation in aqueous phase. The cavitations forces

within particles are suitably high to change drug microparticles into nanoparticles. The

issue with this technique is the necessity of small sample particles before loading and

the fact that many cycles of homogenization are needed [86].

Dissolution rate and bioavailability of poorly soluble drugs such as

spironolactone, budesonide, and omeprazole have been enhanced by decreasing their

particle size by high pressure homogenization [87-89].

2.3.6.4 Combined Precipitation and Homogenization

The precipitated drug nanoparticles have an ability to carry on crystal

development of micron size. These crystals required to be treated with high-energy

forces (homogenization). Problem occur in stability and bioavailability due to totally

amorphous, moderately amorphous or crystalline nature. To avoid this problem

suspension of particles is ultimately homogenized which stabilize particle size achieved

after the process of precipitation.

2.3.7 Supercritical fluid (SCF) process

A new technique for production of nano size particle and solubilization whose

use has increased in past few years is supercritical fluid (SCF) technique. Supercritical

fluids have temperature and pressure above than there critical temperature (Tc) and

critical pressure (Tp), simultaneously having characteristics of liquid and gas. SCFs

close to their critical temperatures, can easily be compressed and permitting slight

variations in pressure to significantly modify density and mass carriage properties of

fluid that mainly govern its solvent ability. As soon as drug constituents are solubilized

17

inside the SCF (generally carbon dioxide), their particle size usually decreased and may

be recrystallized. Particles produced by this method usually have particle size that is

very close to each other mostly in the range of submicrons. Presently SCF methods

have confirmed the ability to produce nanoparticulate suspensions having particles

diameter 5–2,000 nm. A number of pharmaceutical industries including Nektar

Therapeutics and Lavipharm, are focusing in nano size particle manufacturing by using

SCF approach for solubility improvement.

Different approaches of SCF handling have been established to overcome

specific limitations, such as precipitation by using compressed antisolvent method

(PCA), gas antisolvent recrystallization (GAS), solution enhanced dispersion by SCF

(SEDS), fast expansion of supercritical solutions (FESS), supercritical antisolvent

methods (SAS), and aerosol supercritical extraction system (ASES) [90, 91].

2.3.8 Cryogenic techniques

Cryogenic approach has been established to improve dissolution rate of drugs

by producing nanosized amorphous drug particles with large number of pores at low-

temperature. Cryogenic techniques are defined by type of injection means (capillary,

rotary, pneumatic, and ultrasonic nozzle), site of nozzle (above or below liquid level),

and composition of cryogenic liquid (hydrofluoroalkanes, Ar, O2, N2, and organic

solvents). After cryogenic processing, dry powder can be gained by several drying

procedures such as vacuum freeze drying, atmospheric freeze drying, spray freeze

drying, and lyophilization [92-94].

2.3.8.1 Spray Freezing onto Cryogenic Fluids

Briggs and Maxvell developed method of spray freezing using cryogenic fluids.

In this method, drug and carrier (lactose, mannitol, inositol, maltose, or dextran) were

dissolved in water and atomized on surface of boiling agitated fluorocarbon refrigerant.

Sonication probe can be retained in stirred refrigerant to improve dispersion of aqueous

solution [95].

2.3.8.2 Spray Freezing into Cryogenic Liquids (SFL)

Amorphous nano sized particles of drug having greater surface area with

enhanced wetting ability can be produced by using SLF particle technique. This

technique provides direct incorporation of liquid into liquid between automatized feed

18

solution and cryogenic liquid to attain strong atomization into micro droplets and then

ultimately quicker freezing rates. Then dry and free-flowing powder is obtained by

lyophilizing frozen particles [96].

2.3.8.3 Spray Freezing into Vapor over Liquid (SFV/L)

In this technique drug solution is frozen in cryogenic fluid and ultimately

elimination of frozen solvent results in production of drug particles having greater

wetting ability. In SFV/L atomized tiny particles normally initiate to freeze in vapor

form before they interact cryogenic liquid. When solvent freezes then drug become

supersaturated in unfrozen area of atomized particles so small droplets become nucleate

and grow [97, 98].

2.3.8.4 Ultra-Rapid Freezing (URF)

Ultra-rapid freezing is an innovative cryogenic technique that produces nano

sized drug particulate with significantly improved surface area and desired surface

morphology. Presentation of drugs solution to solid surface of cryogenic moiety takes

towards immediate freezing and following lyophilization (for exclusion of solvent)

result into micronized drug having more solubility. Such rapid freezing hampers phase

parting and crystallization of pharmaceutical constituents takes towards complete

mixing of amorphous drug-carrier solid dispersions and solid solutions [99].

2.3.9 Inclusion complex formation-based techniques

Amongst all the solubility improvement methods, inclusion complex

development approach has been used more accurately to enhance aqueous solubility,

bioavailability and dissolution rate of hydrophobic drugs.

Inclusion complexes are made by the addition of nonpolar fragment or nonpolar

part of one moiety (known as guest) into the hole of second moiety or group of

molecules (known as host). The most frequently implied host molecules are

cyclodextrins. The breakdown of starch by using enzyme such as cyclodextrin-

glycosyltransferase (CGT) yields cyclic oligomers, Cyclodextrins (CDs). Basically

these are monomers of glucose linked in a donut like ring containing hydrophobic inner

cavity and hydrophilic outer surface as shown in Figure 2.2. These are crystalline, water

soluble, nonreducing, cyclic oligosaccharides. Three naturally present CDs are α-

Cyclodextrin, β-Cyclodextrin, and γ-Cyclodextrin [100].

19

Figure 2.2: Representations of hydrophobic cavity and hydrophilic outer

surface of cyclodextrin

Cyclodextrin are water soluble due to their surface. Drug cyclodextrin complex

can be formed in 1:1 or 1:2 depending upon the structure and properties of drug particle

as shown in Figure 4.3 and 4.4.

Figure 4.3: 1:1 drug cyclodextrin complexes

Figure 4.4: 1:2 drug cyclodextrin complexes

20

Several techniques have been used to formulate inclusion complexes of low

water soluble drugs with cyclodextrins are summarized below;

2.3.9.1 Kneading Method

This technique is built on soaking CDs with slight quantity of water or hydro

alcoholic solutions to transform into a paste. Then drug is placed into prepared paste

and kneaded for a definite time. The mixture prepared by kneading is dried and passed

through a sieve of specified size. In laboratory, kneading can be accomplished by means

of a mortar and pestle. On industrial level, kneading can be performed by using

extruders and other equipment’s. This is the most frequently used and simplest

technique applied to formulate inclusion complexes and it is very economical technique

[101].

2.3.9.2. Lyophilization/Freeze-Drying Technique

This technique is used to obtain more porous, amorphous particles having

greater extent of complexation between drug and cyclodextrin. In this system, the

solvent from solution is removed firstly using a freezing and following drying of

solution comprising of both drug and CD at low pressure. Thermo labile materials can

be effectively converted into complex form by applying this technique. The

disadvantage of this process is use of particular apparatus, time requiring method, and

produce product having low flowing properties. Lyophilization/freeze drying method

is thought as a replicate to solvent evaporation technique and drug and carrier are mixed

in same solvent [102].

2.3.9.3 Microwave Irradiation Method

This method includes microwave radiation reaction among drug and

complexing material by means of a microwave oven. The drug and CD in certain molar

fraction are dissolved in a combination of water and organic solvent in a definite ratio

into a round-bottom flask. The mixture is allowed to react for some time approximately

1-2 minutes at 60°C in microwave oven. After completion of reaction, suitable quantity

of solvent blend is dissolved into previously prepared reaction mixture to eliminate

remaining unreacted drug and CD. The precipitate gotten is filtered by means of

whatman filter paper, and dried up in vacuum oven at 40°C. Microwave irradiation

21

technology is a unique approach for manufacturing on large scale because it is

advantageous over other due to short reaction time and higher product yield [103].

2.3.10 Micellar solubilization

The use of surfactants to increase dissolution rate of poorly water soluble drugs

is basic, primary, and oldest technique. Surfactants decrease surface stiffness and

enhance dissolution of hydrophobic drugs in aqueous medium. They are also applied to

maintain drug suspensions. When the quantity of surfactants increases than their critical

micelle concentration (CMC, that is in the range of 0.05–0.10% for majority of

surfactants), micelle development happens which capture the drugs inside micelles.

This phenomenon is known as micellization and usual consequence is better solubility

of poorly soluble moieties. Surfactant also increases saturating ability of solids and

increases rate of disintegration of solid into smaller units [55]. Different nonionic

surfactants are frequently used include polysorbates, polyoxyethylated castor oil,

lauroyl macroglycerides, polyoxyethylated glycerides, and mono- and di-fatty acid

esters of low molecular weight polyethylene glycols. Surfactants are frequently used to

stabilize microemulsions and suspensions into which drugs are dissolved [104, 105].

Antidiabetic drugs such as gliclazide, glyburide, glimepiride, glipizide, repaglinide,

pioglitazone, and rosiglitazone are poorly water soluble drugs that form micellar

solubilization [106].

2.3.11 Hydrotrophy

Hydrotrophy is a solubilization method, in which solubility of one solute can be

enhanced by adding a large quantity of another solute called hydrotropic agent.

Hydrotropic ingredients are salts of ionic organic materials having salts of alkali metal

of different organic acids. Materials or salts that increase solubility in given solvent are

called “salt in” and those that decrease solubility are called “salt out” solute. Different

salts that are very soluble in water and having excess number of anions or cations result

in “salting in” of non electrolytes known as “hydrotropic salts”; and this phenomenon

is termed as “hydrotropism.” Hydrotrophy defined as improvement in aqueous

solubility due to the existence of large quantity of additives. The process by which it

increases solubility is more nearly associated with complexation that involve a weak

interaction between poorly soluble drugs and hydrotrophic moiety such as sodium

benzoate, sodium acetate, sodium alginate and urea [107, 108].

22

The hydrotropes are recognized as automatically accumulate in solution. The

grouping of hydrotropes on the basis of molecular structure is challenging, thus large

number of moieties have been described to show hydrotropic properties. Particularly,

ethanol, aromatic alcohols such as resorcinol, pyrogallol, catechol, α and β-naphthols,

alkaloids and salicylates e.g., caffeine and nicotine, ionic surfactants like SDS (sodium

dodecyl sulphate), diacids, and dodecylated oxidibenzene. The aromatic hydrotropes

having anionic head groups are frequently studied compounds. These are high in

number due to isomerism and their active hydrotrope action that may be because of

presence of interactive pi (π) orbital [109].

Hydrotropes having cationic hydrophilic moiety are non abundant like salts of

aromatic amines, such as procaine hydrochloride. In addition to improving aqueous

solubility of compounds, they also improve accumulation of surfactants that results in

micelle formation [110].

2.3.12 Crystal engineering

Particle size is the most important parameter that decide how much surface area

of drug is exposed to dissolution. Thus particle size is very important for drug

dissolution and is based upon various conditions used for crystallization or on

techniques used for comminution such as impact milling and fluid energy milling.

By using comminution method we can yield particles which are very dissimilar

having charge and cohesive, with likely to cause difficulties in downstream processing

and product performance. Hence, crystal engineering methods are established for

precise manifestation of drugs to yield pure powders having clear particle size range,

crystal habit, crystal form (crystalline or amorphous), surface nature and surface energy

[111]. By manipulating the crystallization conditions (use of different solvents or

change in the stirring or adding other components to crystallizing drug solution), it is

possible to prepare crystals with different packing arrangement; such crystals are called

polymorphs.

Due to this reason, polymorphs of same drug differ in their various

characteristics like melting point, dissolution rate, solubility and stability etc. Majority

of drugs have different polymorphs and efforts have been made to formulate multiple

polymorphs of drugs that are more stable under different conditions with improved

solubility. A typical example of the significance of polymorphism on bioavailability is

that of chloramphenicol palmitate suspension. Polymorphs of chloramphenicol

23

palmitate have been made that have low serum concentration as compared to same dose

of metastable polymorph [112]. Another research was carried out on oxytetracycline

and it was observed that polymorph A (have 55% dissolution at 30 min) was dissolved

more slowly than polymorph B (have 95% dissolution at 30 min) [113].

Crystal engineering technique also includes the formation of hydrates and

solvates for improving dissolution rate. Throughout crystallization method, it is likely

to entrap molecules of solvent inside lattice. If water is used as solvent, the subsequent

crystal is a hydrate; if solvent used is other than water then it is termed as solvate. Every

solvate has its own dissolution rate and solubility. Dissolution rate and solubility of a

drug differ significantly for different solvates. For example, toluene and pentanol

solvates of glibenclamide have more solubility as compared to different non solvated

polymorphs [114]. Hydrates may have rapid or slow dissolution rate than anhydrous

form. Most favorable condition for anhydrous form is that it should have fast

dissolution rate rather than hydrate. For example, dissolution rate of anhydrous form of

theophylline was rapid than its hydrate form [115]. In some cases, anhydrous forms of

drug are less soluble than hydrous form and have slow dissolution rate. It was observed

that dihydrate form of erythromycin was more soluble than monohydrate and anhydrate

forms [116]. Normally solvates are not preferably used because organic solvents are

present that can be toxic. Organic solvents are used in formulations upto specified limits

because in human, use of greater concentrations in daily dose are not recommended.

Crystal engineering suggest large number of methods to enhance solubility and

dissolution rate, which can be implemented by having complete understanding of

crystallization procedures and the specific characteristics of active pharmaceutical

moieties. The method comprises of dissolving drug in solvent and precipitation

occurred in a precise mode to yield nanoparticles by addition of an antisolvent generally

it is water [111].

Formation of cocrystals opened new ways in pharmaceutical field to solve the

issues of poorly water soluble drugs. In this technique two or more molecules are linked

together to form new crystals having superior characteristics than alone. Molecular or

ionic drug combined with cocrystal former which is in solid form at specific conditions

to form pharmaceutical cocrystals [117]. Generally these are formed by slowly

evaporating cocrystal former from solution however, sublimation, growth from the

melt, or crushing different solid cocrystal formers in a ball mill are also appropriate

approaches [118].

24

Carbamazepine: saccharin cocrystal was more stable, enhanced dissolution rate

and more rapid absorption in dogs when compared with alone crystals of

carbamazepine [119]. In another study by Childs et al., fluoxetine HCl and succinic

acid cocrystal exhibited approximately two fold enhancement in aqueous solubility

after only 5 min [120]. It was detected that itraconazole L-malic acid cocrystal shown

a related dissolution profile to that of the marketed preparation [121].

Generally, crystallization techniques consist of sublimation, crystallization

from solutions, evaporation, thermal handling, desolvation, or grinding/milling. These

are being substituted with innovative approaches of crystal engineering like SCF

techniques [122, 123] to prepare pharmaceutical solids with preferred dissolution rate

and stability. Melt sonocrystallization is an alternative emerging tool that uses

ultrasonic energy to yield spongy rapid dissolving particles for poorly water soluble

drug moieties [124]. Depending upon recently available literature, it look like that

crystal engineering techniques need to be exploited more for enhancing the dissolution

rate of hydrophobic drugs.

2.3.13 Hydrogel microparticles

A hydrogel is made from a macromolecular hydrophilic polymers that assemble

themselves in a cross-linked complex. These have ability of engrossing a great quantity

of water fluctuating from 10% to many folds of their own weight and eventually

showing swelling activities [125]. When hydrogels are of microscopic size and

arranged in smaller dimensions, they are designated as microgels or hydrogel

microparticles or called as cross-linked polymeric microparticles. When size of

microgels is decreased up to the level of submicrmeters, they are recognized as

nanogels. [126]. Microgels/nanogels have exceptional benefits for polymer-based drug

delivery systems (DDS) over polymer protein conjugates, [127] polymer–drug

conjugates, [128] micelles, [129] and vesicles [130] depending upon amphiphilic and

doubly hydrophilic block copolymers, dendrimers, [131] and submicrometer-sized

particles [132]. Microgels/nanogels’ size range from nanometers to several

micrometers with an enormous surface area for conjugation with drugs [133].

Hydrogel microparticles consist of a hydrophilic mixture, which has the

characteristics of both solid and liquid [134]. The hydrogel structure contains networks

that are formed by random cross-linking of macromolecules [135]. It has three phases:

polymeric-network matrix solid phase, interstitial fluid phase, and ionic phase. The

25

solid phase consists of a network of cross-linked polymeric chains. The cross-linked

polymeric network can be made physicochemically, for example, by Vander Waals

interactions, hydrogen bonding, electrostatic interactions, and physical entanglements

as well as by covalent bonding. The fluid phase imbibes into polymeric network pores

and give wet and elastic properties to hydrogel microparticles. Owing to these

properties, the structure of hydrogels resembles to a living tissue. The ionic phase

consists of ionizable groups that are bounded to polymeric chains and mobile ions

(counter ions and coions). This phase exists due to the presence of an electrolytic

solvent. Hydrogel microparticles can be formed from both natural and synthetic

polymers [136].

Hydrogel microparticles are used for improvement of solubility of hydrophobic

and poorly water soluble drugs [137]. Hydrogel microparticles are employed in solid

dosage forms, i.e. tablets as superdisintegrants, e.g., polyacrylic acid (AA) super porous

hydrogel microparticles [138]. Microgels/nanogels/hydrogel microparticles are used in

diagnostic Imaging [139] and as semiconductor nanocrystals [140]. Hydrogel

microparticles and hydrophilic polymers are used in patterning surfaces, immobilizing

cells, and proteins within the microenvironment of hydrogels [141]. Cell-laden

hydrogels are used as scaffolding materials in tissue engineering [142] and as immune

isolation barriers in microencapsulation technology [143]. Hydrogel microparticles

have been used in fabrication of tunable micro lenses that respond to pH and

temperature changes, for example, N-isopropyl acrylamide hydrogel microparticles

prepared by aqueous polymerization.

2.3.13.1 Drug Loading in Hydrogel Microparticles

In hydrogel microparticles, drug can be loaded by two methods [144];

1. Post loading and

2. In situ loading.

In post loading of drug, first the particles matrix is formed and then drug is

absorbed. The diffusion method drags drug molecules inside the particles. Release of

drugs from these particles is also carried out by diffusion and swelling of hydrogel

microparticles. In in situ loading of drug, the polymer solution is mixed with drug and

drug–polymer conjugates are formed.

In this method, matrix formation and encapsulation of drug molecules is carried

out side by side. Drug release can be determined by diffusion, hydrogel swelling,

26

reversible drug–polymer interactions, or degradation of labile covalent bonds [145,

146].

2.3.13.2 Drug Release Mechanisms

Release of drug from matrix of hydrogel microparticles is based upon

composition of the formulation (type of polymer, drug, monomer, initiator, cross-

linker, etc.), size and shape of particles, method of preparation, and environmental

conditions.

In addition to these factors, the following factors also affect the release of drug

from swollen particles:

Penetration of water into the particles through pores,

In contact or wetting of the particles matrix with release media,

Creation of pores by the entrapped water,

Drug and polymer degradation,

Change in the pH of particles matrix due to degradation of polymers,

Swelling ability of particles,

Presence of acidic or basic environment due to degradation of products,

Amount of drug present in the matrix of hydrogel microparticles,

Diffusion of drug in the fluid,

Absorption and desorption processes,

Hydrostatic pressure created in drug delivery devices, and

Changes occurred in the sizes of pores due to swelling of polymers [146, 147]

By these mechanisms, drug release follows the following phenomenon:

Exterior diffusion,

Interior diffusion,

Desorption, and

Chemical reactions

2.3.14 Fast disintegrating tablets (FDT’s)

According to FDA, a FDT is ‘‘a solid dosage form having active moieties that

disintegrates quickly, mostly within seconds, when employed upon tongue.’’

Commonly dissolution and absorption happens concurrently with disintegration of

27

drug. It can be given with or without water and thus eliminating use of water for oral

drug administration. Food and Drug Agency (FDA) enlisted strategies for development

of FDT’s in pharmaceutical industry to come across quality standard. Preferably, FDT’s

weight should be below than 500 mg to sustain ease of administration and it should

disintegrate in less than 30 seconds in United States Pharmacopeia (USP) using

disintegration test method or some other techniques [148].

About 50% of peoples having chronic illness do not comply with medication

[149]. Patient compliance is most important issue for attainment of all pharmacological

treatment but serious in chronic conditions. Approximately one third of hospitalized

patients in United States are because of noncompliance to medication [150].

Noncompliance in use of medication can lead towards more death rates, and ultimately

increase in the price of health care facilities all over the world. Elements accountable

for noncompliance are physician, patients, health system [151], and formulation

parameters [152]. Taste of formulation is one of the most important parameter to be

considered when discussing patient noncompliance towards medication. Unpleasant

taste of medication is especially considered in case of pediatric and geriatric patients.

Bitter taste can be reduced or even eliminated by using various formulation techniques

used for manufacturing of FDT’s.

Latest advancements in technology have shown new dosage forms for

paediatric, geriatric or non-compliant peoples, those who have problems in chewing or

swallowing solid dosage forms and are reluctant to use solid dosage forms due to

anxiety of choking [150]. Hence, MDT’s tablets are a perfect fit for them.

Superdisintegrants used in these dosage forms increase drug release, thus improvement

in bioavailability of drug [151]. When these tablets are placed in mouth, disintegrate

immediately and drug releases, which dissolves in saliva and become liquid in oral

cavity, without using water [152]. FDT’s have an advantage over convenient dosage

forms especially in case of travelling patients where there is no availability of water

[153].

Furthermore, this dosage form have characteristics of both liquid and solid

formulation. Some drugs have been absorbed from mouth, pharynx and esophagus

when they pass through these parts along with saliva. In these cases, bioavailability of

such drugs have been greatly enhanced as compared to those seen in conventional

tablets. FDT’s permits children, elderly, and as well as to general patients to take their

medications separately wherever required, much eradicating the aspect of patient non-

28

compliance. This provide advantages in terms of patient compliance, quick onset of

action, enhanced bioavailability, and better stability make these tablets popular as a

dosage form in present pharmaceutical market [154, 155].

Table 2.2: Commonly used cross linking agents in hydrogel microparticles [39]

Abbreviation Cross Linker Chemical Structure

EGDMA Ethylene Glycol

dimethacrylate

MBA Methylenebisacrylamide

GP Genipin

GA Gallic Acid

29

Table 2.3: Various Monomers used in the formulation of Hydrogel Microparticles [39]

Abbreviation Monomer Chemical Structure

HPMA Hydroxypropyl

Methacrylate

HEMA Hydroxyethyl methacrylate

HEEMA Hydroxyethoxyethyl

methacrylate

HDEEMA Hydroxydiethoxyethyl

methacrylate

MEMA Methoxyethyl methacrylate

MEEMA Methoxyethoxyethyl

methacrylate

30

MDEEMA Methoxydiethoxyethyl

methacrylate

EGDMA Ethylene glycol

dimethacrylate

NVP N-vinyl-2-pyrrolidone

NIPAAm N-isopropyl AAm

VAc Vinyl acetate

AA Acrylic acid

HPMA N-(2-hydroxypropyl)

methacrylamide

EG Ethylene glycol

31

PEGA PEG acrylate

PEGMA PEG methacrylate

PEGDA PEG diacrylate

PEGDMA PEG dimethacrylate

32

Table 2.4: Polymers used in fabrication of Hydrogel Microparticles [39]

Polymers Abbrevi

ation

Structure

β-Cyclodextrin β -CD

Chitosan CHIT

Polyvinyl pyrollidone PVP

Carboxy methyl cellulose CMC

33

Hydroxy methyl cellulose HMC

Polyvinyl alcohol PVA

Polyhydroxy ethyl

methacrylate

PHEMA

Polyethylene glycol PEG

Pluronic F127 PF127

Poly(N-

isopropylacrylamide)

PNIPA

M

34

Polyacrylamide

PAAm

Polyacrylic acid

PAA

Poly (methyl methacrylate) PMMA

Carrageenan CGN

2.3.15 Other techniques

Other methods that improve the solubility of poorly water soluble drugs

comprise of salt formation, modification in dielectric constant of solvent, Chemical

alteration of drug, use of hydrates or solvates, Soluble prodrug usage, application of

ultrasonic waves, and spherical crystallization.

35

2.4 Cyclodextrins

Cyclodextrins are cyclic oligosaccharides, containing six, seven or eight

glucopyranose units (α, β or γ, respectively) obtained by the enzymatic degradation of

starch. Natural cyclodextrins have limited water solubility. However, a significant

increase in water solubility has been obtained by alkylation of the free hydroxyl groups

of the cyclodextrins resulting in hydroxyl alkyl, methyl and sulfobutyl derivatives.

Complexation of drugs in various host molecules not only works as an operative tool

for carrying drugs to target site, but in various situations is also known to enhance their

therapeutic efficacy. A detailed knowledge of the fundamental laws that manage the

interaction of these complexes is essential for better design of delivery systems

associated with efficient modulation of clinical efficacy.

β-Cyclodextrin (β-CD) is an acyclic oligosaccharide that works as an active

drug carrier for large number of drugs. It consist of D (+)-glucopyranose components

linked by α-(1, 4)-glycosidic association. The molecule shape is just like a torus, with

openings of different sizes at the primary hydroxyl and secondary hydroxyl sides of

cyclic sugar network. The outer side of the molecule is hydrophilic and the

cyclodextrins are soluble in water. However, inner side of the cavity contains a ring of

C–H groups, a ring of glucosidic oxygens, and additional ring of C–H groups; that’s

why inner cavity is comparatively hydrophobic. The interior diameter of cavity is 6.5

°A and depth is 8 °A [156]. Because of its capability to make inclusion complexes with

numerous molecules (guests) of appropriate dimension, leads towards alteration in

some physicochemical characteristics of guest molecules, it has found wide use in the

pharmaceutical industry. Cyclodextrins are able to form complexes with poorly water

soluble drugs and have been shown to improve pharmaceutical properties like

solubility, dissolution rate, bioavailability, stability and even palatability without

affecting their intrinsic lipophilicity or pharmacological properties. The ability of

cyclodextrins to form complexes may also be enhanced by substitution on the hydroxyl

group [157, 158].

The use of cyclodextrins (CD) involves simple and most efficient method.

Cyclodextrins (CD) are a group of structurally-related cyclic oligosaccharides that

have a polar cavity and hydrophilic external surface. Inclusion complexes are formed

by insertion of nonpolar molecule or nonpolar region of one molecule (known as

guest) into the cavity of another molecule or group of molecules (known as host).

Cyclodextrins are most commonly used as guest molecules. Cyclodextrins consisting

36

of 6, 7 and 8 D glucopyranosyl units connected to α-1,4 glycosidic linkages are

known as Q, R, D cyclodextrins, respectively [159].

The derivatives of R-cyclodextrin with increased water solubility (e.g.

hydroxypropyl-R-cyclodextrin HP-R-CD) are most commonly used in pharmaceutical

formulation. Hydrophilic cyclodextrins are nontoxic in normal doses. Co-

administration of water-soluble polymers with drug- CD (cyclodextrins) complex has

increased efficiency of CD in increasing aqueous solubility of drug with using

minimal amount of CD [160].

Figure 2.5: Structure of β-Cyclodextrin

2.5 Methacrylic acid

Molecular formula of methacrylic acid (MAA) is C4H6O2. Its molecular weight

is 86.09 g/mol and IUPAC name is 2-propenoic acid, 2-methyl. According to CEFIC

(European Chemical Industry Council), MAA is used in chemical industry for

manufacturing of a variety of polymers mainly its ester derivatives. Hydrogels

comprises of poly (methacrylic acid) (PMA) grafted with poly (ethylene glycol)

(PEG) exhibit pH responsive behavior [161]. Methacrylic acid controls the hydrolytic

and swelling behavior of hydrogels. High contents of methacrylic acid increase

swelling of hydrogel in alkaline/ intestinal medium and vice versa [162].

37

Figure 2.6: structure of Methacrylic acid

2.6 Acrylic acid

Acrylic acid is a colorless liquid with an acrid odor at room temperature. It is

miscible with water and most organic solvents. Acrylic acid is highly reactive so

polymerize very easily. The polymerization is catalyzed by heat, light, and

peroxides and inhibited by stabilizers, such as monomethyl ether of hydroquinone

or hydroquinone itself. Acrylic acid is hydrophilic and become ionize at high pH due

to carboxylic group. Acrylic acid based hydrogels also having electro sensitive

properties [163]. Hydrogels prepared with acrylic acid broadly used in

mucoadhesive system for drug delivery. Acrylic acid has capability to build up

different intermolecular contacts to produce hydrogels with other polymers. Swelling

capacity mainly depend upon acrylic acid intermolecular forces [164].

Figure 2.7: Structure of acrylic acid

2.7 2 Acrylamido-2-methylpropane sulfonic acid (AMPS)

2-Acrylamido-2-methylpropane sulfonic acid (AMPS) is white crystalline

powder or having granular particles with chemical formula C7H13NO4S. Its molecular

weight is 207.24 g/mol. AMPS is a hydrophilic, very reactive, sulfonic acid acrylic

monomer that is used to change the characteristics of large number of polymers. AMPS

38

has sulfonate group that is very rapidly ionized and due to this property it has gained

great attention from last two decades. Its dissociation is pH independent and completely

dissociated at pH range of 1.2-7.5. Due to this property hydrogels prepared by using

AMPS show pH independent swelling. From different studies it was observed that

polymers having sulfonate groups obtained from AMPS show great swelling in aqueous

solution. Han et al. 2011, studied swelling behaviour of hydrogels prepared from AMPS

and N, N-dimethyl acrylamide (DMAA) and also found that swelling was pH

independent [165].

Figure 2.8: Structure of 2-Acrylamido-2-methylpropane sulfonic acid (AMPS)

2.8 Statins

HMG-CoA reductase inhibitors or statins inhibit conversion of HMG-CoA to

mevalonate which is an early and rate-limiting step in the biosynthesis of cholesterol as

shown in Figure 2.9. By inhibiting this enzyme, statins markedly reduce plasma

concentrations of low density lipoprotein cholesterol (LDL -C) and total cholesterol

(total-C) and to a lesser extent decrease the concentrations of apolipoprotein B (ApoB)

and triglycerides (TGs) and increase the concentration of high-density lipoprotein

cholesterol (HDL-C). Liver is the primary site of action at which statins inhibit

biosynthesis of cholesterol [166].

39

Figure 2.9: Mechanism of action of statins

2.9 Rosuvastatin Calcium

Rosuvastatin Calcium (RST), a poorly water-soluble 3-hydroxy-3-methyl

glutaryl Co-enzyme A (HMG-CoA) reductase inhibitor. This enzyme catalyzes

conversion of HMG-CoA to mevalonate, which is an early and rate-limiting step in

cholesterol biosynthesis, a potent lipid-lower ingredient and used as hypolipidemic

agent. The liver is the primary site of action at which the statins inhibit the biosynthesis

of cholesterol. It is also used in the treatment of osteoporosis, benign prostatic

hyperplasia and Alzheimer’s disease. The molecular formula is C22H28FN3O6S and

Molecular mass is 481.539 [167].

40

Figure 2.10: Structure of Rosuvastatin calcium

2.9.1 Properties of Rosuvastatin

RST is crystalline in nature, so it reduces its aqueous solubility and finally

results in a normal bioavailability of 20%. After oral administration of RST, peak

plasma concentration is reached within 3–5 hr, volume of distribution is 1.1-1.4

liter/kg and plasma protein binding is 90%. RST is extensively metabolized by

oxidation, lactonisation and glucuronidation and metabolites are eliminated by biliary

secretion and direct secretion from blood to intestine [168-170].

Like other statins, rosuvastatin is indicated as an adjunct to diet to reduce

elevated total-C, LDL-C and increase HDL-C concentrations in patients with

primary hypercholesterolemia (heterozygous familial and nonfamilial) and mixed

dyslipidaemia [116]. Rosuvastatin calcium is supplied in tablets in amounts equivalent

to 10 mg, 20 mg, and 40 mg of rosuvastatin. The promotion and availability of 40-

mg dosage/tablets is more limited to discourage any indiscriminate use of the higher

dosage that is associated with greater risks [171].

2.9.2 Pharmacokinetics

The peak plasma concentration (Cmax) of 6.1 ng/ml is reached at 5 hours

after a single oral dose of 20 mg. Prolonged dosing with 20 mg once daily leads

to a steady state Cmax of 9.7 ng/ml which occurs 3-5 hours after dosing. In

pharmacokinetics trials, Cmax and area under the concentration time curve (AUC0–24)

demonstrated an approximately linear relation throughout dosage range of 5 to

80 mg after single and seven daily doses. Steady state tmax ranged from 3 to 5 hours

and it was longer than other currently available statins (with tmax values usually lower

41

than 3 hours) [172, 173]. The elimination half-life (t1/2) is about 19 hours in healthy

volunteers [174].

2.9.3 Dosing information

Rosuvastatin calcium is available in tablets in quantities equivalent to 5 mg, 10

mg, 20 mg, and 40 mg of rosuvastatin. The promotion and availability of 40-mg

dosage/tablets is more restricted and discourage because higher dosages are linked with

greater risks.

In the management of hypercholesterolemia, traditionally recommended initial

dose is 10 mg once a day.

A primary dosage of 5 mg once a day should be recommended for patients who

do not have much higher values of LDL-C. Higher doses of satins are also prone

towards greater chances of development of myopathy.

For patients with marked hypercholesterolemia and aggressive lipid targets, a

starting dosage of 20 mg once a day may be considered.

A dosage of 40 mg once a day should be reserved for those patients who have

not achieved the LDL-C goal at a dosage of 20 mg.

Concomitant drug use - the dosage of rosuvastatin should not exceed 5 mg once

a day in patients who are also being treated with cyclosporine, or 10 mg once a

day in patients also treated with gemfibrozil as these drugs can increase the

levels of rosuvastatin and increase the risk of myopathy.

Kidney failure - Dosage adjustment is not necessary in patients with mild or

moderate renal insufficiency. However, in patients with severe renal

impairment (creatinine clearance less than 30 mL/minute) who are not on

hemodialysis, the initial dosage should be 5 mg once a day, and should not

exceed 10 mg once a day.

Food – doses can be taken without regard to meals.

Time of day – like atorvastatin, doses of rosuvastatin can be taken at any time

of the day. Other statins are taken at night to maximize the action of the statin

by inhibiting the synthesis of cholesterol which takes place during the night

[166, 175].

42

2.9.4 Adverse effects

2.9.4.1 Myopathy

In one trial, 5.5% of patients taking rosuvastatin group had myalgia. Creatine

kinase (CK) levels in almost half of these patients were 10 times the upper limit

of normal (ULN). All patients were taking 8-0mg rosuvastatin daily except for

one patient who was taking 5mg. Another 1% of patients had raised CK levels

but had no symptoms of myopathy. The incidence of these effects was similar

to atorvastatin [176].

In another trial using lower doses of rosuvastatin (5-10mg daily), no cases of

myopathy (defined as CK elevation >10 times the ULN accompanied by muscle

symptoms) were observed [177]. Three patients out of 239 studied had single

transient elevations in CK that were >10 times the ULN without overt muscle

symptoms.

Rare cases of rhabdomyolysis have been reported in clinical trials where doses

>80mg have been used [166].

The Food and Drug Agency (FDA) in the United States has issued a warning

regarding rosuvastatin advising physicians to start doses as low as possible and

that maintenance doses of drug should be based on individual cholesterol goals.

All patients should be informed that statins can cause muscle injury, which in

rare, severe cases, can cause kidney damage and other organ failure and is

potentially life-threatening. Patients should be told to promptly report to their

physician signs or symptoms of muscle pain and weakness, malaise, fever, dark

urine, nausea, or vomiting [178].

2.9.4.2 Liver toxicity

In one trial, 4% of patients taking rosuvastatin had liver enzymes (ALT)

measured at three times the upper limit of normal level. Most of these patients

were taking 80 mg of rosuvastatin. Another trial using lower doses of

rosuvastatin (5-10 mg) recorded that 0.8 % of patients studied had ALT levels

at three times the upper limit of normal level [177].

No cases of liver failure or irreversible liver disease has been reported in clinical

trial [172].

43

2.9.4.3 Pharyngitis

Pharyngitis has been reported as occurring in approximately 9% of patients

taking rosuvastatin. It is not clear why this occurs with rosuvastatin doses. However,

other gastrointestinal reactions have been reported – diarrhea, constipation, stomach

upset and nausea which occur in approximately 3% of patients studied [177].

2.9.5 Drug interactions

2.9.5.1 Cytochrome P450

Rosuvastatin clearance is not dependent on metabolism by CYP 3A4 to a

clinically significant extent, thus rosuvastatin is unlikely to have blood levels increased

by drugs which inhibit CYP 3A4 enzyme system.

2.9.5.2 Cyclosporine

Co administration of cyclosporine with rosuvastatin can elevate rosuvastatin

blood levels 11 times that when rosuvastatin is administered alone. This combination

can lead to the possibility of rhabdomyolysis with such high levels of rosuvastatin.

2.9.5.3 Gemfibrozil

Co administration of gemfibrozil with rosuvastatin can elevate rosuvastatin

blood levels 2 times that when rosuvastatin is administered alone. This combination can

lead to the possibility of rhabdomyolysis with such high levels of rosuvastatin.

2.9.5.4 Antacid

Co administration of cyclosporine with antacids can reduce rosuvastatin blood

levels by 50 per cent. However, when the antacid was administered 2 hours after the

rosuvastatin dose, there were no clinically significant changes in blood levels.

2.9.5.5 Oral contraceptives

Co administration of oral contraceptives with rosuvastatin can elevate

ethinyloestradiol levels by 26% and norgestrel levels by 34%, increasing the possibility

of adverse effects of these hormones [166, 179].

44

3. MATERIAL AND METHODS

3.1 Materials

Rosuvastatin Calcium was obtained as gift sample from Getz pharmaceuticals

Karachi, Pakistan. β-cyclodextrin, methacrylic acid, 2-Acrylamido-2-methylpropane

sulfonic acid (AMPS), Ammonium persulfate, methylene bisacrylamide and methanol

were purchased from Sigma Aldrich Chemie GmbH, Steinheim, Germany. Kyron T134,

sodium starch glycolate, magnesium stearate, lactose and saccharine were obtained

from Warrick pharmaceuticals Islamabad, Pakistan. Acetonitrile, diethyl ether, absolute

ethanol, dichloromethane, chloroform, diethyl acetate were purchased from Sigma–

Aldrich (Oslov, Norway). 0.45 μm sized membrane filters were purchased from

Sartorius, Germany. HPLC grade ultrapure water was prepared by Milli-Q® system

(Millipore, Milford, MA, USA). All the chemicals were used of high purity grade.

3.2 Instrumentation and apparatus

High Performance Liquid Chromatographic1, UV/Visible Spectrophotometer2,

Sonicator3, Dissolution Test Apparatus4, Centrifuge Machine5, pH Meter6,

Ultrasonic Bath7, Electric Balance8, Membrane Filter9, Magnetic Stirrer10, B.P.

Apparatus11, Vacuum Pump12, Distillation Plant13, Ultra-low Freezer14,

Micropipettes15, Filtration Assembly16, Measuring Cylinder17, Beakers18 50, 100, 250,

500 & 1000 mL, Measuring Flasks19 50, 100, 250, 500 & 1000 mL, Centrifuge

Tubes20, Sample Test Tubes21, Disposable Syringes22, Vortex Mixer23, Incubator24,

Centrifuge25, Fourier Transform Infrared Spectroscopy(FTIR) 26, Scanning Electron

Microscopy (SEM) 27, Differential Scanning Calorimeter and Thermo Gravimetric

Analyzer (DSC &TGA) 28, XRD29.

1. Agilent 1100 Series U.S.A

2. UV-1600 Shimadzu. Germany

3. Elma, Germany

4. PTCF II Pharma Test, Germany

5. Model 4000-China 35

6. WTW pH 300-Germany

45

7. Fisher Scientific FS 28 H-Germany

8. PerciaXB120A

9. Sartorius (0.45 μm filters)-Germany

10. Gallen Kamp-England

11. Model No 500-China

12. Rotary Vane Pump ILMVAC-Germany

13. WDA/4 R & M-England

14. Sanyo-Japan

15. Softpet- Finland

16-21. Pyrex-France

22. BD-Pakistan

23. Seouline Bio Scirnce-Korea

24. Velp Scientifica-Italy

25. Hettich-Germany

26. Bruker, Tenser 27-Germany

27. Quanta 250, FEI

28. SDT Q-600 (TA New Castle, DE)

29. Expert pro Panalytical-Germany

46

3.3 Methods

3.3.1 Preparation of solid dispersions

Solid dispersions were prepared by using solvent evaporation technique. In

solvent evaporation technique, drug was dissolved in solubilizing additives at 25 °C.

Then required quantity of β-cyclodextrin was dissolved in hot distilled water. Then it

was added drop wise into drug solution, with continuous stirring for one hour. The

complexes formed were filtered and dried under vacuum. Prepared solid mass was

stored in a desiccator under vacuum. The dried products were removed, pulverized and

passed through sieve No. 60 and finally stored in a closed air tight container for further

analysis [170]. Four formulation S1, S2, S3 and S4 were prepared by this method

containing drug and β-cyclodextrin in 1:1, 1:2, 1:3 and 1:4, respectively.

3.3.2 Preparation of inclusion complexes

Inclusion complexes were prepared by using kneeding technique. In kneeding

technique required quantities of drug and β-cyclodextrin were weighed accurately on

electronic weighing balance (Shimadzu, AUW 220D, Japan) in different ratios. A

homogenous paste of cyclodextrin was prepared in a mortar by adding water: methanol

mixture (1:3) in small quantities. Drug powder added to this paste in portions, with

continuous kneading for three hour. An appropriate quantity of water: methanol mixture

(1:3) was added further to maintain suitable consistency of paste. This paste, then dried

in a hot air oven at 45°C–50°C for 24 hour. The dried complexes were powdered and

passed through sieve No. 60 and stored in air tight containers [171]. Four formulation

K1, K2, K3 and K4 were prepared by this method containing drug and β-cyclodextrin

in 1:1, 1:2, 1:3 and 1:4, respectively.

3.3.3 Preparation of FDT’s

Fast disintegrating tablets were prepared by using direct compression

method. All ingredients required for preparation of mouth disintegrating tablets were

passed through mesh No 60 separately. Then all ingredients were weighed and mixed

in geometrical order and compressed by using 8mm round flat punches on 10 station

rotary tablet machine. Total 15 formulations were prepared by using various

concentrations of two superdisintegrants and β-cyclodextrin, to study enhancement in

solubility of rosuvastatin calcium [172] as shown in Table 1.

47

Table 1: Composition of fast disintegrating tablets F1 –F15

Ingredients

(mg)

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15

Rosuvastati

n

20 20 20 20 20 20 20 20 20 20 20 20 20 20 20

β-

cyclodextrin

- 05 10 15 20 - 05 10 15 20 20 20 20 20 20

Kyron-T134 8 8 8 8 8 - - - - - 8 12 - 16 -

Sodium

starch

glycolate

- - - - - 8 8 8 8 8 8 - 12 - 16

Mg-Stearate 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Talc 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

Lactose 158 153 148 143 138 158 153 148 143 138 130 134 134 130 130

Orange

Flavor

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Saccharine

Sodium

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

Total

Weight

200mg

48

3.3.4 Synthesis of Hydrogel Microparticles

Copolymers were prepared by free radical polymerization. Their composition

is shown in Table 3.2 and 3.3. β-cyclodextrin was mixed with different monomers in

appropriate weights to obtain desired batch. Then desired quantity of cross linker was

added to prepare copolymers. This mixture was dissolved in sufficient quantity of

water taken in a 250 ml beaker at 25 °C. After completely dissolving monomer,

nitrogen gas was purged through well-mixed homogeneous solutions for 30 minute to

remove any dissolved oxygen, a free radical scavenger, which will otherwise act as an

inhibitor. To this solution, ammonium persulfate (APS) (with respect to batch

size) was added with continuous stirring to initiate polymerization. Then reaction

was allowed to continue for 3 hours to form hydrogel and washed three times with

water and methanol solution 50:50 to remove any unreacted species. Hydrogel formed

were freeze-dried at 47 °C for overnight and passed through sieve to get hydrogel

microparticles [173].

3.3.4.1 Drug loading

A known amount of dried hydrogel microparticles were immersed in drug

solution for remote loading. After specified time period microparticles and excess

of drug solution were removed. Loaded microparticles were taken out and dried

under vaccum at low temperature [174].

49

Table 3.2: Composition of (β-CD-g-MAA) hydrogel microparticles

Code β-Cyclodextrin

(g/100g)

Methacrylic acid

(g/100g)

Cross linker

(g/100g)

Initiator

(g/100g)

HM1 2 30 0.4 0.4

HM2 4 30 0.4 0.4

HM3 6 30 0.4 0.4

HM4 4 10 0.4 0.4

HM5 4 20 0.4 0.4

HM6 4 40 0.4 0.4

HM7 4 30

0.6 0.4

HM8 4 30

0.8 0.4

HM9 4 30

01 0.4

Table 3.3: Composition of (β-CD-g-AMPS) hydrogel microparticles

Code β-Cyclodextrin

(g/100g)

AMPS

(g/100g)

Cross linker

(g/100g)

Initiator

(g/100g)

HS1 0.5 2 0.3 0.1

HS2 1 2 0.3 0.1

HS3 2 2 0.3 0.1

HS4 1 4 0.3 0.1

HS5 1 5 0.3 0.1

HS6 1 6 0.3 0.1

HS7 1 4 0.4 0.1

HS8 1 4 0.5 0.1

HS9 1 4 0.6 0.1

50

3.4 Characterization of microparticles

3.4.1 Product yield and entrapment efficiency

Prepared microparticles were dissolved in phosphate buffer of pH 6.8 (10 ml)

to facilitate that microparticles get dissolved. The resulting suspension was allowed to

evaporate for further removal of solvent prior to filtration. Then residue was washed

and diluted appropriately with phosphate buffer of pH 6.8 to determine drug content

and entrapment efficiency. Absorbance of samples were measured in triplicate at 243

nm in double beam UV/vis spectrophotometer. Similar procedure was performed with

20 mg of pure drug to calculate entrapment efficiency. Product yield of various

formulations was calculated to determine efficiency of the process.

𝑃𝑟𝑜𝑑𝑢𝑐𝑡 𝑦𝑖𝑒𝑙𝑑 = 100 − 𝑚𝑎𝑠𝑠 𝑙𝑜𝑠𝑠

Where, M0= Initial weight, M1=Final weight

The encapsulation efficiency (EE) of rosuvastatin-loaded formulations was calculated

as;

𝐸𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 % = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑐𝑜𝑛𝑡𝑎𝑖𝑛𝑖𝑛𝑔 20 𝑚𝑔 𝑅𝑆𝑇

𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 20 𝑚𝑔 𝑝𝑢𝑟𝑒 𝑅𝑆𝑇 𝑥 100

3.4.2 Micromeritics properties

3.4.2.1 Angle of repose

Power blend of each formulation was evaluated for angle of repose by funnel

technique using following equation. Value of angle of repose less than 30° confirms

free flowing of powder blend through hopper.

Where Ɵ = angle of repose, h = height of blend cone, r = radius of base of cone.

100(%)0

10

M

MMLossMass

51

3.4.2.2 Bulk density

Powder mixture was poured in graduated volume measuring cylinder (Pyrex®)

and bulk volume (Vb) was visually noted. After this powder mass (M) was measured

on electronic weighing balance (Shimadzu, AUW220D Japan). Bulk density (ρb) was

determind by using following equation.

3.4.2.3 Tapped density

Measuring cylinder containing known mass (M) of powder material was tapped

for definite number of tapings. Tapped volume (Vt) was noted. Tapped density was

calculated by using following equation.

3.4.2.4 Carr’s compressibility index

Free flowing property of powder was confirmed from compressibility index (I).

It was calculated by using following formula. Carr’s index between 13 -19 % confirms

good flow and if it is more than 21 % it presents poor flow of microparticles.

Where Vb and Vt are bulk and tapped volume, respectively.

3.4.2.5 Hausner ratio

It is another parameter of micromeritics properties determination. It is a ratio

between two densities i.e. tapped (ρt) and bulk (ρb). Value less than 1.25 indicates that

powder particles have good flow while more than 1.25 suggest poor flow.

3.4.3 Particle size analysis

The mean size (volume average particle diameter) and size distribution of

drug loaded microparticles was obtained by using light optical microscopy (Nikon

E200, Tokyo, Japan) equipped with (DCM-35 USB 2.0 and MINISEE IMAGE

software, Scopetek Electric, Hangzhou, China). Size detrmination was made in

52

triplicate by subsequent suspension of microparticles in redistilled water at 25 ºC. To

make the samples for analysis, approximately 2 mg prepared microparticles were

suspended in 5 ml of deionized water and then visualized under optical microscope

[175].

3.4.4 FTIR spectroscopy

FTIR spectra were obtained between 400 and 4000 cm-1 by using IR

spectrophotometer (Tensor 27, OPUS software) to confirm the formation of copolymer.

FTIR spectra of individual ingredients and prepared formulations were taken. Samples

were prepared by finely grinding prepared formulations and exposing to infrared

radiation [172].

3.4.5 Thermal analysis

DSC and TGA analysis of prepared formulations and all ingredients were taken

to determine stability of ingredients in prepared formulations. DSC (Q600 TA USA)

was done by heating the samples from ambient to 400 ºC at heating rate of 10 ºC/min

in a nitrogen atmosphere at flow rate of 10 ml/min. Thermal stability of copolymer was

studied by TGA (Q600 TA USA) in temperature range of 0 ºC to 800 ºC under inert

nitrogen atmosphere [172].

3.4.6 Scanning electron microscopy (SEM)

Scanning electron microscopy was performed to describe shape and

morphology as well as to check particle size. For SEM measurement’s, rosustatin

based prepared formulations were attached on metal stubs using double-sided

adhesive tape. Then it was dried in a vacuum chamber and sputter-coated with a

gold layer of 10 nm thick and viewed under high resolution SEM (JSM-840, Joel

Instruments, Tokyo, Japan) [174].

3.4.7 Transitions electron microscopy (TEM)

Transitions electron microscopy was used to determine internal morphology of

prepared microparticles. For TEM measurement’s JEM-2800 Transmission Electron

Microscope was used. For TEM analysis, rosuvastatin based prepared formulations

were attached on metal stubs using double-sided adhesive tape. Then it was dried

53

in a vacuum chamber and sputter-coated with a gold layer of 10 nm thick and viewed

under high resolution TEM at 120 kV using high magnification [176].

3.4.8 Zeta size measurement

The average particle size of prepared microparticles was measured by using

particle size analyzer (Zetasizer Ver System; Malvern Instruments Ltd., Malvern, UK).

To analyze particle size, microparticles were suspended with filtered (0.22 μm)

ultrapure water [177].

3.4.9 Dissolution studies

Dissolution studies of prepared microparticles and tablets were performed in a

calibrated 6 station dissolution test apparatus equipped with paddles USP type-2

apparatus (Pharma Test Germany) using 900 ml of 6.8 pH phosphate buffer as a

medium. The paddles were operated at 50 rpm and temperature was kept at 37 ± 0.2 ºC

throughout the experiment. 5 ml samples were taken after pre decided time interval and

replaced with equal volume of same dissolution medium to keep volume constant

during experiment. The amount of the drug dissolved was estimated by UV/Vis

spectrophotometer (Pharma Spec 1700 Shimadzu Japan) at 243 nm. The dissolution

studies on each formulation were carried out in triplicate.

Dissolution data was evaluated by means of several model-dependent methods

like zero order, first order, Higuchi, and Korsmeyer–Peppas to evaluate the kinetics of

drug release. Time required for 25% and 50% release of drug (T25% and T50%,

respectively) was also calculated from regression equation of zero-order model. The

zero-order model illustrates system where rate of drug release is independent of initial

concentration of drug:

Mt = ko𝑡

Where ko is zero-order rate constant and Mt is the amount of drug released at

time t. The release of drug from an insoluble matrix is a function of square root of time

as shown in Higuchi equation:

𝑀𝑡 = 𝐾𝐻𝑡1/2

Where KH is Higuchi rate constant, which represents design variables of system.

To determine mode of drug release, the initial 60% drug release values were fitted to

Korsmeyer–Peppas model:

54

Mt/M∞ = 𝐾𝑡𝑛

Where Mt/M∞ is fraction of drug released at time t, K is drug release rate

constant, and n is release exponent. The n value is employed for characterization of

different release modes for cylindrical-shaped solid formulations. Drug release

exponent (n) and drug release mode are related as: n = 0.45, Fickian diffusion; 0.45 <

n < 0.89, Anomalous (non-Fickian) diffusion; n = 0.89, Case-II transport; and n > 0.89,

Super case-II transport. Dissolution data was evaluated using DD Solver [178].

3.4.10 Solubility studies

The solubilities of rosuvastatin calcium and RST- β-CD complex were

determined at pH 1.2 and 6.8 and in distilled water by shake-flask method. Excess

amount of RST and RST- β-CD prepared formulations were placed in 10 ml vials to

which 1.0 ml of two different solutions (pH 1.2, 6.8) and distilled water was added.

These mixtures were shaken in a water bath (MSC-100 China) at 37±0.5 °C at 150 rpm

for 72 hour. All samples were filtered through 0.45 µm membrane filters, diluted with

buffer and analyzed for RST on UV/Vis spectrophotometer at 243 nm [179].

3.4.11 X-Ray diffraction studies

Samples were subjected for powder XRD studies to compare with physical

mixture and pure drug. Powder X-ray diffractograms were taken at 5 to 50 ºC at 2Ɵ to

study crystalline and amorphous nature of rosuvastatin calcium.

3.4.12 In vitro dispersion time

Time needed for complete dispersion of tablet was measured in this test. Six ml

of phosphate buffer having pH 6.80 was taken in a beaker having 10 ml volume. Tablet

was placed in that beaker and dispersion time was observed. The experiment was

repeated thrice for each formulation.

3.4.13 Water absorption ratio

It is the amount of water absorbed by tablet in a unit time. Six ml of water was

taken in a Petri dish of internal diameter 6.50 cm. A piece of tissue paper folded twice

was placed in petri dish. A tablet was weighed before keeping onto tissue paper in petri

dish and time taken by tablet for complete wetting was measured. Wetted tablet was

reweighed and water absorption ratio was calculated [172].

55

3.4.14 Friability

Friability of tablets was determined by using Roche Friabilator (Pharma Test,

Germany). Twenty tablets were weighed and placed in the drum of friabilator and speed

was kept at 25 rpm. Tablets were allowed to revolve, fall freely from height of six

inches for 4 min. Then tablets were de-dusted using muslin cloth and re-weighed [180].

3.4.15 Tablet disintegration

Tablet disintegration apparatus was used. Six tablets were taken and placed

individually in each tube and cover them properly. The temperature of medium was

maintained at 37±2°C. Time taken by the tablet to disintegrate completely was noted

[181].

3.4.16 Wetting time

10 ml solution of phosphate buffer of pH 6.8 as saliva was taken in petri dish.

A circular tissue paper having diameter 8 cm folded twice was placed in petri dish.

Single fast disintegrating tablet was placed on tissue paper and time for complete

wetting was noted [180].

3.4.17 Swellability of hydrogels microparticles

The swelling ability of prepared hydrogel microparticles were determined at

37 °C by introducing known amount of hydrogel microparticles in 100 ml 0.5 M

buffer solutions of pH 1.2 and 7.4. The samples were taken at predefined time

intervals to calculate dynamic swelling, weighed after removing excess of water

by blotting with tissue paper. The swelling studies were continued until equilibrium

weight was achieved. The degree of swelling and equilibrium water content were

determined using following equations [183].

Q=𝑀𝑠

𝑀𝑑

𝐸𝑊𝐶% =𝑀𝑠 − 𝑀𝑑

𝑀𝑑× 100

3.5 In vivo studies

In vivo studies were performed using Agilent 1100 series HPLC system to

calculate different pharmacokinetic parameters in rabbit’s plasma.

56

3.5.1 Study design

18 male albino rabbits of average weight 2.5 ± 0.010 kg were used for this

study. Rabbits were divided into 3 groups of 6 rabbits each (n=6). All the rabbits were

fasted overnight with ad libitum access to water during experiment and animals were

fed 4 for hour after oral dose. Two group of animals had received a single dose of FDT’s

and hydrogel microparticles containing 20 mg of rosuvastatin calcium while 3rd group

had given a 20 mg of commercially available tablet of rosuvastatin. Samples were

drawn at 0, 0.5, 1, 1.5, 2, 4, 6 and 12 hour.

3.5.2 Instrumentation and analytical conditions

Agilent 1100 series HPLC system consisted of LC-10 AT VP pump, DGU-14

AM on-line degasser, Rheodyne manual injector fitted with a 20 µL loop, and SPD-10

AVP UV–VIS detector and a Hypersil BDS C 8 (250 X 4.6 mm) column was utilized

for separation. Chromatographic system was integrated via Shimadzu model CBM- 102

Communication Bus Module to P-IV computer loaded with CLASS-GC software

(Version 5.03) for data acquisition and mathematical calculations. Centrifuge Machine

(Model 4000-China), Vortex Mixer (Seouline Bio Scirnce-Korea), pH Meter (WTW

pH 300-Germany), Ultrasonic Bath (Fisher Scientific FS 28 H-Germany), Electric

Balance, (Percia XB 120A), Membrane Filter (Sartorius, 0.45 µm filters-Germany),

Distillation Plant, Micropipettes (Softpet- Finland), Filtration Assembly (Pyrex-

France), Distillation Plant (WDA/4 R & M England).

3.5.3 Preparation of standard solutions

The stock solutions of rosuvastatin calcium were prepared by dissolving

appropriate amount corresponding to 1.0 mg/ml concentration of working standards in

methanol. All stock solutions were stored at 2–8 °C. The stock solutions were further

diluted with the mobile phase acetonitrile–water (50:50, v/v; pH adjusted to 3.0 with

orthophosphoric acid) to give a series of standard mixtures having a final concentration

in the range of 2.0–500 ng/ml [184].

3.5.4 Sample preparation

A simple, one step liquid–liquid extraction (LLE) procedure was carried out for

the extraction of rosuvastatin from serum samples. A volume (50µl) of working

solution (to give a final concentration of 500 ng/ml) was added to 200µl of serum and

57

mixed for approximately 10 seconds. Then absolute ethanol (600µl) was added and

vortex-mixed for 2 minute for deproteination. Next 1.0 ml of diethyl ether (extraction

solvent) was added, vortex-mixed for 5 minute and centrifuged at 3500 rpm at 0 °C for

5 min. The organic layer was separated, collected in same tube and evaporated to

complete dryness under gentle stream of nitrogen on a heating block maintained at 40

°C. After drying, residue was reconstituted in 500 µl of mobile phase, vortex-mixed for

2 minute and 20 µl sample was injected onto HPLC system [185].

3.5.5 Chromatographic conditions

Chromatographic separation was performed with different proportions of

acetonitrile–water and methanol–water as a mobile phase with different flow rates in

the range of 0.8–1.6 ml/min in an isocratic mode. Injection volume was kept in the

range of 10–50µl. Column oven temperature was varied in the range of 25–35 °C and

eluate was monitored using UV detection at various wavelengths in the range of 220–

280 nm. Various experimental parameters were optimized for determination of

rosuvastatin calcium.

3.5.6 Method validation

The suggested analytical method was validated according to international

guidelines with respect to certain parameters such as specificity/selectivity, linearity,

LLOQ, LLOD, precision, accuracy, sensitivity, recovery and robustness/ruggedness.

3.5.7 Linearity

The linearity of the method was established by spiking a series of standard

mixtures of rosuvastatin (2.0–500 ng/ml), extracting and analyzing in triplicate.

Calibration curves for standard solutions and spiked serum samples were then acquired

by plotting their response ratios (ratios of the peak area of the analytes to internal

standard) against their respective concentrations. Linear regression was applied and

slope (a), intercept (b), correlation coefficient (r) and standard error (Es) were

determined [184].

3.5.8 Precision

Method precision was determined both in terms of repeatability (injection and

analysis) and intermediate precision (intra-day and inter-days reproducibility). Inorder

58

to determine injection repeatability, serum samples spiked with 500 ng/ml of

rosuvastatin were injected 10 times into HPLC system and repeatability of retention

time and peak area was determined and expressed as mean and %RSD calculated from

the data obtained. Similarly, analysis repeatability was verified by analyzing eight

serum samples spiked with 500 ng/ml of rosuvastatin prepared individually, determined

as amount recovered and expressed as mean and %RSD calculated from the data

obtained. For the intermediate precision (intra-day and inter-days reproducibility),

serum samples spiked at three different concentration levels were analyzed three times

a day in triplicate injections over three consecutive days and expressed as mean ± SD

and % RSD calculated from data obtained [185].

3.5.9 Specificity/selectivity

The specificity/selectivity of the analytical method was investigated by

confirming the complete separation and resolution of all the desired peaks of the

analytes in mobile phase and spiked rabbit’s serum.

3.5.10 Accuracy

Accuracy was determined in terms of percent recovery. Blank rabbit’s serum

was spiked with the analytes at five different concentration levels (2.0, 64, 145, 250,

500 ng/ml of rosuvastatin. Another set of standard mixtures at the same concentration

levels was also prepared in the mobile phase (acetonitrile–water, 50:50, v/v; pH

adjusted to 3.0 with orthophosphoric acid). The serum was extracted with the procedure

noted above and injected onto the HPLC system in triplicate. Percent recoveries for

rosuvastatin was calculated using the following formula [185]:

%Recovery =𝐴

𝐵× 100

Where A is the response ratio of the analyte in serum sample, B is the response

ratio of the analyte in standard mixture.

3.5.11 LLOD and LLOQ

Detection and quantification limits were determined through dilution method

using S/N approach by injecting a 20 µl sample. LLOD was considered as the minimum

concentration with a signal to noise ratio of at least three (S/N≈3), while LLOQ was

taken as a minimum concentration with a signal to noise ratio of at least ten (S/N≈10).

59

3.5.12 Stability

The stability studies of rosuvastatin spiked serum samples were carried out over

a period of 48 h at 25 °C (room temperature under laboratory light), 2–8 °C

(refrigerator) and−80 °C (frozen) and standard solutions for one month at 2–8 °C.

3.5.13 Robustness

The robustness of the developed method was investigated by evaluating the

influence of small deliberate variations in procedure variables like column oven

temperature (±1 °C), flow rate (±5%) and pH of the mobile phase (±0.2 units) [186].

3.5.14 Pharmacokinetic analysis

Noncompartmental analysis was used to study pharmacokinetic of Rosuvastatin

calcium in rabbit’s plasma by using the software package kinetic v 4.4. Peak plasma

concentration (Cmax) and time to reach peak plasma concentration (Tmax) were

calculated by visual inspection of plasma concentration-time curves. The area under the

plasma concentration curve (AUC0-t) was measured using trapezoidal rule.

3.6 Acute oral toxicity studies of prepared formulatios

Prepared formulations were administered to different groups of rabbits to

determine toxicity caused by these formulations. For this purpose hematological,

biochemical and histopathological parameters were evaluated. Slides were prepared to

see any histological changes in normal structure of such as heart, liver, lungs, kidney,

spleen, intestine and stomach.

3.7 Statistical analysis

SPSS 18® software was used to analyze the data. All results were expressed as

mean ± standard deviation (SD). The data for various formulations were compared by

one-way analysis of variance (ANOVA). P < 0.05 was considered to be statistically

significant.

60

4. RESULTS AND DISCUSSION

For proper pharmacological response of drug, it’s necessary for drug molecules

to properly dissolve and become soluble in available media to produce desired effect at

targeted area. For this purpose, efforts have been made to prepare Fast Disintegrating

Tablets (FDT’s) and microparticles. FDT’s were prepared by using direct compression

method containing rosuvastatin calcium with β-cyclodextrin and superdisintegrants in

various ratios. Microparticles were prepared by using various techniques such as

kneeding and solvent evaporation techniques to prepare inclusion complexes and solid

dispersions and copolymerization to formulate hydrogel microparticles. All these

formulations were prepared to enhance solubility as well as dissolution profile of

rosuvastatin to achieve desired therapeutic efficacy. This study was divided into three

groups: first was preparation and characterization of FDT’s, in second group

microparticles were prepared through kneeding and solvent evaporation technique

while in third group hydrogel microparticles were prepared by using different

concentrations of polymer along with various monomers. All prepared formulations

were characterized through various parameters and compared with each other as well

as with commercially available conventional formulations. From this characterization

we have determined difference in solubility of prepared formulations with marketed

dosage form. Results of these three groups of formulations are presented under

following broader headings.

1) Fast disintegrating tablets

2) Inclusion Complexes and Solid Dispersions

3) Hydrogel Microparticles

4) In vivo pharmacokinetic studies

5) Acute oral toxicity study of prepared formulations

All of above results are discussed in detail one by one.

61

4.1 Fast Disintegrating Tablets


Fifteen formulations of FDT’s were prepared by varying concentration of β-

cyclodextrin and superdisintegrants. FDT’s were first evaluated for micromeritics

parameters to study flow properties of powder material to be compressed into tablets.

Flow properties are very important for powder or particulate material to compress into

tablets. Materials that do not have good flow properties are difficult to compress into

tablets and ultimately leads towards improper mixing of drug with excipients. To avoid

such issues powder was first evaluated for flow properties such as angle of repose, bulk

density, tapped density, Carr’s index and Hausner’s ratio. Results of these rheological

parameters are present in Table 4.1. Bulk density and tapped density were 0.728 to

0.774 g/ml and 0.860 to 0.891 g/ml, respectively. These values were further used to

calculate Carr’s index and Hausner’s ratio. Calculated values of angle of repose for

various formulations were evaluated as 23.10Ɵ to 26.60Ɵ. These values were lying

within the range given in different pharmacopeia’s i.e. angle of repose should be less

than 30° for powder material to be compressed into tablets. Carr’s index is also another

important parameter to judge flow of powder material. Normally powder material is

considered good for compression into tablets if its Carr’s index value is between 13-

19%. If it exceeds 21% then powder has poor flow. Results of our formulations for

Carr’s index were 14.70% to 16.75%. Similar to angle of repose and Carr’s index

calculated values of Hausner’s ratio were also within acceptable limits of better flow

that were 1.15 to 1.19. These results had favored that our material was suitable for

compression into FDT’s and all ingredients were mixed properly before compression.

These results are similar to another study conducted by Hafeez et al. (2014) [187]. They

also prepared FDT’s by using kyron T134 and evaluated for different rheological

properties. Their results of rheological parameters are in agreement to our findings.

62

Table 4.1: Results of bulk density, tapped density, angle of Repose, Hausner’s

ratio and Carr’s index

Code Bulk density

(g/ml)

Tapped

density

(g/ml)

Angle of

repose

(ϴ)

Hausner’s

ratio

Carr’s

index

%

F1 0.751 0.873 26.40 1.16 15.75

F2 0.765 0.890 24.60 1.16 16.50

F3 0.760 0.877 23.50 1.15 14.70

F4 0.741 0.868 25.40 1.17 16.59

F5 0.774 0.891 24.60 1.15 15.80

F6 0.755 0.873 23.50 1.15 15.74

F7 0.745 0.877 25.50 1.17 15.90

F8 0.740 0.875 26.60 1.18 16.58

F9 0.742 0.860 24.80 1.15 16.20

F10 0.761 0.895 26.60 1.17 15.68

F11 0.738 0.861 25.60 1.16 15.80

F12 0.746 0.872 23.10 1.16 17.60

F13 0.734 0.879 24.70 1.19 16.75

F14 0.728 0.862 26.30 1.18 15.50

F15 0.741 0.873 25.70 1.17 16.86

4.1.2 FTIR studies

Prepared formulations were characterized for different parameters and optimize

formulation was selected based upon theses parameters. Then best formulation from

each type of preparation i.e. inclusion complexes, solid dispersions, rapid disintegrating

tablets and hydrogel microparticles was checked for compatibility of ingredients and

complex formation by performing FTIR, DSC and TGA studies. These tests were

performed only for optimized formulation because all formulations have same

ingredients with variable concentrations.

63

FTIR spectra of drug alone and of various FDT’s formulation were taken to

confirm whether any interaction was present between drug, superdisintegrants and β-

cyclodextrin. Drug spectra had shown that characteristic peeks were present at 3337.90

cm-1, 2968.23 cm-1 and 1435.48 cm-1 corresponding to cyclic amines, C-H stretching,

C=O stretching, O-H bending as shown in Figure 4.1 (a). These were almost same as

reported in monograph for rosuvastatin. Spectrum of β-cyclodextrin had alcoholic O-H

stretching at 3292.74 cm-1, methyl and methylene C-H stretching vibration at 2927.62

cm-1 and C-O ether stretching vibration at 1021.63 cm-1 as shown in Figure 4.1 (b).

FTIR spectra of Kyron T134 had shown characteristics peaks at 3681.16 cm-1 and

2316.65 cm-1 as appeared in Figure 4.1 (c). In FTIR spectra of sodium starch glycolate

characteristics peaks were appeared at 2345.37 cm-1 and 1866.43 cm-1 as shown in

Figure 4.1 (d). FTIR spectra of FDT’s were also taken inorder to compare with pure

spectra of drug. Similar peaks of drug, β-cyclodextrin and superdisintegrants were

present in FTIR spectra of FDT’s as shown in Figure 4.1 (e). It confirmed that no

interactions were present between drug, β-cyclodextrin and superdisintegrants. Our

results are similar to another study conducted by Inayat et al. (2013) where they

concluded that no interaction was present when similar type of peaks were appeared in

different mixture [188].

Figure 4.1 (a): FTIR spectra of rosuvastatin calcium

64

Figure 4.1 (b): FTIR spectra of β-cyclodextrin

Figure 4.1 (c): FTIR spectra of Kyron T134

65

Figure 4.1 (d): FTIR spectra of sodium starch glycolate

Figure 4.1 (e): FTIR spectra of FDT’s

66


Differential scanning calorimetry and thermo gravimetric analysis of prepared

formulations were compared with individual ingredients to check thermal stability of

rosuvastatin calcium. TGA curve of pure Rosuvastatin calcium depicts that gradual

reduction in weight due to thermal degradation. This loss in mass occurred in three

steps as shown in Figure 4.2 (a). Upto 372 °C 48% of mass was lost. Results had also

shown that there was presence of sharp endothermic peak at 123 °C in DSC

thermograms of pure drug as shown in Figure 4.2 (b). TGA curves of β-cyclodextrin

had shown 85% of mass was last at 360 °C as shown in Figure 4.2 (c). In DSC curves

of β-cyclodextrin sharp endothermic peak was present at 106 °C as illustrated in Figure

4.2 (d). TGA spectra of prepared formulations shown that delay in thermal degradation

of drug weight as compared to spectra of alone drug. These observations represent that

drug molecules were complexed with superdisintegrants and β-cyclodextrin and as a

results drug will become more soluble due to more hydrophilic and rapid disintegrating

nature of these ingredients.

Figure 4.2 (a): TGA curves of Rosuvastatin calcium

2.31min81.00°C96.92%

9.86min231.24°C93.54%

16.86min372.01°C52.08%

40

60

80

100

Weig

ht (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: TSize: 2.3280 mg

Comment: T

DSC-TGAFile: D:\TGA\RAY\T.001

Run Date: 15-Apr-2015 11:16Instrument: SDT Q600 V8.2 Build 100

Universal V4.2E TA Instruments

67

Figure 4.2 (b): DSC curves of Rosuvastatin calcium

Figure 4.2 (c): TGA curves of β-cyclodextrin

3.95min109.44°C86.97%

14.86min327.46°C81.65%

16.44min359.21°C15.72%

0

20

40

60

80

100

We

igh

t (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: BSize: 2.3400 mg

Comment: B

DSC-TGAFile: D:\TGA\RAY\B.001



68

Figure 4.2 (d): DSC curves of β-cyclodextrin

In TGA spectra of pure drug there was gradual loss of drug molecules and after

17 minutes at 372 °C approximately half of the drug was lost. While in case of FDT’s

delay in loss of drug molecules because after 17 minutes at 356 °C only 33 % weight

of drug was lost as shown in Figure 4.2 (e). This confirmed that complex was formed

between drug and polymer molecules that ultimately leads towards more stability.

Similarly in case of DSC spectra of drug there was shifting of endothermic peaks

towards higher temperature that confirmed attachment of drug molecules with polymer

and superdisintegrants as shown in Figure 4.2 (f). Due to hydrophilic nature of these

disintegrants, solubility of drug was enhanced. Our results are in agreement with

another study conducted by Razali et al. (2011) where they prepared fast release tablets

of poorly water soluble drug, Tolbutamide by using hydrophilic polymer. From results

of DSC studies they concluded that complex was formed when peaks were shifted

towards higher temperature values [189].

106.89°C

81.25°C0.06192J/g

329.46°C

324.95°C0.003278J/g

382.31°C

0.03110J/g

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

Heat F

low

(W

/g)

0 50 100 150 200 250 300 350 400

Temperature (°C)


Comment: B



Exo Up Universal V4.2E TA Instruments

69

Figure 4.2 (e): TGA curves of FDT’s

Figure 4.2 (f): DSC curves of FDT’s

70

4.1.4 Post compression studies

4.1.4.1 Weight variation and Hardness

After evaluation of precompression studies, powder was compressed into Fast

Disintegrating Tablets. Results of weight variation for all formulations were present

within the pharmacopeial limits as shown in Table 4.3. This represent that tablets were

of proper weight and size. Hardness generally indicates that tablets are able to bear

stress during handling and remain stable during storage. A linear relationship exists

between tablet hardness and disintegration time (DT). If tablets are harder then pores

present on the surface of tablets are so compact that it is very difficult for water to

penetrate within tablets to impart drug release. Increased hardness caused increase in

tablet density by reduction in pores. This results in reduction of number of pores on the

surface of tablets, which hampered liquid penetration and access to disintegrate

particles in FDT’s. As a result reduction in wetting of tablets that ultimately affects the

development of proper force and led to prolongation of DT. The effect of tablet

hardness is shown in SEM photographs in Figure 4.3.

Figure 4.3: SEM image of FDT

71

Table 4.2: Results of weight variation, hardness, thickness, friability, disintegration

time and wetting time

*Average of three determinations; ±SD – (Standard deviation

Parameters

Code Wt. variation

(mg)

Hardness

Kg/cm2

Thickness

(mm)

Friability*

(%)

Disintegration*

Time (Sec)

Wetting*

Time

(Sec)

F1 199.56 3.20 2.70 0.655 ± 0.02 77 ± 1.50 96 ± 1.50

F2 197.51 3.30 2.63 0564 ± 0.03 67 ± 1.73 88 ± 1.70

F3 199.67 3.10 2.66 0.345 ± 0.03 64 ± 1.70 83 ± 0.53

F4 198.87 3.40 2.76 0.564 ± 0.04 59 ± 1.15 76 ± 1.15

F5 198.82 3.30 2.85 0.472 ± 0.00 55 ± 1.50 71 ± 1.70

F6 199.55 3.20 2.76 0.489 ± 0.02 83 ± 1.73 99 ± 1.55

F7 197.75 3.10 2.75 0.645 ± 0.03 73 ± 2.08 93 ± 1.50

F8 197.90 3.40 2.53 0.467 ± 0.04 69 ± 1.30 87 ± 1.15

F9 199.32 3.30 3.07 0.587 ± 0.00 65 ± 1.10 80 ± 1.00

F10 198.80 3.50 2.87 0.618 ± 0.01 61 ± 1.30 77 ± 1.30

F11 196.96 3.20 2.75 0.594 ± 0.04 37 ± 1.50 49 ± 1.50

F12 197.65 3.40 2.92 0.486 ± 0.01 41 ± 1.70 57 ± 2.08

F13 196.02 3.50 2.54 0.564 ± 0.03 47 ± 1.73 63 ± 0.58

F14 199.47 3.40 2.69 0.476 ± 0.00 31 ± 1.50 43 ± 1.15

F15 195.58 3.50 2.94 0.602 ± 0.03 42 ± 1.30 56 ± 1.70

72

In addition to hardness, lubricant concentration also has great influence on DT.

Magnesium stearate is hydrophobic in nature. When high concentration of lubricant is

used this might cause coating of hydrophilic components of tablet formulations,

limiting the wetting of components of formulation especially the disintegrants and drug

carrier which are very vital in the disintegration process of tablet. Subsequently, DT of

tablet had increased. Overall results of hardness indicate that they were within

prescribed limits of FDT’s. Our results are in agreement to previously reported study

conducted by Jha et al. (2010) on orally disintegrating tablets [190]. They concluded

that when higher concentrations of Magnesium stearate were used then it resulted in

coating of hydrophilic moieties. Thus wetting of tablets was decreased. This reduction

in wetting ultimately increased disintegration time of Fast Disintegrating Tables.

4.1.4.2 Friability

Generally tablets having friability less than 1% are considerd good. Friability of

all formulations was less than 0.8% which suggested that tablets had good mechanical

strength as shown in Table 4.2. Tablets did not show any unnecessary breakdown of

the particles when rotated in drum of friabilator. Statistically results of friability were

tested by using one way ANOVA. Results of ANOVA between the groups were

significant having 0.025 p-value.

4.1.4.3 PXRD studies

XRD is one of the most important parameter to be studied when working on

solubility enhancement of some poorly water soluble chemical entity. Changes in

polymorphic characteristics of certain compounds have great impact on solubility,

dissolution as well as bioavailability. Powder X-Ray diffractogram were taken to found

nature of rosuvastatin whether it was crystalline or amorphous. X-ray diffraction

pattern of rosuvastatin showed sharp diffraction peaks at 2θ values of 16.04, 22.45,

and 34.3 while FDT’s showed no such characteristics peaks at 2θ as appeared in

Figure 4.4. The absence of characteristics peak in formulation indicated that drug had

converted from crystalline to amorphous form. As a result solubility of rosuvastatin was

enhanced in FDT’s prepared by using β-cyclodextrin and superdisintegrants because

amorphous forms are more soluble as compared to crystalline form. Similar results were

obtained by Pavan et al. (2014) in their studies [191]. They had applied liquisolid

73

technology for solubility and dissolution profile enhancement of Rosuvastatin calcium

and concluded that characteristics peaks in X-ray diffraction pattern of formulations

were disappeared that were present in X-ray diffraction pattern of pure Rosuvastatin

calcium. Thus drug was converted into amorphous form in liquisolid complex and

crystallinity was lost. Due to this amorphous nature, its solubility was enhanced.

Figure 4.4: X-Ray diffraction patterns of (A) Rosuvastatin calcium, (B) β-

cyclodextrin and (C) FDT’s

4.1.4.4 Scanning electron microscopy

Morphology of FDT’s was determined by using scanning electron microscopy.

SEM images of FDT’s were taken to see the upper surface as well as inner structure.

SEM images had shown that pores were present on surface of tablets as shown in Figure

4.3. These pores had favored in the penetration of water inside of tablets. This

penetrated water helped to fasten the release of drug from tablets and ultimately

solubility was enhanced.

74

4.1.4.5 Wetting time

Wetting time is the indication of hydrophobicity of the ingredients. Lower the

wetting time faster will be the disintegration. Wetting time for all 15 formulations was

less than 2 minute lying between 43 to 96 seconds as shown in Table 4.2. It was lowest

for F14 formulation containing drug and β-cyclodextrin in 1:1 and kyron T134 8% as

superdisintegrant. The results of one way ANOVA had shown that p-value for wetting

time was 0.036. It indicates that results were significant and wetting of tablets was

affected by the nature and concentration of β-cyclodextrin and superdisintegrants.

Akbari et al. (2011) had also prepared fast disintegrating tablets of Rosuvastatin by

using β-cyclodextrin and sodium starch glycolate as superdisintegrant. They concluded

similar results that maximum amount of Rosuvastatin was released when β-

cyclodextrin and drug were used in 1:1 and it had wetting time less than 1 minute [192].

4.1.4.6 Disintegration time

Disintegration time for FDT’s is generally less than 1 minute. Rapid uptake of

water from surrounding media can cause swelling of tablets and ultimately quick

disintegration to produce bursting effect. This results in fast release of drug from its

medium into surrounding environment and hence solubility of loaded drug was

enhanced. Similar to wetting time it was also lowest for F14 as mentioned in Table 4.2.

Similar results were obtained by Madgulkar et al. (2009) in their study where they

prepared mouth disintegrating tablets of Tramadol by using superdisintegrants and

concluded that solubility of drug was enhanced when rapid disintegration of tablets had

taken place [193].

4.1.4.7 In vitro dispersion time

In vitro dispersion test was performed for all of the formulations. This test was

used to find dispersion time for tablets by using petri dish method. Dispersion of tablets

was affected by swelling which was due to disintegrants. Formulation F14 had lowest

dispersion time among all formulations as shown in Table 4.3. These results had shown

that kyron T134 was better choice as superdisintegrant than sodium starch glycolate

because dispersion time was lowest for formulations containing kyron T134.

Comparable results were obtained by Gupta et al. (2010) where they concluded that

rapid disintegrating tablets containing kyron T134 as superdisintegrant shown less in

vitro dispersion time when compared with rapid disintegrating tablets containing

75

sodium starch glycolate and caused rapid release of drug with improved solubility

[194].

Table 4.3: Results of wetting volume, dispersion time, pH of tablet

solution and water absorption ratio.

*Average of three determinations; ±SD – (Standard deviation)

Code Wetting*

volume (ml)

Dispersion*

time (Sec)

pH of

Tablet Sol

Water*

absorption ratio

F1 22 ± 1.50 57 ±0.58 7.10 1.10 ± 0.01

F2 20 ± 0.58 53 ± 1.15 6.80 1.25 ± 0.03

F3 18 ± 1.73 50 ± 1.30 7.00 1.35 ± 0.04

F4 17 ± 1.50 48 ± 1.15 6.80 1.40 ± 0.03

F5 15 ± 1.73 45 ± 0.58 6.80 1.50 ± 0.04

F6 25 ± 1.15 60 ± 1.50 7.00 1.00 ± 0.00

F7 23 ± 1.73 56 ± 1.15 7.20 1.15 ± 0.02

F8 21 ± 1.15 52 ± 1.50 7.10 1.30 ± 0.04

F9 19 ± 0.58 51 ± 0.53 7.20 1.25 ± 0.01

F10 17 ± 0.58 48 ± 1.15 6.80 1.40 ± 0.04

F11 10 ± 1.15 32 ± 1.73 6.90 1.80 ± 0.23

F12 12 ± 0.53 35 ± 1.15 6.90 1.75 ± 0.18

F13 13 ± 1.15 40 ± 0.53 7.00 1.70 ± 0.03

F14 08 ± 0.53 29 ± 0.58 6.80 2.00 ± 0.02

F15 12 ± 0.58 36 ± 1.15 6.80 1.75 ± 0.23

76

4.1.4.8 Water absorption ratio

Water absorption ratio was used to determine that how much water was

absorbed by the tablets which ultimately affect the disintegration of tablets. As more

water is absorbed then more rapid will be disintegration. As value of water absorption

ratio increases it indicates that rapid breaking of tablets and therefore faster

disintegration. This disintegration ultimately affect dissolution rate of tablets that is

directly related with solubility of drug. This parameter favored formulation F14 that

had absorbed water approximately double than its original weight (2.00 ± 0.02) as

shown in Table 4.3. Overall results of water absorption ratio were present well within

specified limits. P-value for water absorption ratio was 0.037 which confirmed that

results were significant. Our results are in agreement to results obtained by Pandya et

al. (2009) where they prepared various formulations of fast dissolving tablets and found

that water absorption ratio had direct relation with drug dissolution rate [195].

4.1.4.9 Dissolution studies

Dissolution studies were carried out at 6.8 pH phosphate buffer to determine in

vitro drug release. Standard calibration curves of Rosuvastatin calcium were obtained

in phosphate buffer of pH 6.8 and in HCl buffer of 1.2 pH as shown in Figure 4.5 (a)

and 4.5 (b), respectively. These standard curves were used to determine concentration

of Rosuvastatin in our samples by comparing their absorbance with absorbance of

standard. Results had shown that there was a marked difference in release of

commercially available tablets of rosuvastatin and FDT’s prepared using β-

cyclodextrin and different superdisintegrants. Maximum release of drug was observed

for FDT’s within 18 minutes but no significant release was observed in case of

commercially available tablets. Maximum 97% of drug was released from formulation

F14 containing 8% kyron T134 and 10% β-cyclodextrin in 18 minutes as shown in Figure

4.5 (c). Overall values of drug release were present between 91-97%. RST

commercially available tablet had released only 43% of drug within 3 hours while

FDT’s released upto 97% of drug in first 20 minutes of dissolution as in formulation

F14. These result may also be due to the increase in pores on surface area of tablets as

shown in Figure 4.3 of SEM image (page-70) that ultimately had absorbed more water

from dissolution media and shown better release than commercially available tablets.

FDT’s had porous structure due to the presence of superdisintegrants that helped in the

penetration of water from surrounding to inside of FDT’s. This penetrated water

77

swollen FDT’s within few minutes and as a results drug was released very rapidly.

These results were similar to another study that was conducted by us. Where

orodispersible tablets of atorvastatin were prepared by using kyron T134 as

superdisintegrant and found that 96% of drug was released within 18 minutes when 8%

of kyron T134 was used [196].

Figure 4.5 (a): Standard Curve of Rosuvastatin calcium at pH 6.8

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 5 10 15 20 25

Ab

sorb

an

ce (

nm

)

Concentration (µg/ml)

78

Figure 4.5 (b): Standard Curve of Rosuvastatin calcium at pH 1.2

Figure 4.5 (c): Cumulative % drug release from different formulations of FDT’s and

RST tablets at 6.8 pH

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 5 10 15 20 25

Ab

sorb

an

ce (

nm

)

Concentration (µg/ml)

0

20

40

60

80

100

120

0 50 100 150 200

Cu

mu

lati

ve

% D

rug R

elea

se

Time (hr)

F1

F2

F3

F4

F5

F6

F7

F8

F9

F10

F11

F12

F13

F14

F15

RST Tab

79

Different kinetic models, i.e. zero order, first order, Higuchi and Korsmeyer-

Peppas were applied on in vitro release data by using DD Solver® excel based Add in

program. Final model selection was made on best fit observations. Higher values of

correlation coefficient i.e. R2 of model proved best fit of that model for explanation of

drug release from formulations. From R2 values of F12 (0.9980) and F14 (0.9990) it

was observed that release pattern followed first order release kinetics as shown in Table

4.4. The results of T25, T50 and T75 had shown that there was rapid release of drug from

FDT’s due to bursting effect because T25 was approximately double than T50. These

results had shown that an increase in concentration of superdisintegrant and β-

cyclodextrin had increased the release of drug from FDT’s significantly (p < 0.05),

which is in accordance with our previously reported study in which FDT’s of Acyclovir

were prepared by using β-cyclodextrin and kyron T134 and concluded that drug release

followed first order drug release kinetic model that was diffusion and erosion controlled

[197].

80

Table 4.4: In vitro drug release kinetics of FDT’s

Zero order

Parameters F12 F14 RST Tab

Ko 0.810 0.826 0.378

R2 0.5648 0.5469 0.8279

T25 30.862 30.252 66.103

T50 61.724 60.504 132.205

T75 92.587 90.756 198.308

First order

K1 0.172 0.193 0.006

R2 0.9980 0.9990 0.8703

T25 1.677 1.487 46.989

T50 4.042 3.582 113.217

T75 8.083 7.165 226.433

Higuchi

KH 10.461 10.713 4.552

R2 0.7278 0.7119 0.9297

Korsmeyer

Peppas

Kkp 46.731 49.726 10.999

R2 0.8902 0.8876 0.9664

n 0.162 0.154 0.303

81

4.1.4.10 Solubility studies

Rosuvastatin calcium is a weak acid. Due to its acidic nature it had more

solubility in basic media as compared to acidic pH. So it become ionized in basic

medium and is therefore soluble in high pH solutions. Solubility studies of pure drug

and prepared FDT’s were conducted both basic as well as in acidic media. Results had

shown that highest solubility of FDT’s were present at pH 6.8 in phosphate buffer when

compared with HCl buffer of low pH (1.2). However, highest solubility was observed

in water in ionic form as shown in Figure 4.6. From solubility studies of rosuvastatin

calcium it was observed that pure drug had very low solubility in acidic media at 1.2

pH buffer while had slightly better solubility in phosphate buffer of basic pH 6.8. In

water pure drug had greater solubility as compared to 1.2 and 6.8 pH buffers.

When solubility studies of FDT’s were performed, it was observed that same

amount of drug loaded in FDT’s had several folds more solubility than pure drug. From

Figure 4.6, it is clear that there was increase in solubility of rosuvastatin calcium in

FDT’s upto 7.42 folds at pH 1.2 while at 6.8 pH solubility was enhanced upto 11.71

folds. In distilled water there was 9 folds increase in solubility of drug occurred loaded

in FDT’s as compared to solubility of pure drug. These results clearly depict that FDT’s

had enhanced solubility of rosuvastatin calcium in different pH media but it varied

based upon the pH of used media. Pure drug had more solubility in distilled water as

compared to HCl buffer of 1.2 pH and phosphate buffer of pH 6.8. The order in which

pure drug had more solubility was distilled water > phosphate buffer of pH 6.8 > HCl

buffer of 1.2 pH. While in case of FDT’s rosuvastatin calcium solubility was increased

in all three media used for solubility studies but it was 11.71 folds increase in phosphate

buffer of pH 6.8 and 9 folds in distilled water. Our results are in agreement to Shilpi et

al. (2015), they prepared different complexes of poorly water soluble drug with β-

cyclodextrin and concluded that solubility of complex was enhanced upto 5.32 folds as

compared to solubility of pure drug [198].

82

Figure 4.6: Solubility studies of pure drug and FDT’s

4.1.4.11 Stability studies

Stability studies of three best formulations were conducted for six month

period. Tablets were kept under accelerated conditions of temperature and humidity 35

± 5оС and 75% ± 5%, respectively. The samples were taken after 1, 2, 3, 4, 5 and at the

end of 6 month. Tablets were evaluated for different parameters. The results had

indicated that there were no significant variations occurred in drug content and in vitro

dispersion time at the end of 6 months. It’s mean that our formulations were stable

under different accelerated conditions of temperature and humidity.

289 351

900

2145

4111

8146

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

pH 1.2 pH6.8 Water

RS

T S

olu

bil

ity (

μg/m

l)

RST

RST RDT

83

4.2 Inclusion complexes and solid dispersions

4.2.1 Product yield and entrapment efficiency

Different formulations of microparticles were prepared by using various ratios

of β-cyclodextrin with drug using solid dispersions and inclusion complexes technique.

Prepared formulations were first evaluated for product yield and entrapment efficiency

inorder to determine that how much our methods were efficient to prepare

microparticles. It was found that all formulations had good product yield 82.25 ± 1.00

- 91.25 ± 0.15% and also had entrapment efficiency 76.20 ± 0.50 - 87.50 ± 0.15% as

represented in Table 4.5.

Formulation F3 containing drug and β-cyclodextrin in ratio of 1:3 prepared by

solid dispersion (solvent evaporation) had maximum entrapment efficiency of 87%.

This may be due to use of higher concentration of β-cyclodextrin present in F3 that had

entrapped greater amount of drug into its cavity as compared to other formulations.

Drug release from F4 was approximately same as that of F3, its mean that further higher

concentration of polymer has no great impact on drug entrapment. Formulation F7

(84.01 ± 0.25) also had same ratio of drug and polymer but prepared by inclusion

complexes (kneeding technique) had good entrapment efficiency but less than F3

(87.50 ± 0.15). Results were found significant because p value was 0.023. So

concentration of polymer had influence on the amount of drug entrapped in

microparticles. In another study microparticles were prepared by Shashank et al. (2015)

using chitosan which is hydrophilic polymer [199]. Due to this hydrophilic nature of

polymer, more drug was entrapped and maximum entrapment was found in formulation

containing polymer and drug upto 3:1 ratio. Their findings also supported our study

because maximum drug was entrapped in our formulation F3 that contain polymer and

drug in 3:1 ratio. Entrapment efficiency is important parameter for administration of

proper dose to achieve desired therapeutic levels.

84

Table 4.5: Entrapment efficiency and product yield of inclusion complexes

and solid dispersions

aAverage of three determinations; ± SD- (Standard deviation)


Prepared inclusion complexes and solid dispersions were evaluated for flow

properties. Results of flow parameters are shown in Table 4.5. For any particulate

material it is necessary to be lying within pharmacopeial limits inorder to formulate in

suitable dosage form. If value of angle of repose is less than 30° is considerd good for

microparticles. All formulations have angle of repose less than 25°. Results of other

flow properties such as bulk density, tapped density, Carr’s index and Hausner’s ratio

were also present within acceptable limits. Overall findings of all parameters of

micromeritics properties were present within the acceptable limits as shown in Table

4.6. These results showed that prepared microparticles had good flow. These are very

important for microparticles to have proper flow inorder to release drug.

Formulations % Entrapment

Efficiencya

Product

yielda (%)

F1 80.00 ± 0.15 85.00 ± 0.35

F2 83.00 ± 0.20 88.00 ± 0.25

F3 87.50 ± 0.15 91.25 ± 0.15

F4 87.00 ± 0.25 90.51 ± 0.25

F5 76.20 ± 0.50 82.25 ± 1.00

F6 79.00 ± 1.00 84.25 ± 0.50

F7 84.01 ± 0.25 87.55 ± 0.20

F8 83.50 ± 0.50 88.15 ± 1.00

85

Table 4.6: Micromeritics properties of microparticles of inclusion complexes and

solid dispersions

4.2.3 Optical microscopy

Microparticles were visualized under optical microscope to determine the size

of prepared microparticles. It was observed that all formulations had particle size in

microns ranging from 10 µm to 200 µm as shown in Figure 4.7. These results represent

that all formulations had desired particle size. For proper release of drug from particles

it is necessary that particles should have size in a narrow range.

Figure. 4.7: Optical microscope images of microparticles

Code

Bulk

density

(g/ml)

Tapped

density (g/ml)

Angle of

repose

(ϴ)

Hausner’s

ratio

Carr’s

index

(%)

F1 0.736 0.845 22.20 1.14 16.84

F2 0.765 0.887 23.40 1.15 15.76

F3 0.756 0.869 22.60 1.14 16.02

F4 0.749 0.871 24.90 1.16 15.95

F5 0.745 0.885 23.30 1.18 16.45

F6 0.748 0.862 24.70 1.15 15.84

F7 0.756 0.886 23.60 1.17 17.00

F8 0.759 0.873 22.40 1.15 15.80

86


Rosuvastatin calcium is a weak acid in nature so it shows ionization in basic

medium and is therefore soluble in high pH solutions. Our results had also shown

highest solubility at pH 6.8 when compared with other buffers of low pH (1.2).

However, highest solubility was observed in water in ionic form. From the solubility

study of both drug as well as prepared microparticles, it was observed that there had

been significant increase in the solubility of drug in water (5.86 fold), pH 1.2

(3.32 fold) and pH 6.8 (8.54 fold) as seen in Figure 4.8. This increase in solubility of

drug in microparticles was due to the complex formation of drug with β-cyclodextrin.

β-cyclodextrin is hydrophilic in nature, when it forms complex with other poorly water

soluble moieties then these water insoluble drug molecules becomes water soluble.

Solubility of prepared microparticles was enhanced several folds as compared to pure

drug in different pH medias. But highest solubility was observed at 6.8 pH. Shilpi et al.

(2015) [198] worked on solubility enhancement of Nelfinavir by preparing complex of

drug with β-cyclodextrin in different ratios and concluded that solubility was enhanced

upto 5.32 folds at 1.2 pH while 3.64 folds at 4.5 pH. Our results had shown more

solubility at higher pH (6.8) as compared to 1.2 pH. This difference in results was due

to the nature of drug. Nelfinavir is basic in nature therefor, it is more soluble in acidic

media as compared to higher pH. While Rosuvastatin calcium is weak acid in nature.

Due to this acidic nature it become more soluble at higher pH (6.8) values as compared

to lower pH (1.2). Rosuvastatin is available in its calcium salt, therefore, it is more

soluble in water as compared to different buffer media. But when Rosuvastatin calcium

complexed with hydrophilic polymers then it became more soluble in buffer media of

higher pH rather than in pure water as shown Figure 4.8.

87

Figure 4.8: Solubility studies of pure drug and microparticles

4.2.5 Dissolution studies

Dissolution studies were carried out at 6.8 and 1.2 pH phosphate buffer and HCl

buffer respectively to determine in vitro drug release studies. Results had shown that

there was marked difference in the release of drug from commercially available tablets

of rosuvastatin calcium and microparticles prepared by using β-cyclodextrin.

Maximum release of drug was observed within 45 minutes for most of microparticles

but no significant release was observed in case of tablets. To evaluate complete release

of drug from formulations we had extended our dissolution studies and observed that

tablets have maximum release of 43% of drug upto 3 hour but microparticles had

released remaining portion of drug in that period. Almost all of drug was released from

microparticles within first hour of dissolution as shown in Figure 4.9 (a), (b). There was

maximum release of drug from S3 that was 92% in 45 minutes. These findings also

supported our solubility studies that was also maximum for S3. Overall values of drug

289 351

900959.48

2997.54

5274

0

1000

2000

3000

4000

5000

6000

pH 1.2 pH6.8 Water

RS

T S

olu

bil

ity (

μg/m

l)

RST

RST-β-CD (µg/ml)

88

release were present between 71-92 %. P value for release studies was 0.032. It justified

that results were significant and release of drug was influenced by concentration of

polymer and also method of preparation. It was also observed that microparticles

prepared by solvent evaporation technique had released more amount of drug as

compared to formulations prepared by inclusion complexes as shown in Figure 4.9 (a),

(b). These result may also be due to the increase in surface area of particles that

ultimately had more contact with dissolution media than tablets and shown better

release. Some previous studies like Fahmy et al. (2008) supported our results [200].

They prepared liquisolid tablets and concluded that prepared formulations had more

solubility as compared to pure drug and ultimately enhanced dissolution rate of drug.

Results of in vitro release studies had shown that rapid release of drug from

microparticles enhance the solubility and bioavailability of drug.

Figure 4.9 (a): Cumulative % drug release from different formulations and

RST tablet at pH 6.8

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Cu

mu

lati

ve

% D

rug R

elea

se

Time (hr)

S1

S2

S3

S4

K1

K2

K3

K4

RST Tab

89

Figure 4.9 (b): Cumulative % drug release from different formulations and

RST tablet at pH 1.2

Based upon the findings of dissolution studies of various formulations, it was

observed that β-cyclodextrin had influenced solubility of rosuvastatin used as model

drug in this study. As a result in vitro dissolution was enhanced. When dissolution

results of various formulations were compared based on method of preparation then it

was found that solid dispersions had better release rate than inclusion complexes

containing same concentration of all constituents. Patel et al. (2008) [201] conducted

solubility enhancement study of Lovastatin by using Locust beam gum and concluded

that formulations prepared by solid dispersion technique had rapid dissolution rate as

compared to inclusion complexes. These findings supported our results that solid

dispersions released more concentration of drug than inclusion complexes.

Dissolution data was analyzed by using zero order, first order, Higuchi and

Korsmeyer-Peppas models. Final model selection was made on best fit observations.

Higher values of correlation coefficient i.e. R2 of model proved best fit of that model

for explanation of drug release from formulations. The kinetic models proved first order

release kinetic of drug from microparticles based upon the values of R2 and T25, T50 and

T75. Drug release profile of prepared formulations was compared with reference product

(RST commercially available tables) and it was observed that our formulation followed

first order release with anomalous diffusion while in case of reference product, release

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

S1

S2

S3

S4

K1

K2

K3

K4

RST Tab

90

was zero order as shown in Table 4.7. Agnihotri et al. (2004) [202] also reported that

rapid release of drug from chitosan based microparticles followed first order release

kinetics that might be anomalous diffusion or erosion controlled. They also reported

that release kinetics can be varied by using different polymers which supported our

findings that β-cyclodextrin enhanced dissolution rate of prepared formulation and

followed first order release kinetics while reference product followed zero order release

kinetics.

Table 4.7: In vitro drug release kinetics of solid dispersions and inclusion complexes

Zero order

Parameters S3 K3 RST Tab

Ko 0.734 0.662 0.259

R2 0.7955 0.8071 0.9936

T25 34.077 37.760 96.438

T50 68.154 75.519 192.875

T75 102.231 113.279 289.313

First order

K1 0.025 0.019 0.003

R2 0.9925 0.9841 0.9982

T25 11.697 15.098 89.357

T50 28.184 36.378 215.299

T75 56.367 72.757 430.598

Higuchi

KH 8.408 7.559 2.744

R2 0.9067 0.9125 0.9756

Korsmeyer

Peppas

Kkp 13.355 11.075 0.600

R2 0.9381 0.9209 0.9978

n 0.399 0.416 0.827

91

4.2.6 FTIR studies

FTIR spectra of drug alone and of various formulations were taken to ensure

complex formation between drug and β-cyclodextrin. Drug spectra had shown that

characteristic peeks were present at 3337.90 cm-1, 2968.23 cm-1 and 1435.48 cm-1 as

shown in Figure 4.10 (a) corresponding to cyclic amines, C-H stretching, C=O

stretching, O-H bending. These were almost same as reported in monograph for

rosuvastatin. FTIR spectra of β-cyclodextrin had alcoholic O-H stretching at 3292.74

cm-1, methyl and methylene C-H stretching vibration at 2927.62 cm-1 and C-O ether

stretching vibration at 1021.63 cm-1 as shown in Figure 4.10 (b). In physical mixture,

similar peeks of drug also appeared as shown in Figure 4.10 (c). These findings

confirmed that no interactions were present between drug and β-cyclodextrin.

Figure 4.10 (a): FTIR spectra of rosuvastatin calcium

92


Figure 4.10 (c): FTIR spectra of Physical mixture of rosuvastatin and β-cyclodextrin

93

FTIR spectra of solid dispersions had shown that characteristic peaks of

rosuvastatin were shifted or disappeared from their original location. Characteristic

peaks present at 3337.90 cm-1 and 1435.48 cm-1 due to C-H stretching O-H bending,

respectively were completely disappeared while slight shifting was observed in peak

present at 2968.23 cm-1 as shown in Figure 4.10 (d). Spectrum of inclusion complexes

had shown slight shifting in peaks from 3337.90 cm-1 to 3317.28 cm-1 and from 2968.23

cm-1 to 2928.4 cm-1. Characteristic peak present at 1435.48 cm-1 was completely

disappeared. These findings had confirmed that there was complex formation between

drug and polymer but no interaction was found. This shifting or disappearance of

individual peaks in FTIR of Rosuvastatin calcium is the indication of complex

formation between drug molecules and β-cyclodextrin. Our results are in agreement to

another study conducted by Pavan et al. (2014) where they used liquisolid technology

for solubility enhancement of Rosuvastatin and from FTIR studies observed that some

characteristics peaks of Rosuvastatin were shifted towards lower values and some were

completely disappeared [191]. From this shifting of peaks they concluded that complex

was formed between drug and polymer.

Figure 4.10 (d): FTIR spectra of solid dispersions

94

Figure 4.10 (e): FTIR spectra of inclusion complexes


Thermal analysis of prepared formulations was performed to check the stability

of complexed formed between drug and polymer. DSC thermogram of rosuvastatin

calcium alone as well as of prepared microparticles were taken. There was presence of

sharp endothermic peak at 123 °C in DSC thermogram of pure drug as shown in Figure

4.11 (a). TGA curve of pure Rosuvastatin calcium depicts that gradual reduction in

mass. This loss in mass occurred in three steps as shown in Figure 4.11 (b). Upto 372

°C 48% of mass was lost. TGA thermogram of β-cyclodextrin also shown that there

was gradual reduction in mass of drug in three steps. At 359 °C only 15.72% of β-

cyclodextrin was left as shown in Figure 4.11 (c). DSC curve of β-cyclodextrin had

shown that sharp endothermic peak was present 107 °C as shown in Figure 4.11 (d).

95

Figure 4.11 (a): DSC curves of Rosuvastatin calcium

Figure 4.11 (b): TGA curves of Rosuvastatin calcium

2.31min81.00°C96.92%

9.86min231.24°C93.54%

16.86min372.01°C52.08%

40

60

80

100

Wei

ght (

%)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: TSize: 2.3280 mg

Comment: T

DSC-TGAFile: D:\TGA\RAY\T.001



96

Figure 4.11 (c): TGA curves of β-cyclodextrin

Figure 4.11 (d): DSC curves of β-cyclodextrin

3.95min109.44°C86.97%

14.86min327.46°C81.65%

16.44min359.21°C15.72%

0

20

40

60

80

100

We

igh

t (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)


Comment: B




106.89°C

81.25°C0.06192J/g

329.46°C

324.95°C0.003278J/g

382.31°C

0.03110J/g

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

Heat F

low

(W

/g)

0 50 100 150 200 250 300 350 400

Temperature (°C)


Comment: B




97

In DSC thermogram of inclusion complexes characteristic peak of rosuvastatin

was disappeared from 123 °C and shifted at 340 °C Figure 4.11 (e). This shifting of

peaks was occurred due to the complex formation between drug and β-cyclodextrin.

Due to this complex formation, there was increase in melting temperature of drug

molecules. Complex formation had supported the results of solubility studies that was

enhanced in case of microparticles because β-cyclodextrin is hydrophilic in nature and

enhances solubility of poorly water soluble drugs when these form stable complex with

it.

TGA curves of rosuvastatin had shown that at 372 °C only 52% of mass was

present while in case of inclusion complexes and solid dispersions 66% and 76% of

mass was left as shown in Figure 4.11 (f), and 4.11 (g), respectively . From these results

it was observed that complex was formed between drug molecules and β-cyclodextrin.

Due to this complex formation greater amount of drug was present. Similarly DSC

curve of solid dispersions had shown that endothermic peaks were shifted from 123 °C

to 340 °C as shown in Figure 4.11 (h). This shifting of peaks towards higher temperature

confirmed complex formation between drug and carrier. Similar results were observed

by Gubbi et al. (2010) where they conducted studies on complex formation of β-

cyclodextrin with different poorly water soluble drugs and observed that in DSC spectra

peaks were shifted towards higher temperature values. This shifting confirmed complex

formation [203].

98

Figure 4.11 (e): DSC curves of inclusion complexes

Figure 4.11 (f): TGA curves of inclusion complexes

99

Figure 4.11 (g): TGA curves of solid dispersions

Figure 4.11 (h): DSC curves of solid dispersions

100

4.2.8 Zeta sizer and zeta potential

Size of microparticles was determined by using zeta sizer and stability of

prepared microparticles was measured by zeta potential as shown in Figure 4.12 (a).

Size of microparticles was in the range of micron and majority of particles had uniform

size. Size of microparticles have great role in the release of drug from carrier system.

As shown in peak size and peak area of microparticles that large %age of particles was

present in micron size maximum upto 500 µm but most of the particles were of 200-

300 µm. Zeta potential of microparticles was neutral as shown in Figure 4.12 (b). It

indicates that prepared formulations were almost stable. Due to this stable nature

microparticles can easily be dispersed and become more soluble because stable

microparticles have more solubility. Findings of Shashank et al. (2015) are in

agreement to our results. They prepared microparticles in their study and concluded

that solubility of microparticles was enhanced when zeta potential was neutral and

formulations became stable [199].

Figure 4.12 (a): Zeta size measurement of microparticles (µm)

0

5

10

15

20

25

30

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Inte

nsi

ty %

Size (μm)

101

Figure 4.12 (b): Zeta potential measurement of microparticles

4.2.9 PXRD studies

PXRD is one of the most important parameters to be studied when working on

solubility enhancement of some poorly water soluble chemical entity. Changes in

polymorphic characteristics of certain compounds have great impact on solubility,

dissolution as well as bioavailability. Powder X-Ray diffractogram were taken to

determine nature of rosuvastatin whether it was crystalline or amorphous. The X-Ray

Diffraction pattern of pure drug (rosuvastatin calcium) showed sharp diffraction

peaks at 2θ values of 16.04, 22.45, and 34.3 while microparticles shown no sharp

peak at 2θ values as shown in Figure 4.13. The absence of characteristics peak in

formulations indicated that drug had converted from crystalline to amorphous form. As

a result dissolution of rosuvastatin was enhanced in microparticles prepared by using

β-cyclodextrin because amorphous forms are more soluble as compared to crystalline

form. Both types of formulations i.e., solid dispersions and inclusion complexes had

shown complete disappearance of peaks in X-ray diffraction pattern. Some previous

studies supported our results where Shashank et al. (2015) reported that characteristic

peaks of drug were disappeared from X-ray diffractogram of prepared nanoparticles.

This disappearance of peaks ultimately supported solubility enhancement because

amorphous forms are more soluble than crystalline [199].

102

Figure 4.13: X-Ray diffraction patterns of (A) Rosuvastatin calcium, (B) β-

Cyclodextrin and (C) solid dispersions (D) inclusion complexes

4.2.10 Scanning electron microscopy

Morphology and size of microparticles was determined by using scanning

electron microscopy. SEM images had shown that particles were of approximately

same size as shown in Figure 4.14. There was slight difference in appearance of some

microparticles that were slightly irregular in shape but overall microparticles were

spherical with uniform shape. This may be due to freeze drying and sieving through

200 µm size sieve. SEM images had shown that microparticles were loaded with drug

having smaller size as well as small pores on their surface. These pores helped in the

penetration of water molecules from surrounding. Due to this penetration of water, drug

molecules were released rapidly from microparticles that were complexed with β-

cyclodextrin. Our results are in agreement with the findings of Raghavendra et al.

(2011), microparticles were prepared for oral delivery of insulin and concluded that

103

presence of small openings on surface of microparticles helped in penetration of water

to promote release of drug [204].

Figure 4.14: SEM images of lyophilized microparticles at various magnifications

104

4.2.11 Transmission electron microscopy

In order to determine shape and size of prepared microparticles, TEM analysis

was performed. TEM analysis was performed to see the internal morphology of

microparticles. Images had shown that microparticles had small uniform size upto the

range of 500-600 um. Drug loaded in microparticles either present in dispersed

molecules or in the form of nano dispersions. Images had clearly shown that drug was

loaded in microparticles both in molecular form as well as in the form of nano

dispersions as shown in Figure 4.15. Due to this fine distribution of drug within

microparticles results in increase in solubility of drug molecules that ultimately

enhanced its bioavailability.

105

Figure 4.15: TEM images of lyophilized microparticles at various magnifications

106

4.2.12 Stability studies

Stability studies of two best formulations were conducted for six month period.

Microparticles were kept under accelerated conditions of temperature and humidity 35

± 5оС and 75% ± 5%, respectively. The samples were taken after 1, 2, 3, and 6 month

time period. These samples were evaluated for different parameters like solubility

studies, dissolution studies, FTIR, thermal analysis etc. The results had indicated that

there were no significant variations occurred in drug content at the end of 6 months.

Approximately similar amount of drug was released even in samples taken after 6

month period. Its means that our formulations were stable under different accelerated

conditions of temperature and humidity. Physical appearance of prepared formulation

was also compared to check any change but no difference was observed in freshly

prepared formulations and for that kept under accelerated conditions for stability

studies.

107

4.3 Hydrogel microparticles

Two different types of hydrogel microparticles were prepared containing

methacrylic acid and Acrylamido-2-methyl propane sulphonic acid in various

concentrations along with β-cyclodextrin, MBA and APS. Total eighteen formulations

of hydrogel microparticles were prepared, out of which nine had methacrylic acid and

other nine had Acrylamido-2-methyl propane sulphonic acid. Prepared formulations

varied from each other by concentration of β-cyclodextrin which is used as polymer,

MBA used as cross linker and methacrylic acid and Acrylamido-2-methyl propane

sulphonic acid which were used as monomers. Prepared hydrogel microparticles were

evaluated for various parameters and studied the effect of polymer, monomer and cross

linker on swelling, release and solubility of rosuvastatin calcium that was used as model

drug. Hydrogel microparticles were first evaluated for micromeritics parameters.

Results of product yield and entrapment efficiency are listed in Table 4.8 and

4.9. Results of these parameters are in acceptable limits. All prepared formulations of

hydrogel microparticles had product yield of 86.50 ± 0.30 to 91 ± 0.50%. Findings of

entrapment efficiency had shown that 82.30 ± 0.25 to 89.00 ± 0.15% of drug was loaded

in various formulations. Entrapment efficiency is vital parameter to determine

efficiency of prepared carrier system to evaluate proper dosage regimen. Our results

had justified that prepared carrier system was suitable for loading of rosuvastatin

calcium to deliver desired quantity of drug at targeted sites.

108

Table 4.8: Entrapment efficiency and product yield of HA1 to HA9

aAverage of three determinations; ± SD- (Standard deviation).

Table 4.9: Entrapment efficiency and product yield of HS1 to HS9

aAverage of three determinations; ± SD- (Standard deviation).

Formulations % Entrapment

Efficiencya

Product

yielda (%)

HA1 82.30 ± 0.25 88.50 ± 0.50

HA2 85.10 ± 0.30 89.80 ± 0.20

HA3 88.90 ± 0.25 90.50 ± 0.50

HA4 89.00 ± 0.15 91.00 ± 0.50

HA5 86.40 ± 0.10 88.50 ± 1.15

HA6 87.05 ± 0.50 86.50 ± 0.30

HA7 86.20 ± 0.30 88.50 ± 0.25

HA8 85.40 ± 0.25 89.10 ± 0.15

HA9 87.80 ± 0.30 87.90 ± 0.25

Formulations Entrapment

Efficiencya (%)

Product

yielda (%)

HS1 83.50 ± 0.30 89.00 ± 1.30

HS2 84.60 ± 1.15 88.90 ± 0.15

HS3 87.50 ± 0.50 89.00 ± 0.25

HS4 88.50 ± 0.25 90.50 ± 1.15

HS5 87.50 ± 0.30 89.10 ± 0.25

HS6 88.50 ± 0.25 87.00 ± 1.30

HS7 87.80 ± 0.25 89.10 ± 0.50

HS8 88.40 ± 0.15 90.50 ± 0.25

HS9 86.60 ± 1.30 88.70 ± 0.30

109

Flow properties are very important for powder or particulate material in

pharmaceutical dosage forms. Materials that do not have good flow properties are

difficult to use and ultimately leads towards improper loading of drug. To avoid such

issues hydrogel microparticles were evaluated for flow properties such as angle of

repose, bulk density, tapped density, Carr’s index and Hausner’s ratio. Results of these

rheological parameters of formulations containing methacrylic acid are present in Table

4.10. Values of angle of repose was present 21.50 Ɵ to 26.10 Ɵ. Its value below than

30 is considered good for proper flow of powder material [187]. Results of rheological

parameters of formulations containing AMPS are also mentioned in Table 4.11. All

results were found within pharmacopeial limits. These results favored that our material

had good flow properties. These findings are in agreement with the findings of a study

conducted by Sudarshan and Sameer (2012) where they prepared sublingual tablets for

solubility enhancement and evaluated flow properties [205].

Table 4.10: Results of bulk density, tapped density, angle of Repose, Hausner’s ratio

and Carr’s index of HA1 to HA9

Code

Bulk density

(g/ml)

Tapped density

(g/ml)

Angle of

repose

(ϴ)

Hausner’s

ratio

Carr’s index

(%)

HA1 0.629 0.737 23.60 1.17 15.75

HA2 0.637 0.729 21.50 1.14 16.50

HA3 0.631 0.733 23.80 1.16 14.97

HA4 0.619 0.719 25.40 1.16 16.45

HA5 0.621 0.727 24.50 1.17 15.50

HA6 0.609 0.719 22.60 1.18 16.40

HA7 0.629 0.723 24.90 1.14 15.98

HA8 0.623 0.751 24.70 1.20 16.57

HA9 0.621 0.730 23.80 1.17 15.82

110

Table 4.11: Results of bulk density, tapped density, angle of Repose, Hausner’s ratio

and Carr’s index of HS1 to HS9

4.3.1 Swelling behaviour

Release of drug from hydrogel microparticles is clearly based upon their

swelling behaviour. If higher swelling is present within hydrogel microparticles, then

more rapid drug will be released. All prepared formulations had shown good swelling

behaviour. Swelling of hydrogel microparticles was pH dependent in case of

microparticles containing methacrylic acid while almost independent in case of

Acrylamido-2-methyl propane sulphonic acid. Hydrogel microparticles containing

methacrylic acid had low swelling index at pH 1.2 when compared with pH 6.8. Low

swelling of hydrogel microparticles may be due to carboxylic groups that remain

protonated and network structure of copolymer was not exposed. As a result segmental

chain mobility hydrogel microparticles was minimum at low pH. Higher swelling at 6.8

pH might be due to greater mobility of polymer chains as a result size of mesh network

increases.

Methacrylic acid had pka value 4.5. When pH increases above the pka then

deprotonation occurs. Due to this deprotonation, anions of carboxylic group repel each

other and extension of cross linked network occurs. Ultimately more water is entered

Code

Bulk density

(g/ml)

Tapped density

(g/ml)

Angle of

repose (ϴ)

Hausner’s

ratio

Carr’s index

(%)

HS1 0.597 0.713 26.10 1.19 15.80

HS2 0.640 0.727 24.50 1.13 17.26

HS3 0.623 0.727 24.80 1.16 15.28

HS4 0.623 0.738 24.70 1.18 16.90

HS5 0.639 0.746 25.80 1.16 14.50

HS6 0.609 0.713 25.60 1.17 16.80

HS7 0.642 0.752 23.58 1.17 15.45

HS8 0.623 0.739 24.06 1.18 16.10

HS9 0.637 0.742 23.84 1.16 14.90

111

inside hydrogel microparticles that favors swelling and increases swelling index. When

methacrylic acid ratio was further increased then it was observed that swelling was

decreased. This might be due to more cross linking of monomer that results in formation

of dense mass. Penetration of water within hydrogel microparticles was reduced due to

this dense structure. When concentration of beta cyclodextrin was increased in

hydrogel microparticles then swelling was also increased. This might be due to

hydrophilic nature of β-cyclodextrin. Due to this hydrophilic behaviour, β-cyclodextrin

attracted more water and retained this water inside the system. Previously, Elliot et al.

(2004) also observed same kind of swelling behaviour from methacrylic acid and

AMPS and concluded that AMPS had pH independent swelling while in case of MAA

swelling was pH dependent and MAA shown more swelling at higher pH values [206].

4.3.2 FTIR studies

FTIR spectra of pure drug, different excipients used in these formulations and

of various prepared formulation of hydrogel microparticles were taken to ensure

complex formation between drug and β-cyclodextrin. FTIR spectra of physical mixture

of drug, polymer and other excipients were also taken to study any interaction between

them. These spectra’s were recorded between 500 cm-1 to 4000 cm-1. Drug spectra had

shown that characteristic peeks were present at 3337.90 cm-1, 2968.23 cm-1 and

1435.48 cm-1 corresponding to cyclic amines, C-H stretching, C=O stretching, O-H

bending as shown in Figure 4.16 (a). These were almost same as reported in monograph

for rosuvastatin. Spectrum of β-cyclodextrin had alcoholic O-H stretching at 3292.74

cm-1, methyl and methylene C-H stretching vibration at 2927.62 cm-1 and C-O ether

stretching vibration at 1021.63 cm-1 as shown in Figure 4.16 (b). In FTIR spectra of

MAA characteristic peaks were appeared at 2972.62 cm-1, 1708.83 cm-1 and 1173.45

cm-1 Figure 4.16 (c). FTIR spectra of MBA was also recorded and observed that

characteristic peaks were present 3305.41 cm-1, 1538.39 cm-1 and 1225.23 cm-1 as

shown in Figure 4.16 (d). In FTIR spectra of AMPS peaks were present at 2986.54 cm-

1 and 1233.49 cm-1 as mentioned in Figure 4.16 (e) while in spectrum of APS

characteristic peaks were located at 3226.21 cm-1, 1415.36 cm-1 and 1145.46 cm-1 as

appeared in Figure 4.16 (f).

112

Figure 4.16 (a): FTIR spectra of Rosuvastatin calcium


113

Figure 4.16 (c): FTIR spectra of methacrylic acid

Figure 4.16 (d): FTIR spectra of MBA

114

Figure 4.16 (e): FTIR spectra of Acrylamido-2-methyl propane sulphonic acid

Figure 4.16 (f): FTIR spectra of Ammonium per sulphate

115

All ingredients` used in formulation of hydrogel microparticles were physical

mixed and FTIR spectra’s were recorded to study any interaction between drug,

polymer monomers, initiator and crosslinker. Physical mixture of β-cyclodextrin and

MAA had shown that similar characteristic peaks were appeared as in alone spectra’s

as shown in Figure 4.16 (g). FTIR spectra of physical mixture of Rosuvastatin calcium

with β-cyclodextrin was taken and observed that same peaks were appeared as in FTIR

spectra of individual ingredients as shown in Figure 4.16 (h). Rosuvastatin calcium was

also physically mixed with APS, AMPS and MBA to see any interaction present

between these ingredients. But results revealed that similar peaks were observed, as

present in individual spectra of Rosuvastatin calcium as shown in Figure in 4.16 (i).

From evaluation of individual spectra’s of different ingredients and from physical

mixtures, it was observed that no physical interaction was present in all ingredients used

in synthesis of hydrogel microparticles.

Figure 4.16 (g): FTIR spectra of Physical mixture of β-cyclodextrin and MAA

116

Figure 4.16 (h): FTIR spectra of Physical mixture of β-cyclodextrin and

Rosuvastatin calcium

Figure 4.16 (i): FTIR spectra of Physical mixture of rosuvastatin, ammonium

persulfate, Acrylamido-2-methyl propane sulphonic acid and

methylene bisacrylamide

117

In case of hydrogel microparticles containing methacrylic acid as monomer,

characteristic peak that was present at 3337.90 cm-1 disappeared while characteristic

peak present at 1435.48 cm-1 was shifted towards lower value at 1306.50 cm-1 as shown

in Figure 4.16 (j). While in hydrogel microparticles that were prepared by using

Acrylamido-2-methyl propane sulphonic acid as monomer there was shifting of

characteristics peaks from 3337.90 cm-1 and 2968.23 cm-1 to 3306.87 cm-1 and 2930.97

cm-1, respectively as shown in Figure 4.16 (k). Peaks present at 1438.03 cm-1 was

shifted towards higher values of 1655.91. These findings had confirmed that there was

complex formation between drug and polymer. Our results are in agreement of study

conducted by Deepak and Reena (2012) where complex formation between drug and

polymer results in shifting or disappearance of characteristics peaks. Their results also

shown that this complex formation with hydrophilic polymer enhances solubility [207].

FTIR spectra’s of physical mixtures were also compared with prepared hydrogel

microparticles. Characteristic peaks of Rosuvastatin were present in physical mixture

of drug and excipients similar to the peaks of drug that were present in FTIR spectra of

pure drug but FTIR spectra of hydrogel microparticles had shown that characteristic

peaks of drug were shifted or disappeared. This was due to the grafting of polymer with

monomer to form a new co-polymer that was complexed with drug molecules. This

attachment of drug molecules with co-polymer network of hydrogel microparticles

results in either shifting of drug characteristics peaks or complete disappearance of

these peaks. This modification in drug’s characteristics peaks confirmed that drug was

complexed with co-polymer network of hydrogel microparticles. These observations of

FTIR studies supported solubility of drug due to the fact that when drug complexed

with hydrophilic carrier system such as β-cyclodextrin then ultimately it became more

soluble than pure form [207].

118

Figure 4.16 (j): FTIR spectra of Hydrogel microparticles containing

methacrylic acid

Figure 4.16 (k): FTIR spectra of Hydrogel microparticles containing

Acrylamido-2-methyl propane sulphonic acid

119


Thermal analysis of prepared formulations was performed to check the stability

of complex formed between drug and grafted co-polymer prepared by using β-

cyclodextrin and two monomers separately that were Acrylamido-2-methyl propane

sulphonic acid and methacrylic acid. DSC and TGA thermogram of rosuvastatin

calcium, β-cyclodextrin, MAA, AMPS, MBA and APS were taken and compared with

different formulations of prepared hydrogel microparticles. There was presence of

sharp endothermic peak at 123 °C in DSC thermogram of pure drug as shown in Figure

4.17 (a). TGA curve of pure Rosuvastatin calcium depicts that gradual reduction in

mass. This loss in mass occurred in three steps as shown in Figure 4.17 (b). Upto 372

°C 48% of mass was lost. TGA thermogram of β-cyclodextrin also shown that there

was gradual reduction in mass of drug in three steps. At 359 °C only 15.72% of β-

cyclodextrin was left as shown in Figure 4.17 (c). DSC curve of β-cyclodextrin had

shown that sharp endothermic peak was present 107 °C as shown in Figure 4.17 (d).

TGA curve of AMPS illustrated that thermal degradation was accomplished in two

steps at 206 °C and 262 °C with weight loss of 2.34% and 48.84%, respectively as

demonstrated in Figure 4.17 (e). In DSC curve of AMPS a sharp endothermic peak was

present at 216.48 °C as shown in Figure 4.17 (f). Thermal degradation of MBA occurred

in three steps at 187.64 °C, 226.51 °C and 344.52 °C with gradual weight loss of 2.81%,

13.09% and 16.64%, respectively as shown in Figure 4.17 (g). In DSC thermogram of

MBA a sharp exothermic peak was observed at 218.98 °C as shown in Figure 4.17 (h).

120

Figure 4.17 (a): DSC curve of rosuvastatin calcium

Figure 4.17 (b): TGA curve of rosuvastatin calcium

121

Figure 4.17 (c): TGA curve of β-cyclodextrin

Figure 4.17 (d): DSC curve of β-cyclodextrin

3.95min109.44°C86.97%

14.86min327.46°C81.65%

16.44min359.21°C15.72%

0

20

40

60

80

100

We

igh

t (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)


Comment: B




106.89°C

81.25°C0.06192J/g

329.46°C

324.95°C0.003278J/g

382.31°C

0.03110J/g

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

Heat F

low

(W

/g)

0 50 100 150 200 250 300 350 400

Temperature (°C)


Comment: B




122

Figure 4.17 (e): TGA curve of AMPS

Figure 4.17 (f): DSC curve of AMPS

8.61min206.13°C97.66%

11.40min262.05°C51.16%

0

20

40

60

80

100

Weig

ht (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: MSize: 2.5450 mg

Comment: M

DSC-TGAFile: D:\TGA\RAY\M.001



216.48°C

208.21°C0.2792J/g

341.79°C

326.84°C0.01355J/g

0.000

0.002

0.004

0.006

0.008

0.010

He

at

Flo

w (

W/g

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: MSize: 2.5450 mg

Comment: M

DSC-TGAFile: D:\TGA\RAY\M.001



123

Figure 4.17 (g): TGA curve of MBA

Figure 4.17 (h): DSC curve of MBA

7.73min187.64°C97.19%

9.64min226.51°C86.91%

15.55min344.52°C83.36%

50

60

70

80

90

100

We

ight (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: OSize: 2.7410 mg

Comment: O

DSC-TGAFile: D:\TGA\RAY\O.001



218.98°C

211.85°C0.02199J/g

344.19°C

334.12°C0.01859J/g

187.05°C

183.58°C0.003379J/g

196.65°C

194.22°C0.003755J/g

0.002

0.004

0.006

0.008

0.010

0.012

He

at

Flo

w (

W/g

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: OSize: 2.7410 mg

Comment: O

DSC-TGAFile: D:\TGA\RAY\O.001



124

From TGA thermogram of rosuvastatin calcium it was seen that there was

reduction in concentration of drug with increase in temperature. At 80 °C there was loss

of 3% of drug after 2.3 minutes and there was gradual reduction in mass of drug that

reached to 52% at 332 °C. When this TGA thermogram of rosuvastatin calcium was

compared with TGA thermogram of prepared hydrogel microparticles containing

methacrylic acid as monomer and loaded with drug. It was observed that a gradual

reduction in weight occured but it was very slow in hydrogel microparticles. First

decline in mass was occurred at 202 °C and only 10 % weight was lost at this high

temperature. Evan at 370 °C only 28% of mass was lost and 72% of mass was present

as such as shown in Figure 4.17 (i). This was much greater than TGA thermogram of

alone drug. These results were observed due to the formation of complex between

polymer and monomer to form grafted co-polymer that also complexed with loaded

drug. Due to this complex formation of drug with co-polymer results in elevation in

temperature required for thermal degradation.

When TGA thermogram of hydrogel microparticles containing 2-Acrylamido-

2-methyl propane sulphonic acid as monomer was studied then it was also observed

that much greater weight of drug intact in hydrogel microparticles as compared to alone

drug, monomer and polymer. Only 35% of mass was degraded at 372 °C as shown in

Figure 4.17 (j).

In DSC thermogram of pure drug, it was observed that presence of sharp

endothermic peak at 123 °C. While in case of hydrogel microparticles containing

methacrylic acid similar type of endothermic peak observed but at greater temperature

297 °C as shown in Figure 4.17 (k). AMPS containing hydrogel microparticles had

shown endothermic peak even at higher temperature 318 °C as shown in Figure 4.17

(l). This shifting of peaks towards higher temperature values confirmed formation of

complex between drug molecules and grafted copolymer. Due to this complex

formation drug had become more water soluble due to the hydrophilic nature of β-

cyclodextrin that was used as polymer in the formulation of hydrogel microparticles.

125

Results of thermal analysis confirmed formation of stable complex between

drug molecules and polymer that ultimately enhanced solubility of drug. It was also

observed that 2-Acrylamido-2-methyl propane sulphonic acid containing hydrogel

microparticles were more strongly complexed as compared to methacrylic acid

containing hydrogel microparticles. Our results are in agreement with a previously

conducted study of Chen et al. (2008) where they prepared chitosan based hydrogel

beads and concluded that disappearance or shifting of peaks is the indication of complex

formation and drugs become more soluble when they form complex with other

hydrophilic moieties [208].

Figure 4.17 (i): TGA of hydrogel microparticles containing methacrylic acid

8.38min201.74°C89.21%

12.95min293.33°C76.78%

16.77min370.11°C61.49%

50

60

70

80

90

100

Weig

ht (%

)

0 50 100 150 200 250 300 350 400

Temperature (°C)

Sample: H S 1Size: 2.0950 mg

Comment: H S 1

DSC-TGAFile: D:\TGA\RAY\H S 1.001



126

Figure 4.17 (j): TGA of hydrogel microparticles containing AMPS

Figure 4.17 (k): DSC of hydrogel microparticles containing methacrylic acid

127

Figure 4.17 (l): DSC of hydrogel microparticles containing AMPS

4.3.4 Powder X-ray diffraction studies

XRD is one of the most important parameter to be studied when working on

solubility enhancement of some poorly water soluble chemical entity. Drugs are either

present in crystalline form or in amorphous nature. Those compounds that have

crystalline nature they have poor water solubility and have typical characteristics peaks

in X-ray diffractograms while in case of amorphous compounds no peaks are present

when X-ray diffractograms has been taken. Changes in polymorphic characteristics of

certain compounds have great impact on solubility, dissolution as well as

bioavailability. Rosuvastatin calcium is crystalline in nature and has poor water

solubility. Powder X-Ray diffractogram of prepared hydrogel microparticles as well as

drug were taken to study these polymorphic characteristics of rosuvastatin, whether it

was crystalline or amorphous.

The X-Ray Diffraction pattern of pure drug (rosuvastatin) showed sharp

diffraction peaks at 2θ values of 16.04, 22.45, and 34.3. These were typical peaks

of rosuvastatin calcium that confirm its crystalline nature. While in case of hydrogel

microparticles these diffraction peaks were disappeared. Both types of hydrogel

microparticles had shown a flat diffractogram as shown in Figure 4.18, C and D.

Absence of characteristics peak in formulation indicated that drug converted from

128

crystalline to amorphous form. As a result solubility of rosuvastatin was enhanced in

hydrogel microparticles because amorphous forms are more soluble than crystalline. In

a previously reported study, Kazimiera (2001) prepared chitosan based microcrystals

to study release rate of drug from carrier system and concluded that drug was converted

from crystalline to amorphous form in X-ray diffraction. It was also reported that

amorphous form had more solubility than crystalline [209]. These findings are in

agreement with our results.

Figure 4.18: Powder X-Ray diffraction pattern of (A) Rosuvastatin calcium, (B) β-

cyclodextrin, (C) hydrogel microparticles of methacrylic acid, (D)

hydrogel microparticles of AMPS

129

4.3.5 Scanning electron microscopy

Morphology and size of prepared hydrogel microparticles was determined by

using scanning electron microscopy. SEM images of different prepared hydrogel

microparticles were taken. SEM images had shown that hydrogel microparticles had

small crystal like shape in most of the microparticles while few had spherical shape as

shown in Figure 4.19. Inside of microparticles small white spots were present which

indicate that drug was loaded in microparticles and distributed within inner side of

microparticles. Images had also shown that small pores were also present on the surface

of these particles that favor penetration of water from outside and help in the release of

loaded drug from pores. Due to this entrance of water inside microparticles to fasten

the release of drug had ultimately enhanced water solubility of drug. Nihar and Patel

(2014) also observed from SEM studies, that release of drug was enhanced by

penetration of water from small pores present on the surface of microparticles [210].

These findings also supported our results.

130

Figure 4.19: SEM images of lyophilized hydrogels microparticles at various

Magnifications.

131

4.3.6 In vitro drug release studies

Dissolution studies were carried out at 6.8 and 1.2 pH phosphate buffer and

HCl, respectively to determine in vitro drug release. Dissolution studies were performed

for various prepared hydrogel microparticles formulations and for commercially

available tablets in order to compare release of drug. Results had shown that there was

a marked difference in release of commercially available tablets of rosuvastatin and

hydrogel microparticles prepared by using β-cyclodextrin and monomers. It was

observed that there was more release of drug in basic media as compared to acidic pH.

This was due to the acidic nature of rosuvastatin calcium. It is weak acid and become

more ionized in basic environment thus soluble in basic environment. As already

discussed in swelling studies of hydrogel microparticles that methacrylic acid (MAA)

containing microparticles had pH dependent swelling while Acrylamido-2-methyl

propane sulphonic acid (AMPS) containing hydrogel microparticles had pH

independent swelling behaviour. For release of drug from hydrogel microparticles, it is

necessary to become swollen first and then release of drug occurred.

In dissolution studies of methacrylic acid containing hydrogel microparticles it

was observed that rapid release of drug had been taken place at 6.8 pH while very slow

release at acidic (1.2) pH as shown in Figures 4.20 (a) and 4.20 (b). At 6.8 pH maximum

amount of drug was released (85.74%) from formulation HM2 containing 4g/100g of

β-cyclodextrin with in 1 hour of dissolution studies. This formulation had MAA

30g/100g and MBA 0.4 g/100g. Approximately same concentration of drug was

released from formulation HM3. This formulation had slight greater amount of β-

cyclodextrin 6g/100g. So from dissolution studies it was observed that maximum

amount of drug was released when 4 g/100g of β-cyclodextrin was used in hydrogel

microparticles. From results of dissolution studies at pH 6.8 of formulation containing

MAA as monomer, it was found that β-cyclodextrin concentration that favored

maximum release of drug from hydrogel microparticles was 4 g/100g.

In different formulations, concentrations of monomer and crosslinker were also

varied and it was found that maximum amount of drug was released when MAA

(monomer) concentration was 30g/100g and MBA (crosslinker) was 0.4g/100g. When

higher concentrations of MBA were used these resulted in formation of more compact

structure that retorted release of drug from polymeric network. When even less

concentration was used then such type of crosslinking was not occurred that can hold

sufficient amount of drug. Thus 0.4g/100g of MBA concentration was considered

132

optimum for synthesis of hydrogel microparticles to enhance solubility of rosuvastatin

calcium. MAA concentration that was found suitable for synthesis of hydrogel

microparticles to improve dissolution profile of rosuvastatin calcium was 30g/100g.

MAA concentration was varied in various formulation but it was found that maximum

release occurred when MAA was used at 30g/100g of formulation. When release profile

of these formulations was compared with commercially available tablets of rosuvastatin

calcium at 6.8 pH then it was observed that within 3 hours only 43% of drug was

released. Thus it become clear that more drug was released approximately double in

case of MAA containing hydrogel microparticles as compared to RST tablets at 6.8 pH

as shown in Figure 4.20 (a).

Dissolution studies of MAA containing hydrogel microparticles at pH 1.2 had

shown that approximately same concentration of drug released when compared with

RST tablets as shown in Figure 4.20 (b). This was due to non swelling behaviour of

MAA at acidic pH. As discussed earlier that swelling is vital for release of drug from

hydrogel microparticles. Secondly, drug is also acidic in nature and when exposed to

acidic media then it remained insoluble.

Figure 4.20 (a): Cumulative % drug release from hydrogel microparticles containing

MAA and from RST tablets at pH 6.8

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Cu

mu

lati

ve

% D

rug R

elea

se

Time (min)

HM1

HM2

HM3

HM4

HM5

HM6

HM7

HM8

HM9

RST Tab

133

Figure 4.20 (b): Cumulative % drug release from hydrogel microparticles containing

MAA and from RST tablets at pH 1.2

In case of AMPS pH independent release of drug had been occurred but more

in basic media as compared to acidic one. At pH 6.8 maximum amount of drug was

released (92%) from formulation HS7 as shown in Figure 4.20 (c). This formulation

had 1g/100g of β-cyclodextrin. Other formulations containing same concentration of β-

cyclodextrin but less amount of drug was released. Its mean that along with β-

cyclodextrin concentration AMPS and MBA had also important role in the release of

drug from microparticles. Various concentrations of AMPS and MBA were studied to

optimize best concentration to enhance dissolution of RST. When 4g/100g of AMPS

was used, it produced best release rate as compared to 2, 5 and 6 g/100g of formulation.

Similarly, MBA was produced best release rate when 0.4g/100g was used. Various

other concentrations of MBA were used but maximum release observed at 0.4g/100g

of formulation. Thus it was found that maximum amount of RST was released from

HS7 containing 1g/100g of β-cyclodextrin, 4g/100g of AMPS and 0.4g/100g of MBA.

When drug release profile of hydrogel microparticles was compared with RST tablets

then better release was observed from hydrogel microparticles. From formulation HS7,

92% of drug was released within 1 hour while from RST tablets only 44% of drug was

released in 3 hours at pH 6.8.

0

5

10

15

20

25

30

35

40

45

0 50 100 150 200

Cu

mu

lati

ve

% D

rug

Rel

eas

Time (min)

HM1

HM2

HM3

HM4

HM5

HM6

HM7

HM8

HM9

RST Tab

134

Slightly less amount of drug was released from hydrogel microparticles

containing AMPS at pH 1.2 but the optimized formulation was HS7 as shown in Figure

4.20 (d). This better release of drug at 1.2 pH from AMPS containing hydrogel

microparticles than MAA containing hydrogel microparticles was due to swelling

behaviour of AMPS. AMPS containing hydrogel microparticles had shown pH

independent swelling but better release was observed at 6.8pH due to weak acidic

nature of RST. Similar to pH 6.8 there was greater amount of drug released at 1.2 pH

from AMPS containing hydrogel microparticles than RST tablets. From dissolution

studies of various formulations of hydrogel microparticles, it was found that hydrogel

microparticles enhanced release rate of drug. Results of another study conducted by

Chen et al. (2005) also supported our study that β-cyclodextrin enhanced dissolution

profile of BCS class II drugs when these form stable complex with β-cyclodextrin

[211]. Based on the findings of dissolution studies of various formulations, we

observed that β-cyclodextrin has improved dissolution profile of rosuvastatin. As a

result, in vitro solubility was enhanced.

Figure 4.20 (c): Cumulative % drug release from hydrogel microparticles containing

AMPS and from RST tablets at pH 6.8

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Cu

mu

lati

ve

% D

rug R

elea

se

Time (min)

HS1

HS2

HS3

HS4

HS5

HS6

HS7

HS8

HS9

RST Tab

135

Figure 4.20 (d): Cumulative % drug release from hydrogel microparticles containing

AMPS and from RST tablets at pH 1.2

In vitro release kinetic models were applied to determine best fit model on

release of drug from hydrogel microparticles. Drug release followed first order kinetic

in case of hydrogel microparticles while RST tablets followed zero order kinetics as

shown in Table 4.12. Formulations HS4 and HS7 followed anomalous diffusion of

drug, which means that dual mode of drug release i.e., diffusion and erosion. This was

due to the presence of β-cyclodextrin in hydrogel microparticles that enhances release

of drug from carrier system.

0

10

20

30

40

50

60

70

80

90

100

0 50 100 150 200

Cu

mu

lati

ve

% D

rug

Rel

ease

Time (min)

HS1

HS2

HS3

HS4

HS5

HS6

HS7

HS8

HS9

RST Tab

136

Table 4.12: In vitro drug release kinetics of hydrogel microparticles

Zero order

Parameters HS4 HS7 RST Tab

Ko 0.720 0.737 0.259

R2 0.7769 0.7990 0.9573

T25 34.726 33.935 89.438

T50 69.452 67.870 192.875

T75 104.178 101.805 289.313

First order

K1 0.025 0.025 0.003

R2 0.9988 0.9884 0.9682

T25 11.684 11.389 89.357

T50 28.153 27.442 215.299

T75 56.305 54.883 430.598

Higuchi

KH 8.286 8.459 2.744

R2 0.8957 0.9131 2.744

Korsmeyer

Peppas

Kkp 14.148 14.154 0.600

R2 0.9108 0.9711 0.9978

n 0.383 0.9265 0.827

137


Rosuvastatin calcium is a weak acid. Due to its acidic nature it has more

solubility in basic media as compared to acidic pH. So it become ionized at higher pH

values and therefore soluble in high pH solutions. Solubility studies of pure drug and

prepared hydrogel microparticles were conducted at 6.8 pH phosphate buffer solution

as well as in HCl buffer solution of 1.2 pH. Results had shown that highest solubility

of MAA containing hydrogel microparticles was present at 6.8 pH phosphate buffer

when compared with buffer solution of low pH (1.2). However, highest solubility was

observed in water in ionic form as shown in Figure 4.21 (a). From solubility studies of

rosuvastatin calcium it was observed that pure drug had very low solubility in acidic

media at 1.2 pH buffer while slight better solubility in phosphate buffer of basic pH 6.8.

In water, pure drug had greater solubility as compared to 1.2 and 6.8 pH buffers. When

solubility studies of hydrogel microparticles were performed, it was observed that same

amount of drug loaded in hydrogel microparticles had several folds more solubility than

pure drug. Increase in solubility of drug from MAA containing hydrogel microparticles

was upto 9.59 folds at 6.8 pH while at 1.2 pH, solubility was remained unchanged. This

was due to non swelling behaviour of MAA at acidic pH. In distilled water, solubility

was increased upto 6.9 folds as compared to solubility of pure drug as shown in Figure

4.21 (a).

138

Figure 4.21 (a): Solubility studies of pure drug and hydrogel microparticles

containing MAA

AMPS containing hydrogel microparticles had shown enhancement in solubility

in both pH solutions as well as in distilled water. This was due to pH independent

swelling behaviour of AMPS. AMPS had more swelling at 1.2 pH than 6.8 pH. But

more solubility was observed in 6.8 pH solution. This more solubility in 6.8 pH

phosphate buffer solution was due to weak acidic nature of RST. Solubility of AMPS

containing hydrogel microparticles was enhanced upto 7.5 folds in 1.2 pH buffer

solution while 10.66 folds solubility was enhanced at 6.8 pH. In distilled water,

solubility of AMPS containing hydrogel microparticles was increased upto 7.30 folds

as shown in Figure 4.21 (b).

These results justified that hydrogel microparticles had enhanced solubility of

rosuvastatin calcium in different pH media but it varied based upon the pH of used

media. Pure drug had more solubility in distilled water as compared to HCl buffer of

1.2 pH and phosphate buffer of pH 6.8. The order in which pure drug had more

289 351

900

280

3368

6212

0

1000

2000

3000

4000

5000

6000

7000

pH 1.2 pH6.8 Water

RS

T S

olu

bil

ity (

ug/m

l)

RST

β-CD-g-MAA Hydrogel

microparticles (µg/ml)

139

solubility was distilled water > phosphate buffer of pH 6.8 > HCl buffer of 1.2 pH.

While in case of hydrogel microparticles, rosuvastatin calcium solubility was increased

in all three media used for solubility studies but it was 10.66 folds increase in phosphate

buffer of pH 6.8 and 7.30 folds in distilled water. Shilpi et al. (2015) enhanced solubility

of Nelfinavir by preparing complex of drug with β-cyclodextrin in different ratios and

concluded that solubility enhanced upto 5.32 folds at 1.2 pH while 3.64 folds at 4.5 pH

[198]. Our results shown more solubility at higher pH (6.8) as compared to 1.2 pH. This

difference in results is due to the nature of drug. Nelfinavir is basic in nature therefor,

it is more soluble in acidic media as compared to higher pH. While Rosuvastatin

calcium is weak acidic drug. Due to this acidic nature, it become more soluble at higher

pH (6.8) values as compared to low pH (1.2). Rosuvastatin is available in its calcium

salt, therefore, it is more soluble in water as compared to different buffer media. But

when Rosuvastatin calcium complexed with hydrophilic polymers then it became more

soluble in buffer media of higher pH rather than in pure water as shown in Figures 4.21

(a) and 4.21 (b).

Figure 4.21 (b): Solubility studies of pure drug and hydrogel microparticles

containing AMPS

289 351

900

2178

3742

6572

0

1000

2000

3000

4000

5000

6000

7000

pH 1.2 pH6.8 Water

RS

T S

olu

bil

ity (

ug/m

l)

RST

β-CD-g-AMPS Hydrogel

microparticles (µg/ml)

140

4.3.8 Zeta size and zeta potential studies

Zeta size and zeta potential of hydrogel microparticles was calculated to

determine the size and stability of prepared formulations as shown in Figure 4.22 (a)

and 4.22 (b), respectively. Values of zeta size had shown that all hydrogel

microparticles were of micron size ranging from 200-500 µm but majority of

microparticles had size of 300 µm. All hydrogel microparticles had uniform size. Zeta

potential of hydrogel microparticles was neutral, its mean that prepared formulations

were almost stable. Due to this stable nature, hydrogel microparticles can be easily

swollen up by absorbing water from surrounding media and drug was released more

rapidly due to this swelling behaviour. Ultimately more amount of drug was released

in lesser time period with improved solubility. The results of a study conducted by

Shashank et al. (2015) are in agreement to our results where they had prepared

microparticles and concluded that net charge on microparticles was neutral. This favors

solubility enhancement as well as stability [199].

Figure 4.22 (a): Zeta size measurement of hydrogel microparticles (µm)

0

10

20

30

40

50

60

0 200 400 600 800 1000 1200 1400 1600 1800 2000

Inte

nsi

ty %

Size (μm)

141

Figure 4.22 (b): Zeta potential measurement of hydrogel microparticles

4.3.9 Transmission electron microscopy

Internal morphology of hydrogel microparticles was determined by performing

TEM studies. TEM images of various formulations of hydrogel microparticles were

taken. Images had shown that microparticles had small uniform size upto the range of

400-500 um. Drug loaded in microparticles either present in dispersed molecules or in

the form of nano dispersions. In solubility enhancement dispersed molecules or more

preferable than nano dispersion. Images had clearly shown that drug was loaded in

microparticles both in molecular form as well as in the form of nano dispersions as

shown in Figure 4.23. In side microparticles, loaded drug was present just like small

white spots. Due to this fine distribution of drug within hydrogel microparticles results

in increase in solubility of drug molecules that ultimately enhanced its bioavailability.

These results are in agreement with the findings of Shilpi et al. (2010) where they

prepared drug complexes with PVA, PEG and HPMC and concluded that TEM images

shown that drug was present in microparticles either molecular dispersion or in the form

of nano dispersions [212].

142

Figure 4.23: TEM images of lyophilized hydrogel microparticles at various magnifications.

143

4.3.10 Stability Studies

Stability studies of three best formulations were conducted for six month period.

Prepared hydrogel microparticles were kept under accelerated conditions of

temperature and humidity 35 ± 5оС and 75% ± 5%, respectively. The samples were

drawn after 1, 2, 3, 4, 5 and at the end of 6 month. Samples were evaluated for different

parameters such as solubility studies, dissolution studies etc. These results indicated

that there was no significant variations occurred in drug contents at the end of 6 months.

It means that our formulations were stable under different accelerated conditions of

temperature and humidity. Samples were also evaluated for physical appearance and

found similar to that of freshly prepared samples.

144

Figure 4.24: Proposed reaction of β-cyclodextrin-g-MMA hydrogel microparticles

145

Figure 4.25: Proposed reaction of β-cyclodextrin-g-AMPS hydrogel microparticles

146

4.4 In vivo Pharmacokinetic studies

4.4.1 Standard curve

Standard curve of Rosuvastatin calcium was constructed by using

known plasma drug concentrations within ranges of 25 ng/mL to 2000 ng/mL and linear

regression was applied to fit straight line. Mean R2 values 0.9996 was also determined

as shown in Figure 4.26.

Figure 4.26: Standard curve of Rosuvastatin calcium in rabbit plasma

4.4.2 HPLC method development

Several organic solvents were tried for the preparation of stock solutions of all

analytes. Methanol was selected due to greater solubility of analytes in it. The

corresponding working solutions of Rosuvastatin calcium were prepared by diluting

their stock solutions with acetonitrile–water (50:50, v/v; pH adjusted to 3.0 with

orthophosphoric acid). Acetonitrile, methanol and methanol–ethanol in different ratios

were tried for protein precipitation but complete protein precipitation was achieved with

y = 0.1935x + 1.677R² = 0.9996

0

50

100

150

200

250

300

350

400

450

0 500 1000 1500 2000 2500

Pea

k a

rea

Concentration (ng/ml)

147

absolute ethanol at least three times the volume of serum. Dichloromethane, ethyl

acetate, chloroform, n-hexane and diethyl ether were evaluated either alone or in

different ratios for the extraction of all analytes from serum. Recovery of rosuvastatin

was better when extracted with diethyl ether. So best results in terms of recoveries were

obtained with diethyl ether (extraction solvent). The residue was reconstituted in 500

µl of mobile phase and 20 µl sample was injected onto HPLC system.

Feasibility of different solvent systems such as acetonitrile–water and

methanol–water mixtures in different compositions, pumped at different flow rates (in

the range of 0.8–1.6ml/min) having variable pH range (2.0–4.0) and at different column

oven temperatures (in the range of 25–35 °C) were evaluated. Best results were obtained

using acetonitrile –water in the ratio of 50:50, v/v (pH adjusted to 3.0 with

orthophosphoric acid) at a flow rate of 1.0 ml/min. While optimizing the composition

of mobile phase, pH was fixed to 3.0 and while assessing the effect of pH of mobile

phase, the mobile phase composition was acetonitrile–water (50:50, v/v).

The retention of all analytes varied considerably by changing the pH of mobile

phase in the range of 2.0–6.0. Since rosuvastatin (pka = 4.6) is acidic compound so its

retention on the column is likely to be pH dependent. When pH of mobile phase was

decreased from 4.0 to 3.0, the retention times of analytes decreased unexpectedly and

with further decrease in pH to 2.0, the retention times increased once again. This

behavior may be due to a change in the solubility of analytes in the mobile phase or

may be due to change in binding of analytes to the stationary phase. Therefore, pH 3.0

was chosen as optimum pH because of the reasonable retention times, resolution and

separation of rosuvastatin. Various detection wavelengths in the UV range of 220–280

nm were tried for monitoring of all analytes. Keeping in view the theoretical values of

molar absorptivity co-efficient of rosuvastatin, the wavelength 243 nm was selected as

the optimum wavelength for determination of rosuvastatin. Typical chromatograms of

HPLC in mobile phase are shown in Figure 4.27.

148

Figure 4.27: HPLC chromatogram of Rosuvastatin calcium in mobile phase

Five different types of columns were used to find better separation of drug.

While selecting best column for analysis all other parameters were kept constant.

Hypersil BDS C 8 (250 X 4.6 mm) column was finally selected for HPLC analysis of

rosuvastatin in this method. Typical chromatograms of rosuvastatin calcium determined

in rabbit’s blank plasma and spiked plasma and samples taken after administration of

20 mg of rosuvastatin calcium are shown in Figure 4.28, 4.22 and 4.30, respectively.

Retention time was found 5.1 minute by using Hypersil BDS C 8 (250 X 4.6 mm). The

response found was linear upto 2-500 ng/ml. correlation co-efficient was 0.998. The

linearity equations and standard errors for the calibration curves of standard mixtures

and spiked serum samples of rosuvastatin were measured. Average percent recoveries

for rosuvastatin was greater than 98.0% while %RSD value for rosuvastatin was less

than 1% indicating accuracy of the reported method. Precision data and intermediate

precision (intra-day and inter-days reproducibility) are summarized in Table 4.13. The

%RSD values for both intra-day and inter-days were less than 2.0%, which indicates

that the proposed method was precise.

149

Figure 4.28: HPLC chromatogram of blanked Plasma

Figure 4.29: HPLC chromatogram of spiked Plasma

150

Figure 4.30: HPLC chromatogram of rabbit’s samples taken after administration of

drug

Table 4.13: Intra-day and inter-day precision data (n = 3)

Known spiked

concentration

Concentration found

Intra-day

(mean ± SD)

%RSD Inter-day

(mean ± SD)

%RSD

32 30.84 ± 0.20 0.56 30.80 ± 0.30 0.86

64 61.68 ± 1.0 0.24 60.98 ± 1.25 1.10

250 243.56 ± 1.10 0.56 244.12 ± 0.50 0.84

500 493.36 ± 0.25 0.34 492.86 ± 0.25 0.54

151

The LLOD for rosuvastatin standard solutions was found to be 12.0 ng/ml,

while LLOQ was found to be 32.0 ng/ml, as shown in Table 4.13. Results from the

stability studies of both spiked serum samples and standard solutions indicated that

spiked serum samples were stable for 72 hour when stored at room temperature (25 °C),

refrigerator (2–8 °C) and frozen (−80 °C), while the standard solutions demonstrated

stability for two month at 2–8 °C. Minor deliberate changes in different experimental

parameters such as column oven temperature (±1 °C), flow rate (± 5%) and pH of the

mobile phase (± 0.2 units) did not significantly affect the recoveries, peak area and

retention time indicating that the proposed method was robust. These findings of our

study are in agreement with previously reported method of Vittal et al. (2006) [213]

developed for Rosuvastatin. From findings, they concluded that developed method was

robust and suitable for determination of RST from spiked plasma as well as from

samples to study pharmacokinetic.

Prepared formulations were evaluated for different in vitro parameters such as

FTIR, DSC, TGA, PXRD, SEM, TEM, in vitro drug release etc. and based upon their

results two best formulations were selected for in vivo studies. Formulation F14

(FDT’s) and HS2 (hydrogel microparticles) were selected for pharmacokinetic studies.

Total 18 rabbits were used for pharmacokinetic studies that were divided into three

groups having 6 rabbits in each (n=6). Group A, B and C had given hydrogel

microparticles, FDT’s and RST commercially available conventional tablets,

respectively of same strength (5 mg/kg/day). After washout period of one week

crossover study design was applied i.e. group A had received hydrogel microparticles,

group C had given FDT’s while RST commercially available conventional tablets had

been administered to group B. this design was applied to study the reproducibility of

results within different groups of rabbits.

4.4.3 Pharmacokinetic evaluation of Rosuvastatin calcium

Different pharmacokinetic parameters such as Cmax (ng/mL), tmax (Hrs), t1/2 (Hrs),

AUC (0 –t) (ng/mL.h), MRT (Hrs), Clast (ng/mL), Tlast (Hrs) were calculated. These

pharmacokinetic parameters of Rosuvastatin calcium were analyzed after

administration of FDT’s, hydrogel microparticles and commercially available tablets of

Rosuvastatin calcium. Noncompartmental pharmacokinetic analysis was performed

from plasma levels in rabbits using software package kinetica v 4.4. The peak plasma

concentration (Cmax) and time required to reach peak plasma concentration (Tmax) were

152

calculated from the visual inspection of plasma concentration-time curves. The area

under the plasma concentration-time curve (AUC0-t) was calculated by using

trapezoidal rule.

Results of mean plasma concentration after administration of FDT’s, hydrogel

microparticles and commercially available tablets of Rosuvastatin calcium had been

calculated. Mean values of different pharmacokinetic parameters of Rosuvastatin

calcium after oral administration of FDT’s, hydrogel microparticles and commercially

available tablets of Rosuvastatin calcium are shown in Table 4.17, 4.18 and 4.19,

respectively. Pharmacokinetic data was statistically analyzed using one way ANOVA

and results are listed in Table 4.26.

Values of Cmax were 92, 90 and 41 ng/ml for FDT’s, hydrogel microparticles

and commercially available tablets of rosuvastatin calcium, respectively as shown in

Tables 4.17, 4.18 and 4.19. These results had shown that there was rapid release of drug

from prepared formulations that ultimately improved bioavailability of drug in rabbit’s

plasma. Similarly AUC0-24 was also higher for prepared formulations. This means that

more drug was released from prepared formulations due to complex formation with β-

cyclodextrin and drug become more soluble in gastric media. Results of Cmax and AUC0-

24 were significant (p < 0.05). FDT’s and hydrogel microparticles had value of Tmax two

hour while RST tablets had 4 hour which means that prepared formulations released

drug more rapidly as compared to RST tablets. This rapid release of drug was due to

more solubility of prepared formulations than RST commercially available tablets. Our

results are in agreement with Shilpi et al. (2010). They prepared different formulations

for solubility enhancement using hydrophilic polymers and calculated various

pharmacokinetic parameters. They concluded that both Tmax and t½ reduced in prepared

formulations as compared to pure drug while both Cmax and AUC0-24 increased [212].

The elimination half-life (t½) of FDT’s and hydrogel microparticles was 3.96

and 4.20 (p < 0.05) hour, respectively while RST commercially available tablets had

half-life 18.80 hour. This confirmed more rapid bioavailability of drug from prepared

formulations as compared to RST commercially available tablets. These results

confirmed that solubility of drug increased in prepared formulations due to

complexation with carrier system that ultimately enhanced its bioavailability.

153

Table 4.14: Mean ± SEM plasma concentration of Rosuvastatin calcium

after administration of FDT’s in rabbits (n=6)

Group A

Time (hr) Concentration (ng/mL)

(Mean ± SEM)

0 0.0000

0.5 24.9730 ± 2.654

1 43.0730 ± 3.521

1.5 68.9230 ± 2.846

2 92.678 ± 4.058

3 77.643 ± 3.986

4 55.645 ± 2.6554

8 27.765 ± 6.726

12 13.728 ± 4.826

Figure 4.31: Plasma profile of Rosuvastatin calcium after administration of

FDT’s formulation

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Con

cen

trati

on

(μ

g/m

l)

Time (hr)

154

Table 4.15: Mean ± SEM plasma concentration of Rosuvastatin calcium after

administration of hydrogel microparticles in rabbits (n=6)

Group B


(Mean ± SEM)

0 0.0000

0.5 25.9540 ± 4.087

1 44.1130 ± 4.265

1.5 64.2680 ± 6.625

2 90.543 ± 5.276

3 79.478 ± 2.654

4 53.678 ± 4.638

8 25.654 ± 3.275

12 14.364 ± 5.162

Figure 4.32: Plasma profile of Rosuvastatin calcium after administration of hydrogel

microparticles formulation

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Con

cen

trati

on

(μ

g/m

l)

Time (hr)

155



(n=6)

Group C


(Mean ± SEM)

0 0.0000

0.5 3.273 ±3.276

1 8.354 ± 4.264

1.5 15.246 ± 2.746

2 22.364 ± 3.729

3 29.3746 ± 5.637

4 41.263 ± 4.164

8 35.653 ± 3.865

12 30.723 ± 4.724


commercially available tablets

0

5

10

15

20

25

30

35

40

45

0 2 4 6 8 10 12 14

Con

cen

trati

on

(μ

g/m

l)

Time (hr)

156

Table 4.17: Mean values of pharmacokinetic parameters of Rosuvastatin calcium

following administration of FDT’s (F14)

Sr. No. Parameters F14 (FDT’s)

1 Cmax (ng/mL) 92.678

2 Tmax ( Hrs) 2.00

3 AUCo-t (ng/mL.h) 493.2395

4 AUCo-inf (ng/mL.h) 571.720

5 AUMC 0-inf 3616.150787

6 MRT (Hrs) 6.3250

7 Clast (ng/mL) 8.658

8 Tlast (Hrs) 4.00

9 t1/2 (Hrs) 3.96

10 Vz (μg/ml) 8.249474054

11 Cl (μg/ml)/h 1.443013425

157


following administration of hydrogel microparticles (HS2)

Sr. No. Parameters HS2 (Hydrogel

microparticles)

1 Cmax (ng/mL) 90.543

2 Tmax ( Hrs) 2.00

3 AUCo-t (ng/mL.h) 480.09

4 AUCo-inf (ng/mL.h) 567.26

5 AUMC 0-inf 3728.439942

6 MRT (Hrs) 6.57


8 Tlast (Hrs) 4.00

9 t1/2 (Hrs) 4.20

10 Vz (μg/ml) 8.82581294

11 Cl (μg/ml)/h 1.454359553

158


following administration of commercially available RST tablets

Sr. No. Parameters Commercially

available RST tablets

1 Cmax (ng/mL) 41.263

2 Tmax ( Hrs) 4.00


4 AUCo-inf (ng/mL.h) 1200.093789

5 AUMC 0-inf 35029.79044

6 MRT (Hrs) 29.18


8 Tlast (Hrs) 12.00

9 t1/2 (Hrs) 18.80

10 Vz (μg/ml) 18.64547677

11 Cl (μg/ml)/h 0.687446271

159

Figure 4.34: Combined plasma profile of Rosuvastatin calcium after administration

of oral drug of FDT’s, hydrogel microparticles and commercially


After washout period all three groups of rabbits were administered alternate

formulations. Again pharmacokinetic parameters calculated and compared with

eachother before and after washout period. This confirmed that different group of

rabbits had shown same pharmacokinetic when alternate formulations were

administered. Results of various pharmacokinetic parameters are shown in Table 4.23,

4.24 and 4.25. In case of FDT’s Cmax was 92 ng/ml and 91 ng/ml before and after

washout period, respectively. Hydrogel microparticles had also shown same Cmax in

both cases i.e., 90.5 and 91.5 ng/ml before and after washout period, respectively. Tmax

was found 4 hours in case of commercially available tablets while it was reduced in our

prepared formulations upto 2 hours. This reduction in Tmax of rosuvastatin confirmed

that drug become more soluble in prepared formulations as compared to commercially

available tablets of RST.

-20

0

20

40

60

80

100

120

-5 0 5 10 15 20 25 30

Con

cen

trati

on

(n

g/m

l)

Time (hr)

FDT's

Hydrogelmicroparticles

RST tablet

160


administration of FDT’s in rabbits (n=6)

Group C


(Mean ± SEM)

0 0

0.5 25.34 ± 4.275

1 42.54 ± 6.184

1.5 67.78 ± 3.853

2 91.436 ± 5.827

3 74.635 ± 4.265

4 54.374 ± 3.825

8 28.364 ± 3.527

12 14.264 ± 5.365


FDT’s formulation

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Co

nce

ntr

ati

on

(μ

g/m

l)

Time (hr)

161

Table 4.21: Mean ± SEM plasma concentration of Rosuvastatin

calcium after administration of hydrogel microparticles in

rabbits (n=6)


hydrogel microparticles

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

Con

cen

trati

on

(μ

g/m

l)

Time (hr)

Group A


(Mean ± SEM)

0 0

0.5 26.28 ± 6.173

1 43.89 ± 5.287

1.5 65.73 ± 6.376

2 91.543 ± 4.873

3 77.735 ± 4.820

4 51.263 ± 5.365

8 26.364 ± 3.628

12 15.263 ± 2.365

162



(n=6)

Group B


(Mean ± SEM)

0 0

0.5 2.98 ± 2.936

1 7.73 ± 3.283

1.5 14.724 ± 3.625

2 21.354 ± 4.753

3 28.765 ± 3.265

4 40.264 ± 5.243

8 34.263 ± 4.276

12 29.936 ± 6.365

Figure 4.37: Plasma profile of Rosuvastatin calcium after administration

of commercially available tablets

0

5

10

15

20

25

30

35

40

45

0 2 4 6 8 10 12 14

Con

cen

trati

on

(μ

g/m

l)

Time (hr)

163

Table 4.23: Mean values of pharmacokinetic parameters of Rosuvastatin

calcium following administration of FDT’s (F14)

Sr No. Parameters F14 (FDT’s)

1 Cmax (ng/mL) 91.436

2 Tmax ( Hrs) 2.00

3 AUCo-t (ng/mL.h) 488.961.45

4 AUCo-inf (ng/mL.h) 574.237118

5 AUMC 0-inf 3766.352477

6 MRT (Hrs) 6.55


8 Tlast (Hrs) 4.00

9 t1/2 (Hrs) 4.14

10 Vz (μg/ml) 8.589122143

11 Cl (μg/ml)/h 1.436688737

164


Calcium following administration of hydrogel microparticles

(HS2)

Sr No. Parameters HS2 (Hydrogel

microparticles)

1 Cmax (ng/mL) 91.543

2 Tmax ( Hrs) 2.00


4 AUCo-inf (ng/mL.h) 579.2660719

5 AUMC 0-inf 4045.830785

6 MRT (Hrs) 6.58


8 Tlast (Hrs) 4.00

9 t1/2 (Hrs) 4.57

10 Vz (μg/ml) 9.404353166

11 Cl (μg/ml)/h 1.424215987

165


calcium following administration of commercially available

RST tablets

Sr No. Parameters Commercially


1 Cmax (ng/mL) 40.264

2 Tmax ( Hrs) 4.00


4 AUCo-inf (ng/mL.h) 1163.081608

5 AUMC 0-inf 33856.16461

6 MRT (Hrs) 29.1090


8 Tlast (Hrs) 12

9 t1/2 (Hrs) 18.70

10 Vz (μg/ml) 19.14526619

11 Cl (μg/ml)/h 0.70932254

166

Figure 4.38: Combined plasma profile of Rosuvastatin calcium after



Table 4.26: ANOVA table for pharmacokinetic parameters of prepared

formulations (HS2 & F14) and RST tablets

df=Degree of freedom

-20

0

20

40

60

80

100

120

-4 -2 0 2 4 6 8 10 12 14 16

Con

cen

trati

on

(n

g/m

l)

Time (hr)

FDT's

Hydrogel microparticles

RST tablet

source df

Sum of

squares Mean square F P-value

Cmax 2 8368 4184 32.264

0.0010

P < 0.05

AUC0-t 2 4709238 2354619 162.539

0.0020

P < 0.05

MRT 2 503 201.50 371.769

-0.0030

P < 0.05

Tmax 2 87.800 43.900 447.062

-0.0010

P < 0.05

167

4.5 Acute oral toxicity study of prepared formulations

During whole period of toxicity studies general conditions of all groups of

rabbits were observed. Body weights, food and water intake, certain common signs of

illness, dermal and ocular irritation and mortality of both control and treatment groups

were noted accordingly. Findings of these parameters are listed in Table 4.27.

Table 4.27: Clinical observations of acute oral toxicity test for different formulations

Observations Group I

(Control) Group II

(FDT’s {F14})

10 g/kg/b.w

Group III

(hydrogel

microparticles

{HS2})

10g/kg/b.w

Signs of illness Nil Nil Nil

Body weight (kg)

Pretreatment

Day1

Day7

Day14

2.08 ± 1.5

2.06 ± 1.4

2.05 ± 0.5

2.06 ± 1.0

1.98 ± 1.0

1.96 ± 1.1

2.03 ± 1.5

2.04 ± 1.25

2.01 ± 0.5

2.00 ± 1.1

2.03 ± 1.5

2.04 ± 1.3

Water intake(mL)

Pretreatment

Day1

Day7

Day14

200 ± 1.1

195 ± 1.3

203 ± 1.5

205 ± 1.0

193 ± 1.0

199 ± 1.5

200 ± 0.5

201 ± 1.5

202 ± 0.5

197 ± 0.8

193 ± 1.0

201 ± 1.1

Food Intake (g)

Pretreatment

Day1

Day7

Day14

70 ± 1.5

73 ± 1.8

69 ± 1.0

75 ± 0.5

67 ± 1.0

63 ± 0.8

68 ± 0.3

70 ± 0.20

73 ± 0.5

70 ± 1.8

79 ± 1.1

74 ± 1.5

Dermal toxicity

Dermal irritation

Nil

Nil

Nil

Ocular toxicity

Simple Irritation or

corrosion

Nil

Nil

Nil

Mortality Nil Nil Nil

All values are expressed as mean ± SEM

4.5.1 Biochemical blood analysis

Biochemical analysis was performed to investigate whether prepared

formulations resulted in any defect in blood system. Blood samples were collected from

rabbits for biochemical blood analysis which was determined by a hematology

Analyzer. We had calculated various variables through blood analysis. These

parameters include red blood cell count (RBC), white blood cell count (WBC),

168

hemoglobin (HB), platelet count, mean corpuscular hemoglobin (MCH), mean

corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)

summarized in Table 4.28 and liver function test, lipid profile and kidney function test

has been shown in Table 4.29.

Table 4.28: Biochemical blood analysis of rabbits

All values are expressed as mean ± SEM

Haematology

Group I

(Control)

Group II

(treated FDT’s

{F14})

1-10 g/kg

Group III

(Treated

hydrogel

microparticles

{HS2})

1-10g/kg

Hb g/dL 14.3 ± 1.1 14.2 ± 0.5 14.1 ± 1.05

pH 7.3 ± 0.85 7.3 ± 0.25 7.2 ± 1.10

WBCs x 109 / l 6.9 ± 0.25 7.1 ± 0.25 7.2 ± 0.05

RBCs x 106 / mm3 6.1 ± 1.0 6.3 ± 0.5 5.9 ± 1.00

Platelets x 109/l 4.1 ± 1.25 4.05 ± 1.5 4.10 ± 1.50

Monocytes% 3 ± 1.0 3.1 ± 1.00 3.2 ± 0.50

Neutrophils% 50 ± 0.85 52 ± 1.10 53 ± 1.00

Lymphocytes% 70 ± 0.25 67 ± 0.75 69 ± 0.50

MCV% 62 ± 1.25 63 ± 0.50 63.5 ± 1.30

MCH pg/cell 22 ± 0.20 21.2 ± 1.50 21.80 ± 1.50

MCHC % 31 ± 1.10 30.8 ± 1.10 31.3 ± 0.50

169

Table 4.29: Liver, kidney and lipid profile of rabbits after administration of

prepared formulations and control

4.5.2 Histopathological study

Acute toxicity was also evaluated through histopathological examination of

major organs. Weight of rabbits and vital organs like stomach, heart, liver, spleen,

kidney and lung of rabbits were measured before and after administration of control

and prepared formulations. Results of various parameters of different organs are

presented in Table 4.30.

Table 4.30: Effect of oral administration of prepared formulations on the organ weight

(gm) of rabbits

Treatment

groups

Heart Liver Lung Kidney Stomach Spleen

Control 4.10 ± 0.2 76.1 ± 0.5 10.74 ± 0.3 12.3 ± 0.5 12.90 ± 0.25 1.1 ± 0.3

Group II 4.25 ± 0.5 69.8 ± 0.7 9.80 ± 0.25 11.9 ± 0.7 11.95 ± 0.16 1.15 ± 0.25

Group II 4..30 ± 0.1 67.1 ± 0.2 10.50 ± 0.5 11.2 ± 0.3 12.60 ± 0.20 1.1 ± 0.10

Biochemical

analysis

Group I

(Control)

Group II

(FDT’s{F14})

1-10 g/kg

Group III

(hydrogel

microparticles

{HS2}) 1-10g/kg

ALT(IU/l) 170 184 166

AST (IU/l) 72 81 84

Creatinine

(mg/dl)

1.30 1.39 1.37

Urea(mmol/l) 14.5 16 15.6

Uric

acid(mg/dl)

3.3 2.9 3.4

Cholesterol(m

g/dl)

76 81 73

Triglyceride(m

mol/l)

1.55 1.59 1.4

170

4.5.3 Acute oral toxicity study of prepared formulations

Mostly unreacted ingredients such as monomers, polymers, crosslinker

initiators etc. left in hydrogel microparticles that causes various toxicities in different

vital organs of body. So, it is necessary to determine toxicity levels of these prepared

formulation by measuring different hematological, biochemical and histopathological

parameters. Acute oral toxicity study of prepared formulations (hydrogel microparticles

(HS2) and FDT’s) were implemented consistent with the “Organization of Economic

Co-operation and Development (OECD) guideline for chemicals toxicity study [214].

The main goal of the current study was to demonstrate broad spectrum safety of

prepared formulation of rosuvastatin calcium to enhance solubility when ingested

orally. Acute oral toxicity studies did not reveal any significant changes for all

examined tissues. In acute dermal toxicity study, there were no signs of dermal

irritation, adverse pharmacological effects or abnormal behavior, biochemical and

histopathological toxicity. Results of toxicity studies confirmed that no toxic effect was

produced within two weeks of study. Absence of toxicologically related variations in

body weight, feed consumption, hematology, clinical chemistry and organ weight were

noted up to 1000 mg/kg/day of Rosuvastatin. Calculation of acute toxicity gives idea

about the toxic dose of drugs. It can also be used to calculate therapeutic index of drugs

(LD50/E D50). In our acute toxicity studies no mortality case of rabbits were observed

till the 14th day of study even at highest doses. Absence of toxicologically relevant

changes in body weight, percent change in body weight, feed consumption,

ophthalmoscopic examination, neurological-functional examination, hematology,

clinical chemistry and organ weight were noted up to 1000 mg/kg/day of Rosuvastatin.

Therefore, under the conditions of this study, the no-observable-adverse-effect-level

(NOAEL) for rabbits was considered to be 1000 mg/kg/day.

4.5.4 Clinical observations

All three groups of rabbits had produced normal results. All vital organs such

as kidney, liver, heart, lung, stomach and spleen were not harmfully affected throughout

the treatment. Results shown in Table 4.27 had indicated that there was no effect of oral

administration of hydrogel microparticles and FDT’s on body weight, nutrients

consumption, and other poisoning related complications. No skin irritation and other

behavioral were observed in all rabbits. Nutrient intake is also important parameter to

judge toxicity levels. In current study not much variations were occurred in food and

171

water intake of rabbits. These findings also confirmed that our formulations were free

from any clinical toxicity. No symptoms of illness (vomiting, eye secretion, running

nose, salivation) were observed after hydrogel microparticles and FDT’s

administration. According to globally harmonized system (GHS), LD50 value of testing

chemical is higher than 2000 mg/kg dose then it will be classified in “Category 5” and

toxicity score will be “zero.” Therefore, prepared formulations can be characterized in

Category 5 and toxicity score is zero.

4.5.5 Biochemical blood analysis

Blood is the most important system of body that is to be studied in case of

toxicology [215]. Different parameters of blood were calculated in control as well as in

treated rabbits and found that these were present in normal range. WBCs, RBCs and

platelets concentration was present in acceptable limits as shown in Table 4.28. Other

parameters like urea, uric acid, cholesterol, triglycerides and creatinine’s calculated

values were also present within normal range as shown in Table 4.29. From results of

biochemical analysis it was concluded that all three groups of rabbits shown similar

values of different parameters. These values were present within normal limits. Our

results are in agreement with Alia et al. (2014) where they conducted toxicology studies

of Arabinoxylan on mice and rabbits and concluded that no changes were observed in

normal biochemical values of different parameters after administration 10 g/kg of dose

[216].

4.5.6 Histopathological Study

Microscopic examination of samples was obtained from control and prepared

formulations treated groups of rabbits revealed that no clear histopathological lesions

found in vital organs (heart, liver, kidney, lungs and small intestine). Figure 4.39

described from the heart showed normal myocardium. No significant pathology was

present with in myocardial tissues. Figure 4.40 revealed hepatic parenchyma with

normal preserved lobular architecture. Figure 4.41 described that there was no

significant pathology present in lungs. Figure 4.42 had shown normal kidney structure

both in control and treated groups. Figure 4.43 illustrated that no significant changes

were observed in small intestine.

Thus, maximal tolerance dose of prepared formulations was assessed to be

higher than 10 g/kg b.w. in rabbits. It was concluded that the tested formulations were

172

nontoxic, safe and biocompatible following oral administration and it might be

promising way to enhance solubility of drug.

The main goal of the current study was to demonstrate broad spectrum safety of

prepared formulation of rosuvastatin calcium to enhance solubility when ingested

orally. Acute oral toxicity studies did not reveal any significant changes for all

examined tissues. In acute dermal toxicity study, there were no signs of dermal

irritation, adverse pharmacological effects or abnormal behavior, biochemical and

histopathological toxicity. Results of toxicity studies confirmed that no toxic effect was

produced within two weeks of study. Absence of toxicologically related variations in

body weight, feed consumption, hematology, clinical chemistry and organ weight were

noted up to 1000 mg/kg/day of Rosuvastatin. Calculation of acute toxicity gives idea

about the toxic dose of drugs. It can also be used to calculate therapeutic index of drugs

(LD50/E D50). In our acute toxicity studies no mortality case of rabbits were observed

till the 14th day of study even at highest doses. Absence of toxicologically relevant

changes in body weight, percent change in body weight, feed consumption,

ophthalmoscopic examination, neurological-functional examination, hematology,

clinical chemistry and organ weight were noted up to 1000 mg/kg/day of Rosuvastatin.

Therefore, under the conditions of this study, the no-observable-adverse-effect-level

(NOAEL) for rabbits was considered to be 1000 mg/kg/day.

Figure 4.39: Histological examination of heart of rabbit of control group I (A), after

FDT’s treatment group II (B), and hydrogel microparticles group III (C)

173

Figure 4.40: Histological examination of liver of rabbit of control group I (A), after


Figure 4.41: Histological examination of lung of rabbit of control group I (A), after


174

Figure 4.42: Histological examination of kidney of rabbit of control group I (A), after


Figure 4.43: Histological examination of small intestine of rabbit of control group I

(A), after FDT’s treatment group II (B), and hydrogel microparticles

group III (C)

175

5. CONCLUSION

In present work three different types of formulations were prepared to enhance

solubility of Rosuvastatin calcium. These three groups include FDT’s, microparticles

prepared by inclusion complexes and solid dispersions and hydrogel microparticles by

free radical polymerization. From results of these prepared formulations it was

concluded that solubility of Rosuvastatin was enhanced to various degrees by using

these techniques.

FDT’s were prepared by using different ratios of superdisintegrants and β-

cyclodextrin had shown that solubility was enhanced upto 11 folds in phosphate buffer

of 6.8 pH. This enhancement of solubility was present in formulation F14 that had 20

mg (10%) of β-cyclodextrin and kyron T134 16 mg (8%). This study also revealed that

kyron T134 was better choice to impart rapid release of drug with enhanced solubility

than sodium starch glycolate. From findings of these solubility enhancement results by

preparing FDT’s, it was concluded that FDT’s are very useful in solubility enhancement

of poorly water soluble drugs.

Inclusion complexes and solid dispersions had also enhanced solubility of

Rosuvastatin calcium several folds. By using these two techniques eight formulations

were prepared and evaluated for various solubility enhancement parameters. From these

results it was concluded that both methods enhanced solubility but solid dispersion

enhanced greater solubility of Rosuvastatin calcium. Similar concentrations of β-

cyclodextrin were used in both techniques and found that there was more solubility

enhancement from solid dispersions as compared to inclusion complexes. Optimized

concentration of β-cyclodextrin was also determined that enhanced maximum

solubility. It was found that drug- β-cyclodextrin (1:3) had enhanced maximum

solubility. Further increase in concentration had no greater impact on solubility. Thus

from such findings we concluded that both techniques are useful tool to enhance

solubility of Rosuvastatin calcium but solid dispersions are better choice than inclusion

complexes and β-cyclodextrin produces best results when used in 3:1 with drug.

Hydrogel microparticles also enhanced solubility of Rosuvastatin calcium upto

9.5 folds in phosphate buffer of pH 6.8. Overall findings of hydrogel microparticles had

shown that MAA containing microparticles enhanced solubility but lesser than AMPS

containing microparticles. Thus from such findings it was concluded that all the

176

techniques used for solubility enhancement in this study had successfully enhanced

solubility.

Pharmacokinetic studies were conducted to study enhancement in solubility of

Rosuvastatin calcium in vivo. These studies had shown that there was rapid response of

drug release in rabbits as compared to RST commercially available tablets. From

pharmacokinetic studies, it was also concluded that solubility was enhanced because

more AUC0-t was observed. Tmax was also reduced in case of prepared formulations

as compared to RST commercially available tablets. Toxicology studies, it was also

observed that no changes occurred in hematological and biochemical values. No

histopathological changes were observed in vital organs such as liver, kidney, heart,

lungs, stomach and spleen.

Thus, from all of these findings it was concluded that FDT’s, microparticles

prepared by inclusion complexes and solid dispersions and hydrogel microparticles

prepared by free radical polymerization provided an efficient and effective tool for

solubility enhancement of BCS class II drugs. Solubility of Rosuvastatin was

successfully enhanced several folds in this study both in vitro and in vivo without any

toxicity as compared to RST commercially available tablets.

177

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Documents

Doctor of Philosophy - Pakistan Research Repository