SOLUBILITY ENHANCEMENT of MODEL COMPOUNDS Lavanya Pitani University of the Pacific, [email protected]

SOLUBILITY ENHANCEMENT of MODEL COMPOUNDS Lavanya Pitani University of the Pacific, Lavanyapitani1@Gmail.Com

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2017 SOLUBILITY ENHANCEMENT OF MODEL COMPOUNDS Lavanya Pitani University of the Pacific, [email protected]

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SOLUBILITY ENHANCEMENT OF MODEL COMPOUNDS

Lavanya Pitani

A Thesis Submitted to the Office of Research and Graduate School In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE

Thomas J. Long School of Pharmacy and Health Sciences Pharmaceutical and Chemical Sciences

University of the Pacific Stockton, California

2017 3

SOLUBILITY ENHANCEMENT OF MODEL COMPOUNDS

Lavanya Pitani

APPROVED BY:

Dissertation Advisor: Bhaskara Jasti, Ph.D.

Committee Member: Xiaoling Li, Ph.D.

Committee Member: Rajendra Tandale, Ph.D

Department Chair: William K. Chan, Ph.D.

Dean of Graduate School: Thomas H Naehr, Ph.D.

SOLUBILITY ENHANCEMENT OF MODEL COMPOUNDS

DEDICATION

This thesis is dedicated to my advisor Dr. Bhaskara Jasti, my father Purushotham Naidu

Pitani, my mother Mahalakshmi Pitani and my fiancé Uday Kiran Pendyala.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude and indebtedness to Dr. Bhaskara Jasti, for his guidance, counsel and support during the progress of this project. You are an inspiration for my life. I thank you from the bottom of my heart for all your patience, continuous encouragement and showing me the right directions in both my career and personal front.

Special appreciation is expressed to Dr. Xiaoling Li for his scientific discussion with valuable suggestions in meetings. His counseling inspired me to be a better student. I would also thank Dr. Rajendra Tandale for his guidance and support with the utmost patience in my project. I thank Dr. Xiaoling Li and Rajendra Tandale for their encouragement and assistance as dissertation committee members.

I extend my appreciation to Formurex for bestowed support and help in my research.

I thank Dr. Shiladitya Bhattacharya for his immense support in the study and Dr. Xin Guo for his invaluable suggestions in lab meetings. Special thanks to my Labmate Zahir for being very generous and helping me troubleshoot problems that I faced while working in the Lab.

I would like to express gratitude to Vijayasri Nannapaneni, Karthik Kondepudi,

Naresh Vutukuru and Arindom Pal for their support and fellowship. I would also like to thank Lynda Davis and Kathy Kassab for their continuous support from when I moved to the USA. Acknowledgements extended to Vincent, Sridhar, Vinay, Sachin, Rohit and

Shiva Sagar for sharing their love and friendship over all these years and for being my 7 family away from home. I thank my younger brother Sathyavardhan Naidu Pitani for all the memories we made since childhood.

My sincere gratefulness to my fiancé Uday Kiran Pendyala, for his endurance, concern understanding, sacrifice and immense support in both my good and difficult times without which I would not have achieved my goals. Finally, I am grateful to my father

Purushotham Naidu Pitani and mother Mahalakshmi Pitani for being a continuous source of unconditional love and sacrifice. Their morale support kept me motivated and without their support, I would not be able to pursue anything in my life. To them, I dedicate this dissertation. 8

Solubility Enhancement of Model Compounds

Abstract

by Lavanya Pitani

University of the Pacific 2017

Solubility is the amount of solute in the solvent system at phase equilibrium with certain temperature and pressure. Many of the new chemical entities are lipophilic molecules that require techniques to enhance solubility. Solubility enhancement can be achieved by either physical and/or chemical modification of the drug. Various techniques are available for solubility enhancement of poorly soluble drugs include particle size reduction, salt formation, solid dispersions, use of surfactants, prodrug, crystal modification, etc.

In this study, the three model drugs belong to BCS class II and IV having low solubility with a certain range of physicochemical properties were studies in solubility enhancement using fusion method, co-precipitation, nano-milling and spray drying techniques. The two different polymers employed for solubility enhancement are PEG

8000 and PVP 40,000. Solubility was determined by Shake Flask method at the temperature of 37±0.1 °C. The objective is to investigate the enhancement of solubility of the three model drugs namely Glipizide, Carvedilol and Furosemide in 1:1, 1:5 and 1:10 drug-polymer ratios and are characterized by Differential Scanning Calorimetry (DSC). 9

The Solubility of Glipizide was enhanced from 11.18 ± 1.78 µg/ml to 35.73 ± 0.04

µg/ml by 219 % increase with nano-milling technique at 1:5 ratio with PEG 8000 as carrier whereas with PVP 40000 as carrier, 286 % increase in solubility to 43.26 ± 7.87 µg/ml was observed at 1:1 ratio by fusion method. The solubility of Carvedilol was enhanced from

5.04 ± 0.55 µg/ml to 17.51 ± 0.94 µg/ml by 246 % at 1:5 ratio by fusion method with

PEG8000 as carrier and 2924 % enhancement in solubility to 152.70 ± 9.09 µg/ml at 1:10 ratio by nano-milling with PVP40000 as the carrier. Furosemide showed an increase in solubility from 55.94 ± 2.48 µg/ml to 164.11 ± 9.18 µg/ml by 193 % at 1:10 ratio by nano- milling technique with PEG8000 as carrier whereas with PVP40000 as carrier, 444 % increase was observed at 1:1 ratio by nano-milling technique with solubility of 304.52 ±

23.11 µg/ml. The data showed that the decrease in percentage crystallinity and enthalpy of fusion of the model drugs upon implementing solubility enhancement techniques with the effect of particle size and the carrier used resulted in the increase of aqueous solubility of the model drugs.

TABLE OF CONTENTS

LIST OF TABLES ………………………………………………………………………12

LIST OF FIGURES …………………………………………………………………… 13

CHAPTER

1. Introduction …………………………………..……………………………….... 16

1.1 Solubility and its Significance in Drug Delivery …………….……………. 16

Poorly soluble drugs …………………………………..…………….16

Biopharmaceutical Classification System (BCS)….……………...... 17

1.2 Factors Influencing Solubility ………………………………...…………….. 19

Melting point ……………………………………..……………….... 19

Partition coefficient ……………………………..………………….. 20

Particle size ……………………………………………...………….. 20

Crystallinity ………………………………………..……………….. 20

1.3 Solubility Enhancement Strategies ..…………………………………….….. 21

Chemical methods …………………………..……………………… 21

Physical methods …………………………..……………………...... 22

1.4 Solid State Transitions…..………………………………………………….. 29

1.5 Estimation of Aqueous Solubility………………………………………...…..30

1.6 Carriers Used in Solubility Studies…………………………………………...31

2. Research Aims………………………………………………………………..… 33

3. Materials and Methods…………………………………………………………..35 11

3.1 Materials……………………………...... ……...…………………..35

3.2 Methods……………………………………….……………………..……...36

4. Characterization of Compositions……………….……………………………...38

4.1 Differential Scanning Calorimetry………………….……………………... 38

4.2 Results ……………..……………………………………….…………….. 38

5. Aqueous Solubility of Model Drugs…………..…………..……………………..53

5.1 HPLC…………………………………………………………...……………53

5.2 Statistical Analysis……………………………………………….…………. 53

Results…………………………...……………………….……………... 54

6. Discussion ………………………………………………………….…………….58

7. Conclusion ……………………………………………………….………………78

REFERENCES ...………………………………………………… ….……………….. 80

LIST OF TABLES

Table Page

1. USP Solubility Chart ……………………………………………………….…17

2. Biopharmaceutical Classification System ………………………………….....18

3. Physicochemical Properties of Model Drugs ……………………………….... 35

4. Parameters for Spray Drying of Model Drugs ……………………………...... 37

5. Chromatographic Conditions for Analysis of Model Drugs …………………..54

6. Comparison of Solubility of Model Drugs ……………….…………………...55

7. Percentage Enhancement of Aqueous Solubility Compared to the Drug…...…76

LIST OF FIGURES

Figure Page 1. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:1 Ratio …………………………….…..……..40

2. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:5 Ratio……………………………..…..……. 40

3. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:10 Ratio.…………………………..….………41

4. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co-precipitation, Spray drying and Nano-milling Methods at 1:1 Ratio …..…………..…………42

5. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:5 Ratio ………………….………...…………..43

6. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:10 Ratio……………………….…...…….…....43

7. Thermograms of Carvedilol-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:1 Ratio.………………….………....….………44

8. Thermograms of Carvedilol-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:5 Ratio.………………………….…………….45

9. Thermograms of Carvedilol-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:10 Ratio……………………..………………...45

10. Thermograms of Carvedilol-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:1 Ratio ..……………….……….46

11. Thermograms of Carvedilol-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio ..……………….…….....47

12. Thermograms of Carvedilol-PVP40000 Composites from Fusion, Co- precipitation, Spray drying and Nano-milling Methods at 1:10 Ratio …..….... 47

13. Thermograms of Furosemide-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:1 Ratio……………………….....48

14. Thermograms of Furosemide-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratios……...………….…….....49

15. Thermograms of Furosemide-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio ...……………………...49

16. Thermograms of Furosemide-PVP40000 Composites from fusion, co- precipitation, spray drying and nano-milling Methods at 1:1 Ratio .…..……...50

17. Thermograms of Furosemide-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio ..………...………...…...51

18. Thermograms of Furosemide-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio ..…………….………..51

19. Glipizide Solubility with PEG8000 as Carrier in 1:1 Ratio ……...…...…...... 54

20. Glipizide Solubility with PVP40000 as Carrier in 1:5 Ratio………………...... 60

21. Glipizide Solubility with PEG8000 as Carrier in 1:10 Ratio …...…………...... 60

22. Glipizide Solubility with PVP40000 as Carrier in 1:1 Ratio …..……….…...... 62

23. Glipizide Solubility with PVP40000 as Carrier in 1:5 Ratio ………...…...... 62

24. Glipizide Solubility with PVP40000 as Carrier in 1:10 Ratio ………...... 63

25. Carvedilol Solubility with PEG8000 as Carrier in 1:1 Ratio ……...... 64

26. Carvedilol Solubility with PEG8000 as Carrier in 1:5 Ratio ……...... 65

27. Carvedilol Solubility with PEG8000 as Carrier in 1:10 Ratio …….……...... 65

28. Carvedilol Solubility with PVP40000 as Carrier in 1:1 Ratio …….……...... 67

29. Carvedilol Solubility with PVP40000 as Carrier in 1:5 Ratio ……..……….....67

30. Carvedilol Solubility with PVP40000 as Carrier in 1:10 Ratio …….…….…...68

31. Furosemide Solubility with PEG8000 as Carrier in 1:1 Ratio ……….…...... 70

32. Furosemide Solubility with PEG8000 as Carrier in 1:5 Ratio ………..…...... 70 15

33. Furosemide Solubility with PEG8000 as Carrier in 1:10 Ratio……….……...... 71

34. Furosemide Solubility with PVP40000 as Carrier in 1:1 Ratio………………...73

35. Furosemide Solubility with PVP40000 as Carrier in 1:5 Ratio………………...73

36. Furosemide Solubility with PVP40000 as Carrier in 1:10 Ratio ...…………….74

Chapter 1: Introduction

Solubility is defined as the amount of drug solute in a given volume of the solvent system at a certain temperature, pressure and pH. [1] According to IUPAC, solubility is defined as the analytical composition of a saturated solution, consisting of a designated solute in a designated solvent system. A saturated solution is a solution in which the dissolved solute is in an equilibrium with solute (solid phase), at an unambiguous temperature. [1]

Solubility is one of the critical physicochemical properties of the drug that influences the rate and extent of absorption. It is important for a drug to be in the form of an aqueous solution at the site of absorption for it to be absorbed by the gastrointestinal (GI) tract. [1]

Solubility conduct of the drugs is one of the most challenging characteristics in formulation development.

1.1 Solubility and its Significance in Drug Development

Poorly soluble drugs. A majority of active pharmaceutical ingredients that are being developed (drug) are lipophilic with limited aqueous solubility leading to problems in preclinical pharmacokinetic and toxicological investigations. Due to rapid advancement in combinatorial chemistry, high-throughput screening this increase of poorly aqueous soluble drugs have been observed [2].

Dose: Solubility ratio, a parameter that is used to identify poorly soluble drugs, is defined as the volume of gastrointestinal fluids required to dissolve the administered dose.

If this volume exceeds available gastrointestinal fluids, then the drug is likely to have solubility issues. [3] The USP has provided terms to describe the solubility parameters, 17 which are quantitatively based on the number of parts of solvent and solute as shown in

Table 1.[3]

Table 1: USP Solubility Chart

Solubility at ambient temperature Parts of solvent for 1 part of solute

Very soluble Less than 1

Freely soluble From 1-10

Soluble From 10-30

Sparingly soluble From 30-100

Slightly soluble From 100-1,000

Very slightly soluble From 1,000-10,000

Insoluble or practically insoluble Greater than or equal to 10,000

Biopharmaceutical Classification System (BCS). Absorption in gastrointestinal tract involves the breaking down of dosage form into primary particles when exposed to gastrointestinal fluids. This step is called ‘disintegration’. The second step followed by disintegration is ‘dissolution’, which involves the drug molecule to leave the solid form of drug and enter into the form of a liquid solution, which is then followed by absorption where the dissolved drug molecules later pass through the membrane of the gastrointestinal tract to systemic circulation to reach its target site for pharmacological effect.[3,4] Due to the importance of the interplay among solubility and permeability, the Biopharmaceutical

Classification System (BCS) was developed in 1995 to classify drugs depending on the 18 absorption conduct. It has been categorized into 4 groups based on solubility and permeability as shown in Table 2. [5] It defines that a drug, at its highest dose, is soluble in

250ml or less of aqueous media. A highly soluble drug has a pH that ranges between pH

1-7.5, while a poorly soluble drug presents an aqueous solubility of less than 100 µg/ml.

Table 2: Biopharmaceutical Classification System (BCS)

BCS class Solubility Permeability

Class I High High

Class II Low High

Class III High Low

Class IV Low Low

Class I: Drugs possess high solubility and permeability to cross bio-membranes. Gastric motility and first pass effect are the only factors that affect the bioavailability of the drugs and the dissolution rate for immediate release formulation of drugs dissolved 85 % in less than 15 min.

Class II: Drugs have high permeability but low solubility as the rate-limiting step for API’s for systemic absorption.

Class III: Drugs have high solubility and low permeability. API’s exhibit good solubility profile and poor permeation across GI membrane having permeation as the rate-limiting step in absorption and bioavailability. 19

Class IV: Drugs have low solubility and low permeability with problems in oral administration.

Increase in the solubility of BSC class II and IV by enhancement techniques results in an increase of bioavailability. [6]

1.2 Factors Influencing Solubility

Melting point. Enthalpy is the thermodynamic measurement of heat content in the system and enthalpy of fusion is defined as the heat required by the substance to change from its solid state to liquid state. The temperature at which the phase transition from solid to liquid state occurs is defined as the melting point. [6]

−훥푆푓 푙표푔 푆 = (푀푃 − 25) 푖 1364

훥퐻푓 where −훥푆푓 is the entropy of fusion ( ) where 훥퐻푓 is the enthalpy of fusion and 푇푓 is 푇푓 the freezing point [7]. The strength of the crystal lattice is indicated by melting point, which is a physical property of the model drugs at a temperature where both the solid and liquid phases are in equilibrium. It indicates the strength of the intermolecular solid-state interactions, which is used to calculate aqueous solubility. Yalkowsky and Roseman predicted solubility in the equation by simplifying the equation [8]:

log 푆푖 = −0.01(푀푃 − 25)

A higher melting point indicates strong solute-solute interactions for stable crystalline compounds [9]. Hence, more energy is required to solubilize stable compounds as they have strong intermolecular bonds with low solubility causing sharp melting points, whereas, amorphous forms do not have a distinct melting point. Studies conducted by

Yalkowsky showed that the solubility is inversely proportional to melting point and enthalpy [10]. 20

Partition coefficient. It is also denoted as log P and characterizes lipophilicity. It is defined as the ratio of mole fraction concentration of solute in octanol and water phase.

Typically, an amount of log P that is greater than 5 indicates that the compound is highly hydrophobic in nature and has an inverse relationship with aqueous solubility [11].

Many unionized organic solvent systems are quite miscible in octanol and give the value of 0.5 upon simplification and the relationship is stated in the equation:

log 푆푤 = 0.5 − log 푃

With this equation, it clearly shows that negative correlation exists between log P and solubility [12]. The solubility of the crystalline state in water is the product of the solubility of solvent in water and the ideal solution given in the equation:

푋푐 log 푆푐 = log (푆 × ) 푤 푤 푋liq

푖푑푒푎l log 푆푤 + log 푋푢

Upon simplification:

log 푆푤 = 0.5 − 0.01Δ푆푓푢푠(푀푃 − 25) − log 푃

Particle size. The increase in surface area by the decrease in particle size, increases the saturation solubility of the drug models. This is anticipated due to the increase of curvature of the particle as interpreted by Ostwald Freundlich equation, where the solubility increases due to the increase in the radius of the particle and applied to the particle size less than 100 nm [13]. The drug with reduced particle size radius transforms from crystalline to amorphous state with an increase in surface area.

Crystallinity. The repeating molecular patterns arranged in an orderly manner are called crystalline forms and the solid materials with local molecular assemblies in absence of crystal lattice are called amorphous materials. The materials have intermolecular forces 21 in which crystalline intermolecular forces are more than that of amorphous forms [14].

Crystalline forms show definite sharp melting points upon transformation from solids to a liquid state. The non-crystalline state is thermodynamically stable with a tendency to entropically drive solid forms to stable crystalline forms of higher free energy and enhanced thermodynamic properties with higher molecular motion compared to crystalline forms [15]. However, the amorphous forms cannot reach maximum solubility under experimental condition due to strong driving force to recrystallize. Studies show that this problem of recrystallization can be avoided by the addition of recrystallization inhibitors

[16].

1.3 Solubility Enhancement Strategies

Many physicochemical and physiological factors influence the saturation solubility of the drug in the gastrointestinal tract. The solubility of a poorly aqueous soluble drug is a challenging aspect of screening studies of new chemical entities and formulation development. [17] Solubility and permeability are main parameters for in-vitro absorption as it can be modified to enhance the solubility of the drug. Techniques like solid dispersions, particle size reduction, eutectic mixtures, modification of crystal habits like co- crystallization, polymorphs and amorphous techniques, using of buffers, complexation, co- solvency, surface acting agents are few ways to increase the solubility by physical and chemical modification. [18]

Chemical methods. Salt forms have shown to have enhanced solubility depending on the pH of the medium. The conversion of acidic or basic drugs into water-soluble salt forms leads to increase in solubility when in a gastric medium where it converts to free acid or base which have low solubility and stability [19]. A complexation is an approach 22 where two or more molecules associate due to various kinds of forces, hydrophobic or hydrogen bonding between molecules to enhance solubility. The most common association is observed between hydrophobic drug molecule with a central cavity to the hydrophilic carrier on the outer surface [20]. Cyclodextrins (CD’s) are the most commonly used complexing agents possessing the ability to take up the guest molecule into the internal hydrophobic cavity of the CD’s, forming inclusion complexes [21].

Another approach is prodrug which is a chemical modification of the drug to overcome barriers that hinder the drug delivery to reach the active site. It is bio-reversible derivative, but the limitation is that it lacks in chemical stability of drug-promoiety linker and degrades to forms secondary degradation pathways [22]. It involves in linking the ionized group to the chemical structure of the drug to increase the solubility of the drug.

For example, Fosphenytoin, which is the prodrug of Phenytoin, shows more bioavailability than Phenytoin [22,23].

Physical methods. Nanosuspension is the preparation of thermodynamically stable insoluble or very slightly soluble solution in a given solvent by using one or more amphiphilic components, which are known as surfactants [24]. The formation of submicron colloidal suspensions with a pure drug that is stabilized by using surfactants are nanosuspension. They enhance solubility due to increase in surface area. Surfactants lower the surface tension at the air and liquid interface, leading to a rise in the surface area that is available for the dissolution of the drug in the solvent. Homogenization, wet milling, spray drying etc., are the various techniques that are used to make nanosuspensions [25].

Micronization is a technique, which increases the surface area with a decrease in particle size causing an increase in the dissolution rate but not the equilibrium [26]. The 23 decrease in the particle size may lead to solubility enhancement, where the Van der Waals attraction that increases the surface area between the hydrophobic molecules causes a decrease in size that may lead to agglomeration. Wettability is a challenge in micronization and hence, the technique is unsuitable for drugs with high-dose numbers [27]. Ball mill, fluidized micronization, ultrasonic size reduction, etc., are various techniques for micronization used in the reduction of particle size. Sono-crystallization is a technique that uses ultrasound frequencies ranging from 20-100kHz to break the crystalline form of the drug [28,29]. This technique not only reduces the particle size but also enhances nucleation rate [30].

Modification in crystal habit is classification of the solids as amorphous or crystalline is done by analyzing the crystal structure. The amorphous form shows increased solubility than that of crystalline due to the energy required to transform the crystal lattice to non-crystalline solids [31]. The substance consisting one or more crystalline forms in it is called a polymorph. The polymorphs that are unstable are called metastable polymorphs, which consists of low melting points and increased solubility profile relative to the stable forms [32].

There are two types of polymorphs based on the characteristics of melting point, hardness and density. Monotropic polymorphs are those that are unstable at any temperature and pressure. Enantiotropic polymorphs are the forms where the change is reversible to other forms with a change in temperature and pressure [33,34].

Another type of polymorph is the form that contains water as solvent called pseudo polymorphs. These are more soluble in water and requires less energy to break the lattice compared to solvates [35]. 24

Drug dispersion in the carrier is one of the most commonly used technique for the enhancement of drug to enhance its solubility is solid dispersion. It is defined as the dispersion of the hydrophobic drug in hydrophilic biologically inert matrix [35-37]. But the common challenge with this method is that there is a chance of possible drug- recrystallization on long-term storage. Additionally, moisture plays a vital role in the enhancement of drug mobility. There are five types of solid dispersion, which is classified as simple eutectic mixture, solid solution, complex formation, amorphous precipitation, glass solution/suspension and any of the combinations. A simple eutectic mixture is nothing but a mixture that is soluble in its liquid state but immiscible in its solid state [38].

Solid solutions are those that, irrespective of the components used, results in a single phase and are continuous and discontinuous solutions, depending on the drug and the carrier. The continuous solutions are miscible when formed with two miscible proportions in any proportions. However, a discontinuous solution is limited to miscibility of only 1 component [39]. Glass solutions/suspensions are the types of solid dispersions, where the drug is suspended or dissolved into a glassy form that entraps the drug molecule into the matrix. These dispersions are stable as they are formed by dispersing the crystalline drug in an amorphous carrier and are of type IV dispersions [40]. Using DSC thermograph, the melting point and glass transition temperature can be observed. Type V dispersions are the amorphous drug clusters that are subsumed into the carrier. These are metastable and if the clusters are too big, they form nuclei that promote rapid crystallization and increase in growth. But, when clusters are small, enough drugs cannot be incorporated to recrystallize.

There are various techniques that can be used to prepare solid dispersion. 25

Fusion solvent method is used for solid dispersion where the drug is first added and dissolved in a suitable solvent when the prepared solvent mixture is in a molten state. Later, it is cooled to acquire the product [41]. Hot melt extrusion is a process similar to fusion method except that the mixing of drug and the carrier is performed by the extruder.

Hot melt/fusion method is the physical mixture of the hydrophobic drug molecule is melted with hydrophilic carrier together and cooled simultaneously in an ice bath. The obtained hard mass is crushed, pulverized, sieved and stored in a desiccator. The stability of both the components are taken into consideration as the melting point depends on the composition including a selection of carrier, the weight fraction of drug and miscibility of both the components in molten forms [42].

The limitations of this method are that it is suitable only if the drug and polymer are compatible with each other and forms a homogeneous mixture at the heating temperature of percent formation of two incompatible liquid phases or suspensions in the heated mixture [43]. This can be overcome by using surfactants. During cooling, when the drug- matrix miscibility changes, the mixture might produce a phase separation that can be solved when done slowly, forming a crystalline drug. Formation of the crystalline drug is a problem that can be overcome by forming an amorphous dispersion by speeding up the cooling rate. It is unsuitable for compositions where the carrier is high-melting solid and is sensitive to heat.

Co-precipitation method is the technique with both the drug and the polymer are soluble in a common solvent. The polymer is first dispersed in the solvent and the drug is added to it, to form a homogeneous mixture. Later, the solvent is removed by evaporation by vacuum, under a temperature higher than the room temperature. The solvent can also 26 be removed by freeze drying or by spray drying [44]. The advantage of this technique is that the solvent can be removed at a lower temperature; it is useful when using thermolabile drugs. However, it is difficult to completely evaporate the solvent from the composition, which is a limitation.

The challenges and limitations faced when performing solvent evaporation technique are that it is difficult to mix drug in the polymer to one solution with different polarity and prevent phase separation in the time of removal of the solvent.

Nano-milling is the technique with the active pharmaceutical ingredient and carrier is reduced to a submicron range and is aqueously dispersed. These are then processed in wet media milling following the principle of attrition [45]. The advantage of this technique is that the solubility and dissolution are improved upon nanosizing. The main challenge is the development of this method, separation of nanocrystals from solution as the particles are in the nanometer range.

Wet-milling is comminuted by milling media in the presence of surface stabilizers and drug. Depending on the number of contact points, the stress intensity particle size is determined. Stress intensity is the function of kinetic energy involved in the process of grinding beads, where the number of contact points is obtained by a grinding media [46].

For example, the number of contact points increases by using smaller grinding media. The presence of stabilizers in wet-milling is mandatory as the high-energy wet mill process leads to a thermodynamically unstable aqueous nanosuspension. The drive shaft attached to the rotating disk provides energy to the zirconium beads to break the crystals, using compression-shear action. This technique is most commonly used for nanosizing. The 27

Dyno-mill consists of a grinding container, feed pump, grinding beads, driving shaft, dynamic gap separator, agitator disc, pressure gauge and a cooling cylinder.

The advantage of nanosuspension preparation by wet-milling is that it improves the rate of dissolution with reduced variability, leading to increased bioavailability. Some studies have shown that the nanoparticles can increase bioavailability and passive absorption [47] and in some, the decrease in dose and increase in bioavailability is compared to micron-sized API [48].

Spray drying technique contain dry powder that is incorporated into nanosuspension that can be used to prepare solid dosage forms like tablets, capsules, pellets etc. It can be achieved by spray drying process where the drug is embedded in a carrier matrix, to enhance solubility and dissolution [49].

Spray drying is based on the principle of pneumatic drying where the drying particles are entrained and carried in a high-velocity gas stream and the solvent is removed from the liquid stream. The liquid droplets reach a temperature higher than the wet bulb temperature of the gas when it comes in contact with the hot gas. [50]. Formation of a tough shell takes place by the evaporation of the surface liquid and the process of drying occurs when the liquid from the interior of the droplet starts to diffuse through the shell at a slower rate than the transfer of heat from the surface of the shell to the droplet’s interior. The liquid below the shell lining evaporates at a rapid rate and causes a buildup of heat beneath it, resulting in internal pressure and swelling of droplets. This produces a thinner shell and faster diffusion of the liquid from the interior of the shell [51]. Hence, spray dried products have intact spheres, rupture hollow spheres or sphere with bulbs and fragments. Non-elastic shells usually erupt producing fragments or bulb-like structures on the spheres [52]. 28

The components of spray dryer include feed pump, atomizer, drying chamber, cyclone separator, a product collector, aspirator and filter. The liquid feed is sent to the atomizer at an adjusted rate so that each droplet is dried completely before it comes in contact with the walls of the drying chamber, preventing excessive heat than necessary in the drying process. The atomizer like pneumatic, pressure or spinning disc, delivers feed into fine droplets into a drying chamber where the feed is broken into droplets with the help of high-velocity gas [53]. These, in return, produce smaller particles and encounters hot gas. This hot air is supplied by blowing air over a heat exchanger and the particles are separated by a cyclone separator where the solid dried product is collected. The dried product on the walls of the drying chamber is called chamber product. It is usually coarser in size and more exposed to heat than usual.

Factors affecting spray drying is the feed rate adjusted accordingly and is indicated by an outlet air temperature and visual inspection. The inlet temperature of drying air is the temperature required to dry the medium when it encounters the feed [54] and the feed temperature determines the ability to dry and remove the solvent in unit time. The outlet temperature depends on the inlet temperature that is measured when the air enters the drying chamber. For example, the change in inlet temperature results in different glassy state products of 4-O-(4-methoxyphenyl) Acetyltylosin [55]. Also, when the feed rate is increased, the outlet temperature drops and the material builds up on the walls of drying chamber [54]. During spraying, drying gas flow rate determines the amount of air required to dry the samples per unit time effecting the drying level and its separation in the separator

[56]. When the drying air flow is low, it requires a longer duration for the particle to dry. 29

Spray drying is commonly used to remove the solvents from the liquid stream and dry even the most thermolabile fluids without degrading the composition of the samples.

This technique is initially used for extracting active raw materials from the plants [44]. The tablet, which is a spray dried lactose, used for making tablets in the direct compression process, in turn, shows good compression properties of the powder [57]. It is used to change the melting points of the APIs to avoid crystallization in the tabletting process. It is also used to obtain particles with desired size and morphology, to enhance aqueous solubility, apparent solubility and dissolution rate. Spray drying produces appropriate particle size that can be administered via ophthalmic, inhalation powders and vaccines and preparation of self-emulsifying systems for a good release and enhanced bioavailability of drugs. They can be used as protective agents for drugs that are unstable in the gastrointestinal environment.

Re-dispersible dry emulsions used in the small intestine to decrease metabolism and increase oral absorption can be prepared by this technique [58]. It is also used to produce modified releasing tablets and to formulate protein drugs. Nevertheless, little success has been achieved as the low final yield is a major problem and aggregation causes proteins to unfold that may lead to denaturation. The potential and applications in protein drying and preparations using spray drying methodology has not been fully explored.

1.4 Solid-State Transitions

Drugs are present in a high degree of order that is the vital property of the physical state. They are present in crystalline, lattice-like and non-crystalline types [59]. This attributes to the higher melting point as they possess strong bonds. The molecular shape of chemical groups with intermolecular bonding like hydrogen bonding, dipole-dipole 30 interaction and charge transfers is responsible for the packing arrangement of these types.

These features account for melting points of these crystals [60]. The components that are hard and brittle contain high melting points as they contain crystal lattice. They are thermodynamically unstable in the amorphous forms but have a tendency to entropically drive to stabilize crystalline conditions. However, the diffusional process slows down the process of recrystallization. The free energy containing amorphous forms is much higher than crystalline forms [61].

Amorphous forms, also known as disordered materials, lack crystal lattice. They contain intermolecular forces but lack periodicity with different physicochemical properties like melting point, solubility, enthalpy, density, etc [62]. The amorphous materials help in retaining the state at room or body temperature, for instance, drugs with polyethylene glycol, polyvinyl pyrrolidone, etc. [63]. Amorphous forms have an advantage of being partially amorphous for preparation of dosage forms.

Influence of free energy on solubility is that amorphous forms have higher Gibbs free energy and do not require additional energy to break crystal lattice whereas, in case of crystalline materials, the drugs need to overcome the energy required to break the crystal lattice arrangement and enter aqueous solution [64]. Hence, this leads to an increase in solubility when the drug is in amorphous form rather than in crystalline. However, the amorphous materials are unstable due to Gibbs free energy and hence try to alter back into crystalline stable forms over some period of changing the properties.

1.5 Estimation of Aqueous Solubility

The therapeutic efficacy is determined by the aqueous solubility and it is one of the vital physicochemical properties based on the non-experimental structural parameter. Low 31 solubility APIs have been used to obtain solubility enhanced formulations, by increasing and implementing solubility enhancement techniques [65]. Aqueous solubility provides an estimation of enhanced solubility information prior to performing solubility enhancement techniques, in addition to its influence on the factors or parameters that helped in the enhancement [66]. This provides a general idea of the compound and excipient selection, with suitable properties and conditions, to acquire the desired solubility which benefits economically reduces the wastage of supply in the early development.

1.6 Carriers Used for Solid Dispersion

Carriers are polymers or recrystallization inhibitors, which are hydrophilic substances used for solid dispersions, primarily classified as polymers of sugar polyols, surfactants, organic acid and their derivatives [67]. Examples of polymers of sugar polyols contain mannitol, sorbitol and chitosan with its derivative chitin. Apart from PEG and PVP polymers, which are most commonly used, Eudragit, Hydroxypropyl cellulose and

Hydroxypropyl methylcellulose are other examples of polymers. Surfactants, which include Sodium Lauryl Sulfate (SLS) or Tween 80, are amphiphilic in nature and contain both hydrophilic and hydrophobic moieties that help in enhancing solubility with limited toxicities. [68].

Polyethylene glycols (PEG) are ethylene oxide oligomers that can be used as a carrier to enhance solubility, wettability and dissolution of the active pharmaceutical agents. Due to their low melting point that ranges from 53 °C to 63 °C, they can be used easily to produce solid dispersions. PEG is available in different grades, depending on the molecular weights that range from 200 to 300,000, which helps in differentiating them from polyoxyethylene and polyethylene oxide [69]. PEGs with the molecular weight 600 are 32 liquids, and those that range from 800 to 1500 are semisolids; PEGs with a molecular weight that range from 2000-6000 are waxy and above 6000, are crystal, at the room temperature. The molecular weights above 1500 are usually utilized in solid dispersion preparation as the aqueous solubility and viscosity is in direct relationship with the molecular weight of PEG. Viscosity is directly proportional to the molecular weight of

PEG and molecular weight is inversely proportional to aqueous solubility. The advantage of PEG is that it can be soluble in almost all the organic solvents and hence can be used as a pharmaceutical excipient [70].

Polyvinyl pyrrolidone (PVP) are the hydrophilic carriers, which are synthetic linear polymeric lactam, used in gastrointestinal preparations. They are soluble in almost all organic solvents and are formed by polymerization of vinylpyrrolidone. They are amorphous and are prepared in a variety of molecular weights, expressed as K-value, based on the viscosity measurement. Molecular weights of PVP is directly proportional to glass transition temperature (Tg) of linear PVP.

PVP shows anti-plasticizing effect causing surface absorption and a steric hindrance for crystal growth and nucleation inhibiting recrystallization of the drugs [71]. Hence PVP shows good solubility and wettability, which makes it a common choice of carrier to be used in solid dispersions.

Chapter 2: Research Aims

The solubility of poorly soluble drugs has been a challenging factor in the development of the drug, although several techniques are established to enhance the aqueous solubility of the drug at laboratory scale. Due to the limitation in the utility in industrial-scale production, there is a necessity to identify robust, reproducible and reliable technology that can be applied to an insoluble drug. Although solid dispersions and particle size reductions have been showing improved solubility, the basic mechanism underlying for the increase has not been explained elaborately.

The primary objective of this study is to prepare the compositions and physical mixtures of the model drugs with polymer stabilizers, to increase the aqueous solubility using solid dispersions and particle size reduction techniques. The aim is to determine the aqueous solubility using solid dispersion techniques. During these experiments, all the parameters except the model drugs and composition of the polymers were maintained constant. Methanol was used as the common solvent in co-precipitation technique.

The secondary objective is to compare the techniques and characterize the solid-state characters of composites using DSC, to investigate the reasons for the increase in solubility.

The solubility studies were conducted at 37 °C in filtered distilled water. The enthalpy of compositions was compared to a model pure drug from DSC thermograms and the change in crystallinity was determined.

The third objective is to determine the method that exhibits significant enhancement of aqueous solubility of each model drug. For this, the aqueous solubility of Glipizide, 34

Furosemide and Carvedilol determined from the compositions with fusion method, co- precipitation, nano-milling and spray drying techniques and was compared.

The significance of this research is to determine the influence of method of preparation and properties of the excipients used on the increase in the aqueous solubility.

The increase in aqueous solubility over the physical mixtures indicates that the techniques improve the solubility of the drug.

Chapter 3: Materials and Methods

The first objective of the research was to prepare a solid dispersion by using three model compounds with a range of properties and determine their solubility.

3.1 Materials

Glipizide, Furosemide and Carvedilol were obtained from Spectrum Chemicals (NJ,

USA), Polyethylene glycol 8000 from Dow Chemical Company, Polyvinyl pyrrolidone

40000 from Fisher Scientific (NJ). Methanol used was of analytical grade. Deionized water was used for saturated solubility studies and 0.22µm nylon syringe filters were used to filter the supernatant solution from solubility studies. Furosemide, Glipizide and Carvedilol are the three model drugs selected from BCS class II and IV drugs. The physicochemical properties of the model drugs are shown in Table 3.

Table 3. Physicochemical Properties of Model Drugs

Model drugs BCS Class Molecular Log P pKa MP (°C)

weight

Furosemide IV 330.7 2.03 4.25 206

Glipizide II 445.5 1.91 5.9 215.89

Carvedilol II 406.4 4.19 8.74 116.64-120.24

3.2 Methods

Different compositions like physical mixtures were prepared and techniques like fusion method, co-precipitation, nano-milling and spray drying were used, in addition to 2 polymers namely PEG 8000 and PVP 40,000 at 1:1, 1:5 and 1:10 drug-polymer ratios.

The physical mixtures compositions of Glipizide, Furosemide and Carvedilol were prepared by thoroughly mixing of drug with the polymer in a mortar until a homogeneous mixture was obtained.

Fusion method is a technique in which the drug-polymer ratios of 1:1, 1:5 and 1:10 were weighed and triturated in to achieve a proportionately mixed physical mixture. The mixture was then melted on a hot plate above the melting points of the model drugs. This resulted in the formation of a molten mixture, which was then rapidly cooled by placing over an ice bath for 5 minutes and stirring vigorously, until they solidify. All the experiments were carried out in triplicates (n=3) and analyzed further for solubility.

Co-precipitation technique is a technique in which the co-precipitates were prepared by first weighing the drug and polymer at predetermined ratios and then transferring them into the beaker containing methanol. The polymer was added to methanol and mixed well, before adding the drug. The drug was then added, transferred to glass scintillation vial and methanol (solvent) evaporated in a rotary evaporator (Rotavapor R300, Buchi,

Switzerland) at a reduced pressure and temperature. The resulting co-precipitate compositions were kept on a hot plate at a low temperature for the evaporation of the residual solvent. The final product was scraped, pulverized and stored in a desiccator for analysis of solubility. 37

Nano-milling contains nanosuspension prepared by media milling using 120 ml of

Zirconium oxide beads of size 0.5 mm in Dyno mill Multilab (Glenmills, Clifton, NJ) at a speed of 4180 rpm for 1 hour. The nanosuspension was then frozen overnight at -80 °C and dried to yield dry nanoparticle powder for saturated solubility studies.

Spray drying technique is performed where the PVP 40,000 polymer and the drugs were weighed accurately in the desired ratios of 1:1 of Glipizide and Furosemide; 1:10 of

Carvedilol was dissolved in methanol. The solution was then spray dried using SONOtech nozzle system Buchi B290 spray dryer. The drug loading was 5 % (w/v) of Glipizide, 7.5

% (w/v) of Furosemide and 7.5 % (w/v) of Carvedilol. The following table shows the parameters of the different formulations:

Table 4. Parameters for Spray Drying of Model Drugs

Spray Parameters Glipizide Furosemide Carvedilol

Drying air (m3/min) 0.5 0.2 0.45

Inlet temperature (°C) 150 160 145

Outlet temperature 90 89 90

(°C)

Feed flow rate 4.3 5.4 3.6

(ml/min)

Atomizing air (MPa) 0.15 0.2 0.15

Chapter 4: Characterization of Compositions

4.1 Differential Scanning Calorimetry (DSC)

The compositions and physical mixtures were subjected to thermal analysis and compared to pure drug using DSC 6200, SII EXSTAR 6000 with Muse measurement

COM2 software. To calibrate the temperature and enthalpy values of samples, high purity indium was used and the samples were sealed in aluminum pans. An empty aluminum pan was used as the reference standard. The samples of approximately 3 mg were weighed and were scanned and analyzed at a temperature range of -10 °C to 350 °C with a heating rate of 10 °C/min using nitrogen as the blanket gas.

4.2 Results

To determine solid changes and compatibility studies, DSC was extensively used and was a sensitive method used for the detection of solid phase transition. The melting point of crystalline compounds depends on the intermolecular forces between molecules that hold together in forming a crystal [72-73]. When the intermolecular bonds break, it shows an endothermic peak obtaining a melting point of the sample, whereas, in non-crystalline samples, the formation of an endothermic peak is not observed. The crystallinity of the compound is determined by the presence or absence of endothermic peak [74]. The polymer

PEG 8000 showed an endothermic peak at 67.24 °C with an enthalpy of fusion of -301.4

J/g whereas PVP 40,000 showed broad spectrum and melting point at 175.55 °C with an enthalpy of fusion of -29.5 J/g due to the presence of residual moisture in PVP. 39

The DSC of Glipizide showed melting of the drug with a characteristic endothermic peak at 216.53 °C with an enthalpy of fusion of -120.8 J/g, indicating that Glipizide was in a crystalline state in a pure solid form, which was consistent with the literature [75]. The physical mixtures of Glipizide with PEG 8000 in 1:1 ratio showed an endothermic peak at

204.52 °C whereas 1:5 and 1:10 ratios of physical mixtures did not show any endothermic peaks at the temperature of Glipizide as shown in Figure 1 and 3. The melting point decreased, which may attribute to PEG 8000 acting as an impurity to Glipizide in 1:1 ratio.

In the fusion method, drug peak was observed at 204.55 °C and 186.24 °C for 1:1 and 1:5 respectively, with an enthalpy of -19.3 J/g and -5.2 J/g but without a peak in 1:10 ratio as shown in Figures 1-3. In co-precipitation technique, DSC showed an endothermic peak at

205.0 °C with an enthalpy of -44.1 J/g in 1:1 ratio and 207.57 °C with an enthalpy of -2.7

J/g for 1:5 ratio. In nano-milling, 1:1 ratio showed an endothermic peak of the drug at

194.36 °C with an enthalpy of -4 J/g and 1:10 had a drug peak at 197.39 °C with an enthalpy of -6.5 J/g as shown in Figure 3. There was an absence of an endothermic peak in 1:10 ratio and in nano-milled samples and also in 1:5 ratio of co-precipitation. The disappearance of the peak may be due to the high concentration of PEG 8000 in the sample, which leads the drug to below the detection limit and more dilute drug in the polymer or in the formation of amorphous form from crystalline. The absence of endothermic peaks confirms that the lack of crystalline drug in significant amounts. 40

Figure 1. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:1 Ratio

Figure 2. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:5 Ratio

Figure 3. Thermograms of Glipizide-PEG8000 Composites from Fusion, Co-precipitation and Nano-milling Methods at 1:10 Ratio

The physical mixture of Glipizide and PVP 40,000 showed the melting point of

Glipizide to be at 204.76 °C, 204.58 °C and 210.31 °C, at drug-polymer ratios of 1:1, 1:5 and 1:10 respectively. In fusion method, Glipizide and PVP 40,000 with a ratio of 1:1 showed drug melting peak at 192.75 °C with an enthalpy of -6.9 J/g as shown in Figure 4 and 1:10 with -0.5 J/g at 163.46 °C endothermic peaks as shown in Figure 6. In co- precipitation technique with 1:5 ratio presented a peak at 175.03 °C with an enthalpy of -

10 J/g as shown in Figure 5 and 1:10 ratio drug endothermic peak at 179.6 °C with an enthalpy of -8.6 J/g. The endothermic peak at 169.01 °C and 165.6 °C with an enthalpy of

-10 J/g and -7.3 J/g for 1:5 and 1:10 ratios respectively. In 1:1 ratio of nano-mill technique followed by degradation, there was no drug endothermic peak observed whereas in co- precipitation there was an absence at 1:1 ratio and at 1:5 ratio in fusion method. In spray 42 drying technique, Composites at 1:1 ratio showed an endothermic peak at 146.13 °C with enthalpy -9 J/g.

Figure 4. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co- precipitation, Nano-milling and Spray drying Methods at 1:1 Ratio

Figure 5. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio

Figure 6. Thermograms of Glipizide-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio 44

The thermogram of Carvedilol showed an endothermic peak at 120.2 °C with an enthalpy of -97 J/g consistent with the literature [76]. The physical mixtures of Carvedilol and PEG 8000 with ratios of 1:1 showed an endothermic melting peak at 116.88 °C and enthalpy of -22 J/g, whereas, at 1:5 and 1:10 ratios, composited did not show any drug melting peak. In fusion method composites at 1:1 ratio as shown in Figure 7, the endothermic peak was observed at 102.46 °C with an enthalpy of -23.4 J/g and in coprecipitation at 114.41 °C with an enthalpy of -29 J/g. The melting peak of Carvedilol was absent in 1:5 and 1:10 of fusion method and co-precipitation as shown in Figure 8 and

9. The composited from nano-milling technique at all the ratios did not exhibit any endothermic peak.

Figure 7. Thermograms of Carvedilol-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:1 Ratio

Figure 8. Thermograms of Carvedilol-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio

Figure 9. Thermograms of Carvedilol-PEG8000 composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio

The physical mixture of Carvedilol with PVP 40,000 of ratios 1:1 and 1:5 showed an endothermic peak at 116.88 °C and 118.09 °C, with an enthalpy of fusion of -4.1 J/g and -3.3 J/g respectively. No endothermic peak was observed at 1:10 ratio. In fusion method, co-precipitation, nano-milling and spray drying techniques, no drug peak was detected as shown in Figures 10-12.

Figure 10. Thermograms of Carvedilol-PVP40000 composites from Fusion, Co- precipitation and Nano-milling Methods at 1:1 Ratio

Figure 11. Thermograms of Carvedilol-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio

Figure 12. Thermograms of Carvedilol-PVP40000 composites from Fusion, Co- precipitation, Nano-milling and Spray drying Methods at 1:10 Ratio 48

The thermogram of Furosemide showed an exothermic peak at 226.57 °C with an enthalpy of fusion of -2 J/g. The formation of exothermic peak could be the reason of recrystallization of melted furosemide. The melting, recrystallization, and degradation of

Furosemide were observed at 229.65 °C with an enthalpy of 6.7 J/g [77]. The DSC thermograms of a physical mixture of Furosemide and PEG 8000 exhibited an absence of drug peak. In fusion method, 1:1 ratio presented an endothermic peak at 213.47 °C with an enthalpy of -5.4 J/g. At 1:5 and 1:10 ratios showed no endothermic peak. For Furosemide, no endothermic peaks were observed in fusion method, co-precipitation, and nano-milling as shown in Figures 13-15.

Figure 13. Thermograms of Furosemide-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:1 Ratio

Figure 14. Thermograms of Furosemide-PEG8000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio

Figure 15. Thermograms of Furosemide-PEG8000 composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio

The physical mixture of Furosemide and PVP 40,000 did not show any endothermic peaks. In co-precipitation method at 1:1 ratio, an endothermic peak was observed at 142

°C with an enthalpy of -0.8 J/g, whereas, 1:1 ratio of spray dried technique showed a peak at 141.2 °C with an enthalpy of -1.8 J/g as shown in Figure 16. There was an absence of an endothermic peak in fusion method, nano-milling and co-precipitation at 1:5 and 1:10 ratios as shown in Figures 17 and 18.

Figure 16. Thermograms of Furosemide-PVP40000 Composites from Fusion, Co- precipitation, Spray drying and Nano-milling Methods at 1:1 Ratio 51

Figure 17. Thermograms of Furosemide-PVP40000 Composites from Fusion, Co- precipitation and Nano-milling Methods at 1:5 Ratio

Figure18. Thermograms of Furosemide-PVP40000 composites from Fusion, Co- precipitation and Nano-milling Methods at 1:10 Ratio

The complete absence of endothermic peak indicated that the drug could not be detected as the drug quantity is small when compared to the polymer. The other possibility is that a modification took place in the crystalline structure of the drug when compared with the physical mixture and compositions obtained with different techniques.

Chapter 5: Aqueous Solubility of Model Compounds

Solubility was determined by traditional shake flask method, where the samples were added to 10 mL of distilled water in scintillation vial until saturation and the existing solid were observed. These were maintained at a temperature of 37 °C and were constantly shook inside a water bath. Samples were withdrawn and filtered using 0.45 µm nylon filters and the filter sample was then analyzed using HPLC until the constant concentration of drug that is dissolved was obtained. This experiment was done in a triplicate.

5.1 HPLC

HPLC analysis was performed using isocratic gradients and columns, namely C8 and C18. HPLC conditions of different model drugs are mentioned in Table 5. The flow rate of all the formulations was 1 ml/min and a UV detector, suitable for model drugs, set at different wavelengths, was used for detection. The collected solubility samples were diluted and analyzed. The peak area was recorded, and the concentration was determined.

5.2 Statistical analysis

Analysis of Variance and student t-test was performed using Microsoft Excel 2016 and the significance level which is (α = 0.05) was based on the probability value (p < 0.05)

Table 5. Chromatographic Conditions for Analysis of Model Drugs

Model drug Mobile phase Wavelength Retention Column

(nm) time (min) dimensions /

temperature

(°C)

Glipizide pH 6 Monobasic sodium 225 6.79 C18 column

phosphate buffer: Methanol 4.6x150mm

(55:45) 3.5µm

/ 25°

Carvedilol ACN: (pH 2 monobasic 240 5.38 C8 column

potassium phosphate) (31:69) 4.6x100mm

3.5µm/ 55°

Furosemide TFA: water: glacial acetic acid 272 4.35 C18 column

(30:70:1) 4.6x150mm

3.5µm / 25°

Results. The saturated solubility studies were determined in distilled water at 37 °C.

Carvedilol, BCS class II drug, had a very low solubility when compared to Glipizide and

Furosemide. The solubility of physical mixtures was compared to pure drug solubility studies.

Table 6. Comparison of Solubility of Model Drugs

PEG8000 PVP40000

Composition Glipizide Carvedilol Furosemid Glipizide Carvedil Furosemid

(µg/ml) (µg/ml) e (µg/ml) (µg/ml) ol e (µg/ml)

(µg/ml)

Drug R 11.18±1.7 5.04±0.55 55.94±2.4 11.18±1.7 5.04±1.2 55.94±2.4

8 8 8 2 8

Physical 1:1 12.38±0.9 5.23±0.07 55.98±1.0 14.32±2.6 6.38±1.2 61.98±2.5

mixture 9 1 2 8 2

1:5 13.95±2.2 5.37±0.22 60.97±2.2 12.88±0.4 5.74±0.2 70.17±3.1

7 9 3 7 1

1:10 14.33±1.6 6.03±0.67 63.89 ± 11.30±1.2 6.59±0.3 67.42±5.7

9 5.29 1 3 6

Fusion 1:1 12.90±2.1 16.16±0.8 64.21 ± 43.26±7.8 29.46±6. 132.36±9.

method 4 0 3.48 7 53 94

1:5 25.57±1.9 17.51±0.9 69.17 ± 39.15±1.2 48.28±6. 128.42±24

1 4 2.43 8 06 .78

1:10 24.66±2.5 12.7±0.55 76.10 ± 19.96±2.8 62.44±1 100.70±5.

9 5.24 9 7.69 50

1:1 27.03±4.3 11.87±1.4 74.56 ± 25.30±3.7 24.41±4. 93.53±7.3

3 8 4.10 7 48 3 56

1:5 27.56±2.9 11.20±1.8 72.59 ± 43.00±7.1 53.41±6. 281.37±39 Co- 7 2 4.81 0 06 .68 precipitati 1:10 35.39±2.4 11.59±0.3 72.86 ± 22.81±2.1 104.30± 192.48±30 on 6 0 6.61 5 9.28 .67

Nano- 1:1 23.73±1.5 1.95±0.21 134.96 ± 40.37±4.8 2.80±0.3 304.52±23

milling 4 1.20 3 6 .11

1:5 35.73±0.0 2.05±0.23 163.86±10 31.08±4.7 60.78±1 287.48±11

4 .60 2 1.99 .72

1:10 24.11±0.9 2.14±0.28 164.11±9. 8.23 ± 152.70± 232.21±24

1 18 1.22 9.09 .04

Spray 1:1 - - - 15.97±3.1 - 182.52±6.

drying 1 95

1:10 - - - - 67.42 ± -

1.22

The aqueous solubility of the model drugs as shown in table 6, indicated that the

optimum solubility enhancement was achieved at different (drug: polymer) ratios. A

further increase of polymer excipient did not enhance the drug solubility, at times reduced

it [78]. PEG8000 and PVP40,000 were selected for this study since both were soluble in

water and various organic solvents [79-80]. Selection criteria also involve melting points and

Molecular weights as they are different. 57

The solubility was determined at 37 °C in deionized water. The drug concentrations were determined from the calibration curves analyzed using HPLC. In a physical mixture of the drug models with PEG 8000 in 1:1, 1:5 and 1:10, the three model drugs showed that there was no significant enhancement in solubility when compared to the model drug

(p>0.05). However, with all the compositions, the enhancement of solubility was significant compared to the pure drug (p<0.05).

Chapter 6: Discussion

The solubility of model drug Glipizide is 11.18 ± 1.78 µg/ml and the physical mixture with the three ratios in the presence of carriers showed no significance when compared to the pure drug (p>0.05). However, all the techniques used showed an enhancement in solubility (p<0.05) when compared to the physical mixture as shown in table 6. PEG8000 as carrier showed an increase in solubility with an increase in carrier ratio as shown in Figures 19-21. Fusion method with PEG8000 as a carrier at 1:5 ratio, showed a 2.28-fold of increase from the physical mixture was observed as the crystallinity of the drug endothermic peak from DSC thermograph was decreased as shown in Figure

2. There was a decrease in percentage crystallinity to 4.3% with an enthalpy of fusion of -

5.2 J/g, when compared to drug enthalpy of -120.8 J/g, which resulted in an increased solubility. It was observed that there were 1.4-fold of increase in solubility with an enthalpy of -44.1 J/g and 36 % of crystallinity with the techniques at 1:1 ratio, when compared to the drug (p<0.05) and significantly observed in Coprecipitation, as shown in Figure 1.

The solubility of Glipizide using nano milling technique, used in 1:1 ratio, increased the solubility over physical mixture with 2.1 folds with an enthalpy of -4 J/g. In 1:5 ratio, compared to all methods, nano-milling showed an increase of 3.1 folds when compared to pure drug and physical mixture, as shown in Figure 2. The particle size reduction took place in an increased surface area because of the polymer’s effect. The particle sizes were

624nm, 439nm and 633nm for 1:1, 1:5 and 1:10 ratio, respectively. In 1:10 ratio, co- precipitation technique showed an enhanced solubility, as shown in Figure 3. Among the 59 ratios, the enhancement in solubility was significant with all the techniques and 1:5 ratio showed an optimum increase in solubility. Nano-milling showed a high solubility of 35.73

± 0.04 µg/ml, attributing to the reduction of particle size to 439 nm and an absence of crystallinity was observed in the thermogram. The other possibility was because of the carrier that helped in preventing recrystallization [81].

10 Solubility (µg/ml) Solubility 5

0 Glipizide Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 19. Glipizide Solubility with PEG8000 as Carrier in 1:1 Ratio

10 Solubility (µg/ml) Solubility 5

0 Glipizide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 20. Glipizide Solubility with PEG8000 as Carrier in 1:5 Ratio

Glipizide Solubility with PEG8000 as carrier in 1:10 ratio 40

10 Solubility (µg/ml) Solubility 5

0 Glipizide Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 21. Glipizide Solubility with PEG8000 as Carrier in 1:10 Ratio

The solubility of Glipizide with PVP40000 as carrier showed enhanced solubility with the pattern, and it was observed that an increase in carrier ratio leads to a decrease in solubility. All the techniques showed enhancement in solubility when compared to the drug and physical mixture with p<0.05 as shown in Figure 22-24. In 1:1 ratio, fusion method showed a 3-fold increase when compared to the drug. The crystallinity was reduced to 5% obtained from DSC thermogram with a reduced enthalpy of -5 J/g. Nano-milling showed significant solubility in 1:1 ratio, when compared to 1:5 and 1:10 ratios as the solubility increased 3.6 folds when compared to a drug with no crystallinity of drug. It resulted in an amorphous state with a particle size of 435 nm, which was the lowest size among the ratios.

Among 1:1 ratio, it was observed that fusion method showed an enhanced solubility, as presented in Figure 4. The co-precipitation method in 1:5 ratio showed an enhanced solubility where the crystallinity reduced to 7% and enthalpy of -8.7 J/g with an increase of 3.8 folds from the drug, greater than all the techniques shown in Figure 5. In 1:10 ratio, among all the techniques, it was observed that 2-fold of increase in solubility occurred with co-precipitation technique due to the decrease in crystallinity to 8% from drug and the solubility of technique, as shown in Figure 6.

20 Solubility (µg/ml) Solubility

0 Glipizide Physical mixture Fusion method Co-precipitation Nano-milling Spray drying Techniques

Figure 22. Glipizide Solubility with PVP40000 as Carrier in 1:1 Ratio

20 Solubility (µg/ml) Solubility

0 Glipizide Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 23. Glipizide Solubility with PVP40000 as Carrier in 1:5 Ratio

10 Solubility (µg/ml) Solubility

0 Glipizide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 24. Glipizide Solubility with PVP40000 as Carrier in 1:10 Ratio

The solubility of Carvedilol was 5.04 ± 0.55 µg/ml and the solubility of physical mixtures showed no increase (p>0.05) and was comparable to a pure drug as shown in

Figure 25-27. In 1:1 ratio of PEG8000 as a carrier, fusion method showed an increase of 3 folds and in 1:5 ratio and 1:10 ratios, an increase of 3.4 and 2.5-fold was observed when compared to the physical mixture, as shown in Figure 7-9. In the nano-milling technique, the particle size was increasing with a polymer as an agglomeration was taking place. This led to the decrease in the solubility when compared to drug and physical mixture. In co- precipitation technique, no difference in solubility was observed among the ratio of carriers. The trend observed was that, due to the absence of drug endothermic peak in the

DSC thermogram, the polymer ratio was not showing any effect on solubility but the 64 optimum increase in solubility was found to be with 1:5 ratio with PEG8000 as a carrier.

This states that the reason could be due to the transformation of crystalline from a solid state to amorphous. Fusion method with 1:5 drug-polymer ratio was observed to be significant in different ratios to test for solubility of Carvedilol in the presence of PEG8000 as shown in Figure 26.

6 Solubility (µg/ml) Solubility 4

0 Carvedilol Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 25. Carvedilol Solubility with PEG8000 as Carrier in 1:1 Ratio

20 18 16 14 12 10 8

6 Solubility (µg/ml) Solubility 4 2 0 Carvedilol Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 26. Carvedilol solubility with PEG8000 as Carrier in 1:5 Ratio

4 Solubility (µg/ml) Solubility

0 Carvedilol Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 27. Carvedilol Solubility with PEG8000 as Carrier in 1:10 Ratio

In the case of Carvedilol with PVP40000 as a carrier, the pattern observed was that as the ratio of polymer increases, the solubility increases. In 1:1 drug-polymeric ratio, fusion method showed an increase of 4.6 folds, co-precipitation with 4.8 folds and nano- milling with 0.5 folds of increase, when compared to physical mixture, as shown in Figure

28. It was observed from DSC thermogram that the absence of drug endothermic peak results in an increase in solubility as the form may be in amorphous as shown in Figure 10.

In 1:5 ratio, the increase in solubility of Carvedilol was observed to be 10.5 folds in nano- milling technique comparable to a physical mixture as shown in Figure 29 whereas, in co- precipitation and fusion method, there were 9 folds and 8 folds of enhancement respectively, as shown in Figure 11. In 1:10 ratio, 23 folds enhancement of solubility of the drug with nano milling method, 12 folds with co-precipitation and 9 folds with fusion method respectively were observed, comparable to the physical mixture, as shown in

Figure 12. The nano-milling technique is more suitable for a polymer with 1:10 ratio of

PVP40000 as the particle size reduced when compared to pure drug shown in Figure 30.

This lead to increase in solubility as there was an increase in particle curvature. The effect of a change in the solid state also played a vital role as the drug endothermic peak in DSC thermogram was absent due to the change in crystalline to an amorphous form of the drug.

Solubility (µg/ml) Solubility 10

0 Carvedilol Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 28. Carvedilol Solubility with PVP40000 as Carrier in 1:1 Ratio

20 Solubility (µg/ml) Solubility 10

0 Carvedilol Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 29. Carvedilol Solubility with PVP40000 as Carrier in 1:5 Ratio

180

160

140

120

100

60 Solubility (µg/ml) Solubility 40

0 Carvedilol Physical Fusion method Co-precipitation Nano-milling spray drying mixture Techniques

Figure 30. Carvedilol Solubility with PVP40000 as Carrier in 1:10 Ratio

The solubility of model drug Furosemide was found to be 55.94 ± 2.48 µg/ml. When physical mixtures were compared to the pure drug, no enhancement in solubility was observed (p>0.05). There was a trend observed with PEG8000 as a carrier that as the polymer concentration increased, the solubility of the drug increased. In 1:1 ratio, the solubility, when compared with drug and physical mixture, presented 1.1, 1.3 and 2.4-fold increase for fusion method, co-precipitation, and nano-milling techniques respectively, as shown in Figure 13. In 1:5 ratio, there was 1.2 folds increase with fusion method and co- precipitation as shown in Figure 32. An increase of 2.9-fold in solubility was observed with nano-milling technique when compared to physical mixture, as shown in Figure 14.

In 1:10 ratio, the increase in solubility was 1.1 folds in fusion and co-precipitation, in 69 contrast to 2.5 folds with the nano-milling technique, as shown in Figure 15. In fusion method, as the polymer ratio increased, there was solubility enhancement observed in 1:10 ratio as shown in Figure 33. The increase occurred due to the absence of drug crystallinity determined in thermogram of DSC and the drug could be in an amorphous form. In co- precipitation method, there was no significance observed among the change in the ratio of the carrier. There was 1.3-fold increase when compared to the drug and physical mixture in 1:1 ratio as shown in Figure 31. In nano-milling technique, there was an increase in solubility when compared to drug and physical mixture (p<0.05), where there were 2.9 folds of enhancement observed with 1:10 ratio. This is due to the reduction in the particle size of the drug, transforming from crystalline form to amorphous form. The particle size of the drug-polymer ratio was 995 nm, 980 nm, and 929 nm of 1:1, 1:5 and 1:10 ratios respectively. The particle size of 1:10 ratio was comparatively the lowest among the ratios with increased solubility.

160

140

120

100

60 Solubility (µg/ml) Solubility 40

0 Furosemide Physical mixture Fusion method Co-precipitation Nano-milling Techniques

Figure 31. Furosemide Solubility with PEG8000 as Carrier in 1:1 Ratio

200 180 160 140 120 100 80

60 Solubility (µg/ml) Solubility 40 20 0 Furosemide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 32. Furosemide Solubility with PEG8000 as Carrier in 1:5 Ratio 71

200 180 160 140 120 100 80

60 Solubility (µg/ml) Solubility 40 20 0 Furosemide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 33. Furosemide Solubility with PEG8000 as Carrier in 1:10 Ratio

A decrease in solubility was observed with increase in the ratio of polymer

Furosemide with PVP40000 as a carrier. The physical mixture, when compared to the drug, was insignificant in solubility whereas all the methods with PVP4000 as a carrier were significant. In 1:1 ratio, fusion method displayed 2.1-fold, 1.5-fold increase with co- precipitation, 4.9-fold of increase with nano-milling and 2.9-fold increase with spray drying in solubility when compared to the physical mixture (p<0.05), as shown in Figure

16. In 1:5 ratio, fusion method presented a 1-fold increase in solubility whereas a 4-fold increase was observed in co-precipitation and nano-milling when compared to physical mixture, as shown in Figure 17. In 1:10 ratio, there was a 1-fold increase in fusion method, 72

2-fold in co-precipitation and 3-fold in nano-milling technique solubility, when compared to the physical mixture (p<0.05), as shown in Figure 18.

Furosemide at 1:1 ratio with PVP40000 as carrier, presented enhanced solubility compared to other ratios but in co-precipitation method, 1:5 ratio displayed an enhancement in solubility shown in Figure 35. Enhanced solubility was significantly observed in a nano-milling method in 1:1 ratio as it was the effect of particle size reduction when compared to 1:5 and 1:10 ratios as the particle size were 114 nm, 126 nm, and 132 nm, where the lowest particle size shows significant solubility along with the effect of the polymer as shown in Figure 34-26. In spray drying method, there was percentage crystallinity decrease which was observed in 1:1 ratio of DSC thermogram, when compared to drug percentage crystallinity. This provided an increase in solubility of spray drying method when compared to the pure drug (p<0.05). The increase in solubility was confirmed due to the conversion of crystalline form to an amorphous form of the model drug.

350

300

250

200

150

100 Solubility (µg/ml) Solubility

0 Furosemide Physical mixture Fusion method Co-precipitation Nano-milling spray drying Techniques

Figure 34. Furosemide Solubility with PVP40000 as Carrier in 1:1 Ratio

350

300

250

200

150

Solubility (µg/ml) Solubility 100

0 Furosemide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 35. Furosemide Solubility with PVP40000 as Carrier in 1:5 Ratio

300

250

200

150

100

Solubility (µg/ml) Solubility 50

0 Furosemide Physical Fusion method Co-precipitation Nano-milling mixture Techniques

Figure 36. Furosemide Solubility with PVP40000 as Carrier in 1:10 Ratio

Among all the techniques, the highest solubility was observed for Furosemide:

PVP40000 (1:1 ratio) 304.52 ± 23.11 µg/ml. This is attributed because of all the techniques, model drugs and carrier ratios, 1:1 nano-milling technique showed an enhancement in solubility with the lowest particle size (114nm). The carrier also played a vital role in wettability of PVP40000, causing more solubility. The thermogram from DSC revealed the absence of drug endothermic peak, resulting in the change of drug from crystalline to amorphous form. This helped in the increase of solubility to a greater extent. The lowest solubility was observed in Carvedilol nano-milling technique with PEG8000 as a carrier with 1:1 ratio (1.95 ± 0.21 µg/ml). This attributes to the reason that the carrier was not providing stabilization of the drug molecules from recrystallizing. Particle agglomeration occurred as the particle size increased from than that of pure drug. The increase in particle 75 size led to a delay of solubility, which caused least interactions with the carrier and aqueous environment. Similarly, among each model drug, the carrier and technique with a change in ratio showed the effect of enhancement of solubility and the percentage enhancement was shown in table 7. It could be because of the better miscibility of the model drug in the polymeric carrier in these techniques that depended on the solid-state transitions.

Table 7. Percentage Enhancement of Aqueous Solubility Compared to the Drug

Percentage PEG8000 PVP40000

enhancement

Composition Ratio Glipizide Carvedilol Furosemide Glipizide Carvedilol Furosemide

(%) (%) (%) (%) (%) (%)

Fusion 1:1 15 220 14 286 484 136 method 1:5 128 246 23 250 857 129

1:10 120 151 36 78 1138 80

Co- 1:1 141 135 33 126 384 67 precipitation 1:5 146 121 29 284 959 402

1:10 216 129 30 104 1969 244

Nano- 1:1 112 -61 141 261 -44 444 milling 1:5 219 -59 192 178 1105 413

1:10 115 -57 193 -26 2929 315

Spray 1:1 - - - 42 - 226

Drying 1:10 - - - - 1237 -

Summary

Solubility enhancement of 3 model drugs with selected carriers was performed by fusion method, co-precipitation, nano-milling and Spray drying techniques, at 3 ratios of carriers. PEG8000 enhanced the solubility for Glipizide was achieved 128 % by fusion 77 method at 1:5 ratio, 216 % by coprecipitation at 1:10 ratio and 219 % by nano-milling method at 1:5 ratio, while PVP enhanced the solubility by 286% by fusion at 1:1 ratio,

284% by co-precipitation at 1:5 ratio, 261 % by nano-milling method at 1:1 ratio and 42 % by Spray drying at 1:1 ratio. PEG8000 enhanced solubility of Carvedilol was achieved 246

% by fusion method at 1:5 ratio, 135 % by coprecipitation at 1:1 ratio but decrease in solubility by 61 % by nano-milling method at 1:1 ratio, while PVP enhanced the solubility by 1138 % by fusion at 1:10 ratio, 1969 % by co-precipitation at 1:10 ratio, 2929 % by nano-milling method at 1:10 ratio and 1237% by Spray drying method at 1:10 ratio.

PEG8000 enhanced the solubility for Furosemide was achieved 36 % by fusion method at

1:10 ratio, 33 % by coprecipitation at 1:1 ratio and 193 % by nano-milling methods at 1:10 ratio, while PVP enhanced the solubility by 136 % by fusion at 1:1 ratio, 402 % by co- precipitation at 1:5 ratio, 444 % by nano-milling method at 1:1 ratio and 226% by Spray drying method at 1:1 ratio. The results indicated that the change in the melting temperature and enthalpy of the thermograph along with the particle size reduction leading to solid- state transitions of crystalline to amorphous forms resulted in an increase in solubility. The percentage of crystallinity depending upon the polymer used resulting in enhancement of the model drugs aqueous solubility.

Chapter 7: Conclusion

Several techniques were applied to determine the enhancement of solubility among which this study relied on fusion method, co-precipitation, nano-milling and spray drying techniques. The increase in aqueous solubility over the physical mixtures indicated that the techniques improved the solubility of the drug. This can be applied to the model drugs with different structural and properties under a defined range of experimental conditions.

The first objective of this dissertation was to prepare the mentioned model drugs using PEG8000 and PVP40000 in 1:1, 1:5 and 1:10 drug-polymeric ratios. The compositions were compared to physical mixtures, to determine the increase in solubility which successfully enhanced by the techniques over the physical mixture and drug

(p<0.05) except carvedilol with nano-milling technique. The parameters except the model drugs were kept constant and solubility was quantified with HPLC.

The solubility of Glipizide has been enhanced from 11.18 ± 1.78 µg/ml to 43.26

±7.87 µg/ml by fusion method in 1:1 ratio. The solubility of Carvedilol was enhanced from

5.04 ± 1.22 µg/ml to 152.70 ± 9.09 µg/ml by 1:10 ratio with nano-milling technique and the solubility of Furosemide was enhanced from 55.94 ± 2.48 µg/ml to 304.52 ± 23.11

µg/ml with 1:1 ratio using the nano-milling technique with the influence of decrease in particle size. The enhancement of solubility depended on enthalpy decrease from the pure drug with the possibility of conversion of crystalline form to amorphous solid-state transition. With this, the second objective was successfully achieved. 79

The third objective was to determine the method of each model drug that showed significant enhancement in solubility. For Glipizide, the enhancement of solubility was achieved with 1:1 ratio using PVP40,000 with all the techniques, among which fusion method showed enhancement in solubility. Hence, 1:1 drug-polymer ratio is suitable for the ratios and fusion method is the most suitable technique for Glipizide to increase solubility. The solubility of Carvedilol was significant with 1:10 ratio by nano-milling technique and the solubility of Furosemide had increased with 1:1 ratio by nano-milling technique. Hence, this study reports the suitable techniques for enhancement of solubility with an optimum polymeric ratio that can be used in early stages to estimate solubility enhancement of model drugs subjected to fusion method, co-precipitation, nano-milling and spray drying techniques.

All the techniques, except nano-milling exhibited solubility enhancement with

PVP40000 and PEG8000, with optimum ratios for each model drug. There was a decrease in solubility with PEG8000 as the carrier at all ratios in nano-milling of Carvedilol, whereas with PVP40000 as the carrier, the decrease in solubility was observed at 1:10 ratio of

Glipizide and 1:1 ratio of Carvedilol with nano-milling technique. In conclusion, although nano-milling technique showed the decrease in solubility with certain drugs and polymer ratios due to agglomeration as one of the drawbacks, all the other techniques showed significance in solubility with all the model drugs at 1:1, 1:5 and 1:10 ratios with PEG8000 and PVP40000 as carriers. The enthalpy decreased for all the composites when compared to the pure drug, resulting in enhancement of solubility.

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