For Sustained Vasodilation in Pulmonary Arterial

INHALABLE FORMULATIONS OF PROSTAGLANDIN E1 (PGE1)

FOR SUSTAINED VASODILATION IN PULMONARY

ARTERIAL HYPERTENSION (PAH)

VIVEK GUPTA, B.Pharm

A DISSERTATION

PHARMACEUTICAL SCIENCES

Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center in Partial Fulfillment of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Advisory Committee

Fakhrul Ahsan (Chairperson) Young Min Kwon Xinli Liu Reza Mehvar Thomas Thekkumkara

Accepted

Thomas A. Pressley, Ph.D. Interim Dean of the Graduate School of Biomedical Sciences Texas Tech University Health Sciences Center

December 2010

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

ACKNOWLEDGEMENTS

I would like to take this opportunity to express my deepest gratitude and heartfelt thanks to my doctoral advisor Dr. Fakhrul Ahsan for his incredible mentorship, support and for being there at each and every step for me. I am totally blessed to be a part of his lab. He has been an ideal mentor for his excellent guidance, caring, patience; for providing me with an excellent research atmosphere and for allowing me to work independently. I am thankful to him for his continuous input throughout the project. It was only his perseverance that I could be able to learn the process of grant writing and write and publish manuscripts. I feel greatly indebted to Dr. Ahsan for providing me with an opportunity to work under his able guidance.

I am grateful to all my committee members, Dr. Young Min Kwon, Dr. Xinli Liu, Dr. Reza

Mehvar, and Dr. Thomas Thekkumkara for their constant guidance and input and for bringing out the best in me. I sincerely thank each one of them for all the time, suggestions and guidance they have given me for the completion of this doctoral project.

It is a pleasure to thank those who have helped me immensely in making this dissertation work possible. It is an honor for me to me to show my gratitude to our collaborator, Dr. Eva Nozik-Grayck at University of Colorado at Denver. I deeply appreciate her for helping me with establishing the critical PAH animal model in our lab.

A special mention goes to Dr. Amit Rawat, a friend and former colleague. He has been a constant source of knowledge throughout this project. He is the one who trained me in the basics of controlled and novel drug delivery systems. I would like to specially thank two of my former colleagues Dr. Shuhua Bai and Dr. Chandan Thomas for introducing me to basics of research in our lab and for their inputs during my PhD. I would also like to thank my current colleagues Nilesh Gupta, Kamrun Nahar, and Brijesh Patel, who

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 have been instrumental in helping me with some critical experiments, and also in routine lab maintenance chores.

I would like to take this opportunity to thank people who have helped me in a number of ways toward the completion of my PhD. Dr. Imam Shaik, Dr. Sunny Guin, Dr. Ridhi

Parasrampuria, Kaushik Shah, Mohd. Shahriarul Absar. Their help and guidance has been instrumental in completing this dissertation work. Further I would like to thank all the other faculty members, postdocs, technicians, graduate students, the office and research staff for all their support and help during my PhD program.

My parents; Dr. Satish Chandra Gupta and Sushma Rani Gupta, have always been my inspiration and I would like to express my deepest thanks and sincere love and gratitude towards them for all the support and love they have showered on me all these years. A very special thanks goes to my brother Vikas Kumar Gupta and sister-in-law Richa

Gupta for being guiding forces in my life, and for playing an important role in shaping me as an individual. I would also take this opportunity to acknowledge all my immediate family members, and all my friends for all their support and love.

Lastly, I offer my regards and blessings to all of those who supported me in any respect during the completion of the project.

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………………ii ABSTRACT………...... vi LIST OF TABLES……………………………………………………………………………..viii LIST OF FIGURES……………………………………………………………………………...ix LIST OF ABBREVIATIONS……………………………………………………………………xi

CHAPTERS

1. INTRODUCTION………………………………………………………………………..1

References…………………………………………………………………….23

2. FEASIBILITY STUDY OF AEROSOLIZED PROSTAGLANDIN E1 MICROSPHERES AS A NON-INVASIVE THERAPY FOR PULMONARY ARTERIAL HYPERTENSION………………………………………………………..39

Introduction…………………………………………………………………….39

Materials and Methods……………………………………………………….41

Results and Discussion………………………………………………………49

References…………………………………………………………………….61

3. PEI-MODIFIED PLGA MICROSPHERES OF PGE1 FOR NONINVASIVE TREATMENT OF PULMONARY ARTERIAL HYPERTENSION………………..73

Introduction…………………………………………………………………….73

Materials and Methods……………………………………………………….76

Results and Discussion………………………………………………………84

References…………………………………………………………………….95

4. INHALABLE PLGA MICROPARTICLES ENCAPSULATING PROSTAGLANDIN E1-HYDROXYPROPYL-β-CYCLODEXTRIN (PGE1-HPβCD) COMPLEX FOR PULMONARY ARTERIAL HYPERTENSION (PAH) TREATMENT…………...110

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Introduction…………………………………………………………………..110

Materials and Methods……………………………………………………...112

Results and Discussion……………………………………………………..122

References…………………………………………………………………...134

5. PLGA PARTICLES OF PGE1 AMELIORATES SYMPTOMS OF PAH IN A RAT MODEL FOLLOWING ACUTE AND CHRONIC ADMINISTRATION VIA THE PULMONARY ROUTE………………………………………………………………151

Introduction…………………………………………………………………..151 Materials and Methods……………………………………………………...153 Results and Discussion……………………………………………………..158 References…………………………………………………………………...163

6. CONCLUSIONS……………………………………………………………………...175

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

ABSTRACT

Pulmonary arterial hypertension (PAH) is a rare and debilitating disorder of the

pulmonary circulation that afflicts the lives of thousands Americans. The major clinical

features of PAH include increase in mean pulmonary arterial pressure (MPAP), right

ventricular enlargement, increased pulmonary vascular resistance and smooth muscle

hypertrophy in pulmonary arterioles. Several anti-PAH therapies targeting various

pathways involved in PAH progression have been approved by the FDA. However, many

of the currently available anti-PAH drugs suffer from a number of limitations including

short biological half-life, poor pulmonary selectivity and infection or hematoma due to

administration via the intravenous or subcutaneous route. Prostaglandin E1 (PGE1) is a

potent vasodilator with selectivity toward the pulmonary circulation when administered

via the pulmonary route. However, PGE1 has a very short half-life of 5-10 minutes. This dissertation investigates the feasibility of polymeric microparticles of PGE1 for administration via the non-invasive pulmonary route in order to develop a formulation that would produce long-term and selective vasodilation of pulmonary arteries. Toward this end, first, we developed poly (lactide-co-glycolide) (PLGA) microparticles to investigate the suitability of the polymeric delivery system for PGE1. Second, with the aid

of polyethyleneimine (PEI) as a porosigen, we developed large porous PLGA

microparticles of PGE1 to accomplish improved drug entrapment and enhanced deep lung deposition. In the third set of studies, we have evaluated the efficacy of hydropropyl-β-cyclodextrin (HPβCD) as a complexing agent to increase hydrophilicity of

PGE1 and increase its absorption across the respiratory epithelium. Finally, the formulations that showed the most favorable pharmacokinetic and deposition patterns were tested in a monocrotaline-induced rodent model of PAH. We used this animal model to study the efficacy of the formulations in reducing pulmonary arterial pressure

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

and ameliorating pathological conditions associated with PAH following acute and

chronic administration of the formulations. Results from these studies suggest that PGE1 can be encapsulated in PLGA microparticles to modify its systemic availability and extend its half-life. The incorporation of PEI in microparticulate formulations resulted in porous microparticles that showed excellent drug entrapment and favorable aerodynamic properties for efficient deposition in the lungs. Further, PGE1-HPβCD encapsulated microparticles showed sustained release of PGE1 and remarkable increase in relative bioavailability. Hemodynamic studies in PAH-rats demonstrate that the optimized formulations were efficacious in providing a continual reduction in pulmonary arterial pressure and improvement in the morphometry of pulmonary vasculature. Overall, this study is first to show that PLGA-based particles of PGE1 is a feasible inhalable formulation that produces selective and prolonged vasodilation of pulmonary arteries and ameliorates morphological changes of the pulmonary vasculature associated with PAH.

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

LIST OF TABLES

Table 1.1. Updated clinical classification of pulmonary hypertension (Dana Point, CA, 2008)……………………………………………………………………………….30

Table 1.2. Advantages and limitations of prostacyclin analogues for PAH treatment….31

Table 2.1. Composition and polydispersity indices (PDIs) of the microparticulate formulations………………………………………………………………………..65

Table 2.2. Pharmacokinetic parameters of PGE1-loaded PLGA microspheric formulations………………………………………………………………………..66

Table 3.1. Compositions and polydispersity indices (PDIs) of different large porous microspheric formulations……………………………………………………….100

Table 3.2. Compositions of actual lung fluids and simulated lung fluids………………..101

Table 3.3. Pharmacokinetic parameters of PGE1-loaded large porous PLGA microparticles…………………………………………………………………….102

Table 4.1. Compositions of various microparticulate formulations………………………139

Table 4.2. Physical characteristics of PGE1-HPβCD complex encapsulated microparticles…………………………………………………………………….140

Table 4.3. Composition of simulated interstitial lung fluid (SILF) (Moss formula)……..141

Table 4.4. Pharmacokinetic parameters of PGE1-HPβCD complex encapsulated PLGA microspheric formulations……………………………………………………….142

Table 5.1. In-vitro and In-vivo characterization of PGE1 encapsulated microparticulate formulations………………………………………………………………………165

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

LIST OF FIGURES

Fig. 1.1. Maintenance of vascular tone in pulmonary circulation………………………….32

Fig. 1.2. Pathophysiology of pulmonary arterial hypertension (PAH)…………………….33

Fig. 1.3. Cellular mechanisms of PAH progression………………………………………...34

Fig. 1.4. Treatment strategies for PAH………………………………………………………35

Fig. 1.5. Prostaglandin E1 (PGE1) and its biosynthesis…………………………………….36

Fig. 1.6. Non-invasive delivery of anti-PAH medications…………………………………..37

Fig. 1.7. Polyethyleneimine, hydroxypropyl-β-cyclodextrin, and PLGA…………………..38

Fig. 2.1. SEM images of PLGA microparticles……………………………………………...67

Fig. 2.2. Physical characterization of microparticulate formulations……………………...68

Fig. 2.3. In-vitro release studies………………………………………………………………69

Fig. 2.4. In-vivo absorption studies…………………………………………………………..70

Fig. 2.5. Metabolic degradation studies……………………………………………………..71

Fig. 2.6. In-vitro and In-vivo safety studies………………………………………………….72

Fig. 3.1. SEM images of PEI modified large porous PLGA microparticles……………..103

Fig. 3.2. Physical characterization of PLGA microparticles………………………………104

Fig. 3.3. Physiosorption-based surface area characteristics…………………………….105

Fig. 3.4. Entrapment efficiency and In-vitro release pattern……………………………..106

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Fig. 3.5. In-vivo absorption profile…………………………………………………………..107

Fig. 3.6. Metabolic degradation studies of large porous microspheres…………………108

Fig. 3.7. In-vivo and In-vivo toxicity studies………………………………………………..109

Fig. 4.1. SEM images of PGE1-HPβCD encapsulated PLGA microparticles…………..143

Fig. 4.2. Physical characterization of PLGA microparticles………………………………144

Fig. 4.3. Physiosorption-based surface area characteristics…………………………….145

Fig. 4.4. In-vitro release profile of PGE1-HPβCD encapsulated PLGA microparticles……………………………………………………………………….146

Fig. 4.5. In-vivo absorption profile of PGE1-HPβCD encapsulated PLGA

microparticles……………………………………………………………………….147

Fig. 4.6. Metabolic degradation studies for PGE1-HPβCD encapsulated PLGA microparticles……………………………………………………………………….148

Fig. 4.7. In-vivo toxicity studies in Bronchoalveolar lavage studies……………………..149

Fig. 4.8. In-vitro toxicity studies in Calu-3 cell lines……………………………………….150

Fig. 5.1. % decrease in MPAP and MSAP following single dose administration of plain

PGE1………………………………………………………………………………...166

Fig. 5.2. % decrease in MPAP and MSAP following administration of PLGA microparticles……………………………………………………………………….168

Fig. 5.3. Duration of vasodilatory effects of plain and microparticle encapsulated

PGE1…………………………………………………………………………………170

Fig. 5.4. Effect of chronic treatment on MPAP and MSAP……………………………….171

Fig. 5.5. Determination of right ventricular hypertrophy after 10 days chronic

treatment…………………………………………………………………………....173

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Fig. 5.6: Determination of MMP-2 and MMP-9 by gelatin zymography……………….174

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

LIST OF ABBREVIATIONS

ACI: Anderson Cascade Impactor

AI: Apoptotic Index

ALP: Alkaline Phosphatase

ANOVA: Analysis of Variance

ATP: Adenosine Tri Phosphate

AUC: Area under the Curve

BAL: Bronchoalveolar Lavage

BET: Brunauer-Emmett-Teller

BJH: Barrett-Joyner-Halenda

BMP: Bone Morphogenetic Protein

BMPR2: Bone Morphogenetic Protein Receptor Type II cAMP: Cyclic Adenosine Monophosphate

COX: Cyclooxygenase

DCM: Dichloromethane

DGLA: Dihomo-γ-Linolenic Acid

EAP: External Aqueous Phase

ECM: Extracellular Matrix

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

ELISA: Enzyme Linked Immuno-Sorbent Assay eNOS: Endothelial Nitric Oxide Synthase

ET-1: Endothelin-1

FPAH: Familial Pulmonary Arterial Hypertension

FPF: Fine Particle Fraction

HPβCD: Hydroxypropyl-β-cyclodextrin

IAP: Internal Aqueous Phase

IPAH: Idiopathic Pulmonary Arterial Hypertension

LDH: Lactate Dehydrogenase

LMWH: Low Molecular Weight Heparins

LPS: Lipopolysaccharide

LV+S: Left Ventricle + Septum

MCT: Monocrotaline

MMADa: Actual Mass Median Aerodynamic Diameter

MMADt: Theoretical Mass Median Aerodynamic Diameter

MMP: Matrix metalloproteinase

MPAP: Mean Pulmonary Arterial Pressure

MSAP: Mean Systemic Arterial Pressure

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

MTT: [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]

OP: Organic Phase

PAH: Pulmonary Arterial Hypertension

PCNA: Proliferating Cell Nuclear Antigen

PCWP: Pulmonary Capillary Wedge Pressure

PDE: Phosphodiestrase

PDGF: Platelet Derived Growth Factor

PDI: Polydispersity Index

PEI: Polyethyleneimine

PGE1: Prostaglandin E1

PGI2: Prostacyclin I2

PH: Pulmonary Hypertension

PI: Proliferative Index

PLA: Poly Lactic acid

PLGA: Poly (lactide-co-glycolide)

PVA: Polyvinyl Alcohol

PVR: Pulmonary Vascular Resistance

RV: Right Ventricle

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

RVH: Right Ventricular Hypertrophy

RVSP: Right Ventricular Systemic Pressure

SEM: Scanning Electron Microscopy

SDS: Sodium Dodecyl Sulfate

SILF: Simulated Interstitial Lung Fluid

TGF: Transforming Growth Factor

TXA2: Thromboxane A2

WHO: World Health Organization

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

CHAPTER 1

INTRODUCTION

Cardiopulmonary disorders are one of the major genres of diseases that adversely affect

the functionality of both heart and lungs. A World Health Organization report shows that

cardiopulmonary diseases, including coronary heart disease, atherosclerosis, bacterial

endocarditis, cardiomyopathy and pulmonary arterial hypertension are among the four

leading causes of morbidity worldwide. Of these diseases, pulmonary arterial

hypertension (PAH) has attracted a lot of attention because of its complex and relatively

unknown etiology and pathophysiology. It is a very rare disease affecting approximately

50,000 – 100,000 people in the United States. PAH affects about 1-2 persons in a million

population that translates to 300-500 new cases each year in the United States (Rubin,

1993). Although it affects a small number of people, PAH is a debilitating and chronic

disorder of the pulmonary circulation affecting people of all ages and ethnic

backgrounds. The Centers for Disease Control and Prevention (CDC) data suggest that

there were 260,000 hospital visits from PAH related complications and 15,668 patients

died of PAH in 2002 in the United States. Various published reports suggest women

being 2-3 times more susceptible to PAH than men. CDC also reports that 807,000

patients were hospitalized due to PAH between 2000 and 2002; of those 61% were

women and 66% were 65 years or older (Hyduk et al., 2005). Survival among the PAH

patients has been a major concern. Prior to 1995, the mean survival time for a PAH

diagnosed patient was 2.8 years in adults and less than a year in children (Traiger,

2007). Because of the approval of newer drug therapy, there has been a significant

improvement in the mean survival time and quality of life among PAH patients. However,

Modified from Gupta and Ahsan (2010), Critical ReviewsTM in Therapeutic Drug Carrier Systems (In press).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 there is still no cure for this disease and more importantly, currently used therapy suffers from a variety of disadvantages.

History and Classification of Pulmonary arterial hypertension (PAH):

Pulmonary arterial hypertension (PAH) was first identified by a German physician, Ernst

Van Rohmberg in 1891 as “Pulmonary Vascular Sclerosis” but due to inability to directly measure pulmonary arterial pressures, it was not until 1951 that PAH was first reported in the literature. However, the development of drug therapy for PAH and progress toward long-term management of the disease have been relatively slow, perhaps because the disease affects only a small number of persons compared to other cardiovascular disorders. The National Institutes of Health Registry defines PAH as an elevated mean pulmonary arterial pressure (MPAP) of more than 25 mm Hg at rest, or 30 mm Hg after exercise (compared to normal MPAP of 12-16 mm Hg) (Rich et al., 1987). Increased

MPAP, the principal manifestation of PAH, can be correlated to several other complications, including vascular wall remodeling, increased pulmonary vascular resistance, smooth muscle hypertrophy, intimal hyperplasia, in situ thrombus formation,

and right ventricular hypertrophy (RVH), as all of them contribute to narrowing of the

lumen of the small pulmonary arteries.

The symptoms of PAH are rather nonspecific—dyspnea, fatigue, nonproductive cough,

syncope, angina pectoris, peripheral edema, and hemoptysis—which is one of the main

reasons that the disease can remain undiagnosed for many years. Furthermore,

progression of PAH is usually slow; the symptoms take 2-3 years to develop. Thus,

patients may delay seeking medical attention, which further complicates management of

the disease. Moreover, such delay also contributes to the high mortality of PAH patients,

due to right ventricular failure, due to right ventricular enlargement.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Primary classification of pulmonary hypertension (PH) as a disease was first done by

The World Health Organization (WHO) in 1973. In the WHO classification, the term

‘PAH’ includes all types of PH caused by diseases proximal to pulmonary capillaries.

Later, in 1998, at the Second World Conference on Pulmonary Hypertension held in

Evian, France, the classification was modified based on pathophysiology, clinical presentation, and therapeutic management. The classification was then further modified at the Third World Conference on Pulmonary Hypertension held in Venice, Italy in 2003.

The Venice conference divided PH into 5 classes: Group 1 PH, known as pulmonary arterial hypertension (PAH), is the largest of all the groups and comprises several subgroups: (i) idiopathic PAH (IPAH), which includes primary PAH without any known etiology; (ii) familial PAH (FPAH), which clusters within a family; (iii) associated with PAH

(APAH), which is associated with many other disease conditions; (iv) PAH associated with significant venous or capillary involvement; and (v) persistent PAH of the newborn.

Group 2 PH is known as pulmonary venous hypertension (PVH), with disorders in the left side of the heart (atrial or ventricular, and/or valvular heart disease). Group 3 PH comprises hypoxia associated PH and is correlated to various lung diseases and/or hypoxemia. Group 4 PH comprises pulmonary hypertensive disorders due to chronic thromboembolic disease. Finally, Group 5 includes miscellaneous disorders that are very rarely associated with PH such as histiocytosis X, fibrosing mediastinitis, and sarcoidosis

(Simonneau et al., 2004).

At the Fourth World Congress on Pulmonary Hypertension in 2008 (Dana Point,

California), the Evian-Venice classification was again modified to include information published over the previous 5 years and also to clarify ambiguity in the Venice classification (Simonneau et al., 2009). Table 1.1 shows the Dana Point classification and the recent changes included in the Evian-Venice classification.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Pathophysiology of PAH and Associated Cellular Changes

The pathogenesis of PAH can be attributed to several factors, including exposure to toxins, inflammatory mediators and genetic predisposition. The mediators that regulate vasodilation and proliferation in the pulmonary circulation include prostacyclins, nitric oxide, adenosine, and endothelium-derived hyperpolarizing factors, whereas the vasoconstricting factors are thromboxane A2, endothelin-1, hypoxia, serotonin and interleukin. Imbalance among these mediators of vascular tone causes constriction of pulmonary vascular smooth muscle (Fig. 1.1). Due to intermingling of all the vasoactive mediators, it is suggested that pulmonary vasoconstriction can both be a cause or a secondary response to PAH development. In addition to disturbed vascular tone, pulmonary endothelial cell dysfunction also promotes the triad of vasoconstriction, thrombosis, and cellular proliferation.

One of the characteristic changes that occurs in the pulmonary vasculature of PAH patients is remodeling of blood vessels that are <500 μm in diameter. The blood vessels of the pulmonary vasculature are thinner than those of the systemic circulation, and the remodeling occurs in all layers of cells of the vascular wall and includes hypertrophy, hyperplasia and reduction in the number of small vessels. In addition to the above changes, remodeling also stimulates deposition of connective tissue matrix substances such as fibronectin, collagen, and elastin and thus causes narrowing of the arteries. The possible causes of remodeling include higher MPAP, hypoxia, viral infection, lack of apoptosis, endothelial cell injury, and lack of anti-proliferative factors. The development of in situ thrombi in small pulmonary arteries due to thrombin deposition in the lumen of arteries is another important characteristic of PAH pathogenesis. Thrombi develop because of the pro-coagulative environment created by interactions between platelets

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 and growth factors. In situ thromboses may also contribute to the narrowing of the lumen of the pulmonary vessel.

In more advanced stages of the disease, occlusive changes in the pulmonary arteries and development of plexiform lesions can also occur because of progressive intimal hyperplasia and enhanced distal arterial muscularization. These events lead to development of aberrant channels in the lumen and adventitia of the vascular wall due to proliferation of apoptosis-resistant endothelial cells. Additional features of PAH include venous hypertrophy, thickening of pulmonary adventitia, macrophage infiltration, and increased expression of matrix proteins and transforming growth factor (TGF)-β. Overall, the common features of PAH include intimal fibrosis, increased medial thickness, vascular remodeling, pulmonary arteriolar occlusion, and plexiform lesions that are summarized in Fig. 1.2 (Rabinovitch, 2008).

Although PAH is characterized by increased PAP and pulmonary vascular resistance

(PVR), as well as decreased cardiac output, the left-sided pressures are reported to be normal, as represented by pulmonary capillary wedge pressure (PCWP) of ≤15 mm Hg

(Gaine and Rubin, 1998). As the disease progresses, there is an increased degree of vasculopathy, vascular remodeling, smooth muscle hypertrophy, hyperplasia, and in situ thrombus formation. All these pathological changes can cause narrowing of the pulmonary arteries and increased right ventricular afterload that eventually leads to

RVH, dilatation and subsequent RV failure and death.

In addition to the above mentioned stimulants, there are several reports suggesting involvement of some genetic factors as well in the PAH pathogenesis. Familial PAH

(FPAH) has been reported to be an autosomal dominant disease. A gene associated with PAH was localized to a 3-cM region of chromosome 2q31-33 (Morse et al., 1997).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Furthermore, mutations associated with the development of FPAH were determined to affect the gene encoding bone morphogenetic protein receptor II (BMPR II) (Deng et al.,

2000). To date, >46 mutations have been reported in the BMPR II gene, 60% of which may be responsible for the development of PAH. Bone morphogenetic protein (BMP) is a member of the TGF-β superfamily and is synthesized by both endothelial and smooth muscle cells. It is responsible for inhibiting smooth muscle proliferation and inducing apoptosis. Mutation of BMPR II may disrupt the receptor’s kinase activity, thus blocking the signaling pathway and resulting in excessive cell proliferation and vascular structural remodeling. About 10-25% cases of IPAH and >50% of cases of FPAH have been identified to have mutations in BMPR II (Newman et al., 2004).

As discussed earlier, small pulmonary arterial remodeling is the main pathological feature of PAH. In PAH-afflicted patients, all three layers of the pulmonary vascular wall—inner, media, and outer—undergo profound structural changes. Below is a brief discussion of the changes that may occur in cells of the pulmonary vascular wall during

PAH development.

Endothelial Cells: Under normal physiological conditions the vascular endothelium is responsible for maintaining the structure of blood vessels and the integrity of smooth muscle cells by secreting various vasoactive substances. However, when endothelial cells are damaged by hypoxia, inflammation, shear stress or toxins, the link between smooth muscle cells and the barrier function of vascular endothelial cells becomes disrupted, which can lead to vascular injury, vasoconstriction and in situ thrombosis. As a result, smooth muscle cells proliferate and cause remodeling of the pulmonary vasculature. It has been reported that 90% of endothelial cells in PAH-associated lesions do not express TGF-β2 receptors, suggesting involvement of tumor-inhibiting genes in

PAH pathogenesis (Yeager et al., 2001).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Smooth Muscle Cells: The smooth muscle cells in the pulmonary vessels are normally in a state of relaxation because of the presence of an opposing cascade of pulmonary vasodilators and vasoconstrictors. In PAH patients, smooth muscle cells proliferate and enlarge; thus the normally static tunica media layer of the pulmonary vasculature becomes hypertrophic. The intermediate cells and pericytes also begin to differentiate into new smooth muscle cells. Non-muscular and partially muscular arteries start to muscularize and new muscular arteries develop. This phenomenon has been confirmed by reports of increased proliferative index (PI), apoptotic index (AI), and PI:AI ratio, which is an indicator of imbalance between hyperplasia and apoptosis contributing to pulmonary vascular remodeling. Also, various vasoactive substances secreted by smooth muscle cells may participate in pulmonary vascular remodeling and PAH progression.

Fibroblasts: Fibroblasts are present in the outer layer of blood vessels. Fibroblast proliferation, connective tissue deposition, and changes in extracellular matrix (ECM) can also contribute to pulmonary vascular remodeling. Up-regulation of collagen I and II and procollagen I and III mRNAs has been reported in the pulmonary arteries of PAH- induced rats. Furthermore, elevated levels of other ECM components, including elastin, fibronectin, collagen degradation regulatory enzymes, and metalloproteinase-I, have also been observed in the pulmonary arteries of these same rats (Junbao et al., 2005).

Fig. 1.3 represents schematic representation of various cellular changes that occur during PAH pathogenesis.

Treatment Strategies using Prostaglandins and Prostacyclin Analogs

As discussed above, PAH has a multifaceted pathophysiology and there is no single factor that can be knocked down to stop the progression of the disease. The major

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 clinical manifestation of PAH is elevated MPAP, which is attributed to vasoconstriction in distal pulmonary arteries due to remodeling of the vessel wall in pulmonary circulation, and increased right ventricular afterload (Humbert et al., 2004). Various signaling pathways are down regulated in PAH progression including: (i) imbalance between prostacyclin and thromboxane A2 (TXA2) signaling which results in decreased production of prostacyclin due to reduced prostacyclin synthase activity and increased secretion of TXA2, (ii) increased expression of potent vasoconstrictors such as endothelin-1 (ET-1) (Giaid et al., 1993) and various growth factors such as platelet derived growth factor (PDGF), iii) reduced expression of endothelial nitric oxide synthase

(eNOS) and increased production of cyclic nucleotide phosphodiestrase, mainly PDE5,

(Schermuly et al., 2005; Wharton et al., 2005) in the pulmonary circulation and thus disrupts nitric oxide mediated cGMP signaling cascade (Fig. 1.4). In this dissertation, we focus on targeting the prostacyclin pathway to develop an inhalable and long acting formulation for PAH treatment.

Prostacyclins are members of eicosanoid family, a group of 20 essential fatty acids, derived from arachidonic acid and have a variety of physiological effects including vasodilatory, anti-proliferative, anti-aggregation effects. Prostacyclins exert their biological activity through cAMP dependent activation of protein kinase A by acting on various prostacyclin receptors, especially IP and EP2, G protein-coupled receptors, via stimulation of adenylate cyclase (Badesch et al., 2004). Prostacyclins also inhibit vascular smooth muscle cells proliferation and platelet aggregation.

Prostaglandin I2 (PGI2) and prostaglandin E1 (PGE1) are two naturally occurring prostanoids that are synthesized by the endothelial cells in the pulmonary circulation via arachidonic acid metabolism in the presence of a constitutive enzyme, prostacyclin synthase (Kerins et al., 1991). Briefly, activation of prostaglandin pathway results in

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

decreased intracellular calcium and thus muscle relaxation and vasodilation. Both PGI2 and PGE1, two of the most potent pulmonary vasodilators ever known, work locally in the pulmonary circulation causing smooth muscle relaxation and inhibition of platelet aggregation., A major reduction in prostacyclin synthesis, prostacyclin synthase activity, and physiological levels of prostacyclin is reported in lungs and pulmonary arteries of

PAH patients (Tuder et al., 1999; Chan and Loscalzo, 2008). There have also been reports indicating decreased expression of prostacyclin receptors in the lungs of PAH patients (Hoshikawa et al., 2001). However this finding has not been validated in several studies. Several prostacyclin analogues have been investigated and are currently approved for treatment of PAH, which include epoprostenol, treprostinil, and iloprost.

® Epoprostenol (Flolan ), a short-acting PGI2 analogue, was the first drug to be approved

by US Food and Drug Administration (FDA) for PAH treatment via intravenous infusion.

Epoprostenol has shown improved hemodynamic function (including reduced MPAP,

reduced PVR, and improvement in 6 minute walk test), exercise capacity, and survival in

patients in both short term and long term treatment (Barst et al., 1996). However, the

acceptability of this drug is hampered by problems and adverse effects associated with

unfavorable drug characteristics and complicated delivery systems. Therefore, a more

stable long-acting prostacyclin analogue can address some of these problems and

improve the prospects of long-term pulmonary vasodilator therapy.

® Iloprost (Ventavis ) is a stable PGI2 analogue, which was approved by FDA for PAH

treatment via pulmonary delivery of aerosol solution. Various clinical studies have

demonstrated both preventive and protective effects of iloprost in PAH patients

(Higenbottam et al., 1998; Ewert et al., 2007). Treprostinil (remodulin®) is another long- acting, stable and FDA approved PGI2 analogue, with a duration of action up to four

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

hours, is available as a subcutaneous infusion. The safety and efficacy of treprostinil has

been demonstrated in various clinical trials (Simonneau et al., 2002; Barst et al., 2006).

On the whole, the prostacyclin pathway has long been investigated to develop an

efficacious treatment for PAH, which was indeed a significant improvement in anti-PAH

drug therapy. However, there are several limitations associated with the use of currently

approved treatment options, which hinder their widespread acceptance among PAH

patients. Various FDA approved prostacyclin analogues and the limitations associated

with them are summarized in Table 1.2.

Side-effects and Limitations of Currently Approved PAH Therapies:

Despite the fact that PAH treatments, currently approved by FDA, are capable of

alleviating the symptoms, and thus improving the quality of life for PAH patients, there

are several limitations and side-effects associated with their continuous usage. One of

the very important limitation of the anti-PAH medications is very short biological half-life

of the commercially available first-line therapeutic agents (especially prostacyclin

analogs) (Table 1.2), thus making it indispensable to be administered via intravenous or

subcutaneous infusion. There are several limitations associated with the delivery

systems used for continuous infusion as well, which include pain at the site of injection,

risk of infection and thrombosis due to use of catheters, and risk of cardiovascular

collapse due to infusion pump malfunction; which lead to noncompliance with the

therapy. Furthermore, both the method of catheterization and need to carry the

cumbersome pump everywhere eventually lead to noncompliance with the therapy.

Moreover, long-term safety and efficacy studies revealed that systemic administration of

prostacyclin analogues is associated with a wide range of complications, including

systemic hypotension, reduction in right coronary blood flow, deterioration of right

ventricular performance, and increase in shunt function with worsening oxygenation.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

To bypass these limitations associated with usage of continuous infusion pumps, an

inhalable solution of iloprost has recently been approved by FDA (Ventavis®), which

makes it easy for the patients to take the prescribed dosage. However, due to short half-

life (20-30 minutes) of iloprost, it becomes mandatory to take 9-12 inhalations every day

to keep the drug levels in the therapeutic window (Gupta and Ahsan, 2010).

Talking about the physiological side-effects of currently approved PAH therapeutics; the

risk of systemic side-effects associated with pulmonary vasodilation is the biggest threat

to the clinical community in prescribing long-term therapeutic regimen. Systemic side-

effects of anti-PAH therapies include systemic hypotension, reduction in right coronary

blood flow, increase in shunt function, reduced oxygenation, and deterioration of right

ventricular performance.

Since PAH is a disease of small pulmonary arteries but not of the systemic circulation,

currently approved drugs are not intended to target the pulmonary circulation specifically

or affected pulmonary arteries. All currently FDA approved therapies elicit their effects in

the entire circulatory system when administered parenterally in addition to the entire

pulmonary vasculature. In addition, when administered via the noninvasive pulmonary

route, the effects are evident on the entire pulmonary vasculature along withthe systemic

circulation to some extent.

To overcome the above described limitations, there is an urgent need to develop a drug

delivery system that is (i) be capable of delivering the medications over a prolonged

period of time so as to provide sustained pulmonary vasodilation and also (ii) direct the

drug to the diseased area of the lungs and thereby bypass systemic side-effects.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Prostaglandin E1 (PGE1) – A Selective Pulmonary Vasodilator and Molecule of

Interest for PAH Treatment

Prostaglandin E1 (alprostadil, PGE1) is a 20-carbon fatty acid with a cyclopentane ring and a single trans-13 double bond (Fig. 1.5A). PGE1, a naturally occurring eicosanoid, is

synthesized from 20-carbon polyunsaturated fatty acid dihomo-gamma-linolenic acid

(DGLA; 20:3, ω-6) via cyclooxygenase-1 and -2 (COX-1 and COX-2) pathways (Raz et

al., 1977; Fan and Chapkin, 1998) (Fig. 1.5B). PGE1 is a member of series-1

prostaglandin class and produce a number of physiological effects. It is primarily known

for possessing anti-inflammatory and vasodilatory effects. It is also reported to inhibit

platelet aggregation, vascular smooth muscle proliferation and collagenase activity.

(Kloeze, 1969; Zurier and Quagliata, 1971; Salvatori et al., 1992; Levin et al., 2002). Due

to its vasodilating and smooth muscle relaxing properties, PGE1 has been approved by the FDA for the treatment of erectile dysfunction (Caverject®, Muse®; trabecular muscles) and patent ductus arteriosus (Prostin VR® Pediatric). In pulmonary circulation,

PGE1 is synthesized primarily by the cells of the vascular endothelium and smooth muscle cells.

Following intravenous administration, PGE1 is rapidly distributed and metabolized with an estimated half-life of 5-10 minutes. It has a low pKa value of 6.3 (Cox et al., 1988; van Heerden et al., 2000). Lungs serve as the primary site for metabolism of PGE1 and

can metabolize as much as 80% of systemically absorbed PGE1 in a single pass

(Moncada and Higgs, 1988). Metabolism occurs in pulmonary vascular bed and is an oxygen-dependent process (Stone et al., 2006). Majority of the metabolites of PGE1 are

unstable and do not exert any biological activity except 13,14-dihydro PGE1, which is relatively stable in blood and is responsible for retaining anti-aggregatory activity of

PGE1 (Ney et al., 1991). Oxidation of PGE1 at trans-13 double bond is catalyzed by

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

+ NAD dependent 15-hydroxy prostaglandin dehydrogenase. Other metabolites of PGE1 include 15-keto-PGE1 and 15-keto-dihydro-PGE1 (Nakano et al., 1973). Metabolites of

PGE1 are primarily excreted via the kidneys and the excretion is complete within 24 hours. There are no evidences of tissue retention and of excretion of unchanged PGE1 in the urine.

Prostaglandins including PGE1 are normally paracrine lipidic mediators and interact with a distinct family of G-protein coupled prostanoids receptors that are composed of 8 subtypes (EP1-4, IP, FP, TP and DP) (Wright et al., 2001). Of these 8 subtypes, EP2,

EP4, DP, and IP induce smooth muscle relaxation and vasodilatory effects (Narumiya et al., 1999). It interacts specifically with EP2 and thus activates G-protein Gαs, responsible for cAMP-mediated downstream signaling (Regan et al., 1994). As discussed above, PGE1 exerts its pharmacological effects by cAMP-mediated downstream signaling. In pulmonary vascular smooth muscle cells, PGE1 stimulates

adenylate cyclase, which later converts adenosine triphosphate (ATP) to cyclic

adenosine monophosphate (cAMP), resulting in protein kinase mediated cAMP-induced

decrease in intracellular calcium leading to smooth muscle relaxation and vasodilation

(Badesch et al., 2004). This mechanism is different from cardiac striated muscles, where

the troponin complex is responsible for regulation of muscle contractions.

PGE1 has also considered as a potential treatment option for PAH because of its high pulmonary clearance (70-90%) and pulmonary-selective vasodilating effect (Meyer et al.,

1998). Intravenous PGE1 has been used in newborns with PAH (Kunimoto et al., 1997;

Shen et al., 2005). However, intravenous administration of PGE1 results in side-effects,

such as systemic hypotension and low cardiac output, similar to those produced by other

short-acting prostacyclin analogues (Meyer et al., 1998). An attractive approach to utilize

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the pulmonary vasodilatory effects of PGE1 would be to deliver the drug via the

respiratory route.

However, there are little or no studies concerning potential use of PGE1 as an anti-PAH medication. To our knowledge, aerosolized PGE1 was first studied by a Japanese group

in 1998. It was shown to be effective in the treatment of PH and hypoxia in oleic acid-

induced lung injury in Japanese white rabbits. The authors reported that aerosolized

PGE1 along with partial liquid ventilation improved gas exchange, relaxed the pulmonary

circulation, and reduced PVR without causing systemic hypotension or reduction in

cardiac output (Nakazawa et al., 1998). Sood et al. also reported a Phase I/II clinical trial

of inhaled PGE1 for the treatment of hypoxemic respiratory failure and neonatal PAH

associated with respiratory failure. In this study, a significant improvement in

oxygenation was observed following 3 hours treatment with aerosolized PGE1 at a dose

ranging from 100-300 ng/kg/min in 20 newborn babies with hypoxemic respiratory

failure, thus establishing the short-term safety and efficacy of inhaled PGE1 in neonates

with hypoxemic respiratory failure (Sood et al., 2004). More recently, Della Rocca et al.

documented the efficacy of inhaled PGE1 in improving pulmonary hemodynamics and

oxygenation in a clinical trial enrolling 18 patients undergoing lung transplantation. The

study reported that PGE1 administered at a low dose produced a reduction in PAP and improvement in oxygenation without impairing systemic hemodynamics (Della Rocca et al., 2008). Thus, there is compelling evidence that PGE1 is a pulmonary-selective vasodilator and can be used as an inhalable alternative to the current therapy for PAH.

However, there are no studies that attempted to develop a long acting inhaled formulations of PGE1.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Non-Invasive Inhalable Delivery of Prostacyclin Analogues and Local Targeting to

the Pulmonary Circulation

One of the major limitations of investigational and commercially available anti-PAH

prostacyclin analogues is that they are required to be administered via I.V. and S.C.

injection, or via multiple inhalations for Ventavis®. Although these agents are intended to

produce vasodilation in the pulmonary vasculature, parenteral formulations produce both

pulmonary and systemic vasodilation. Because of these limitations of injectable

prostacyclin analogues, several noninvasive anti-PAH drugs have been developed. An

orally active prostacyclin analogue, beraprost sodium, is now available in the market. But

this drug fails to provide hemodynamic effects past 6 months (Barst et al., 2003). This

has propelled the development of drug-delivery systems that can provide selective

pulmonary vasodilation and overcome side-effects associated with the use of infusion

pumps. One of the approaches that would address many of the complications

associated with systemically administered anti-PAH drugs, is to deliver inhalable

formulations directly to the lungs.

For many years, macromolecular and small-molecular-weight drugs have been

administered as aerosolized formulations to the lungs to achieve both local and systemic

effects. The lungs offer several advantages over other routes of administration, including

(i) a large surface area (about 140 m2) available for drug absorption, (ii) high blood flow that bypasses the clearance mechanisms present in the liver, (iii) thin epithelial surface

(0.5 to 1.0 μm) for better absorption than any other mucosal route of administration and importantly (iv) accessibility for self-administration of the therapeutic agents (Labiris and

Dolovich, 2003; Patton et al., 2004; Scheuch et al., 2006). Moreover, lungs have a lower drug metabolizing and efflux transporter activity than the gut or liver, thus drugs remain in intact form in the lungs for a longer period of time (Keith et al., 1987; Tronde et al.,

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

2003). These are the main factors that contribute to the enhanced bioavailability of drugs

administered via the pulmonary route.

In the case of PAH, an ideal delivery system should provide selective pulmonary

vasodilation over a long period of time. As currently there are no medications that

provide pulmonary-selective vasodilation (i.e. medications affecting the hemodynamics

of pulmonary circulation only), attempts have been made to deliver commercially

available agents via the pulmonary route so as to target the pulmonary circulation only.

The advantages that inhaled anti-PAH drug can offer include (i) targeted delivery of the

medications to the diseased pulmonary circulation, (ii) avoidance of cumbersome

I.V./S.C. infusion, (iii) minimal systemic side-effects, and (iv) avoidance of right-to-left

shunt blood flow (Gomberg-Maitland and Olschewski, 2008). The pulmonary route

promotes drug deposition and activity in well-ventilated areas, thus minimizing

ventilation-perfusion mismatch. Because of the close proximity of the airways to the

small pulmonary arteries, anti-PAH medications administered via the pulmonary route

produce localized vasodilation in the pulmonary arteries. In fact, it has been shown that

prostacyclin analogues act directly on the pulmonary arterial wall from the adventitial

side but not upon recirculation of the drugs from pulmonary or bronchial arteries

(Olschewski et al., 2003). The inhalable route of administration for prostacyclin

analogues is shown in Fig. 1.6 (Gomberg-Maitland and Olschewski, 2008).

Two of the currently FDA-approved prostacyclin analogues have been studied for direct

delivery to the lungs. One of them, inhaled iloprost, has already been approved by the

FDA for treatment of NYHA class III and IV [Based on Functional classification of PAH

by New York Heart Association (NYHA)/World Health Organization (WHO)] patients

under the brand name Ventavis® (Olschewski et al., 2002). Results of a randomized

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

placebo controlled Phase-III clinical trial (TRIUMPH-1) for long-term safety and efficacy

of inhaled treprostinil have recently been published (McLaughlin et al., 2010).

Despite being a promising pulmonary vasodilator, the shortcomings and limitations

associated with PGE1 usage have constrained its potential as an inhalable anti-PAH medication. No or very little research has been done to investigate the feasibility of PGE1 as a long-term PAH treatment via the pulmonary route. Moreover, being more like an orphan drug for PAH treatment, PGE1 has tremendous potential for investigations

looking into development of long acting inhalable drug delivery systems for sustained

pulmonary vasodilation in PAH patients. The major focus of this dissertation is to

develop long acting inhalable drug delivery systems for PGE1, and to investigate their efficacy in PAH treatment via the respiratory route.

Despite being a promising and patient-compliant route for delivering anti-PAH medications, respiratory route has often been neglected as an alternative approach for

PAH treatment, which may be due to (i) lack of pulmonary specificity for any of the currently marketed molecules, (ii) difficulty in designing of dosage forms due to hydrophobic nature and chemical instability, and (iii) lack of an ideal delivery system for exploiting the inhalable route for PAH treatment. In this dissertation, we propose to overcome the above mentioned problems by developing inhalable polymeric poly (lactic- co-glycolic) acid (PLGA) (Fig. 1.7C)microspheres of PGE1 for sustained pulmonary

vasodilation. Moreover, effects of various absorption enhancers including

polyethyleneimine (PEI) (Fig. 1.7A), and hydroxypropyl-β-cyclodextrin (HPβCD) (Fig.

1.7B) will also be studied.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Polyethyleneimine (PEI):

Polyethyleneimine (PEI) is a cationic polymer (Fig. 1.7A) which has been extensively

evaluated for its ability as a non-viral gene delivery vehicle, and also as an absorption

enhancer in pharmaceutical formulations. PEI is a highly branched copolymer of

ethylene imine, having protonable amino nitrogen at every third carbon of the polymeric

backbone, thus making it a highly positively charged copolymer (Boussif et al., 1995;

Kircheis et al., 2001). PEI, due to high positive charge, can interact with negatively

charged cell membrane and thus promotes the transmucosal absorption of therapeutic

agents. PEI has been used at very low concentrations to facilitate drug absorption of

various negatively charged molecules such as low molecular weight heparins (LMWH)

following pulmonary administration (Yang et al., 2006). However, there are no data

available that shows the efficacy of PEI in enhancing absorption of negatively charged

small molecular weight molecules via inhalable route.

In this dissertation project, PEI is used as an absorption enhancer to facilitate the

absorption of PGE1, a negatively charged small molecule, when administered via the

pulmonary route. Moreover, PGE1 being a negatively charged molecule may form an

electrostatic complex with PEI thus neutralizing drug’s surface charge and enhancing

absorption via the mucosal routes. Complexation with PEI may also result in charge

dependent increase in biological half-life of PGE1.

Respirable Particulate Carriers for Pulmonary Delivery of PGE1 for PAH Treatment

Inhalable polymeric particulate carriers have attracted interest of scientific community for many years. They can serve as effective carriers for local delivery of drugs into the lungs for treatment of various respiratory disorders including cystic fibrosis, inflammation, lung cancer; and also for prolonged release of chemically unstable/poorly soluble drugs in the

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

systemic circulation. Particulate carriers can either be Nanoparticles (diameter <1μM) or

Microparticles (1-1000μM). Poly-lactic acid (PLA) and Poly-(lactic-co-glycolic) acid

(PLGA) are two of the most commonly used polymers for preparation of inhalable

particulate carriers due to their favorable biocompatibility and safety profile and are FDA

approved.

PLGA microparticulate formulations represent one of the most investigated approaches

for inhalable delivery of pharmaceuticals. However, for effective delivery of therapeutic

agents into the lungs, microparticles need to be deposited into the deep lung regions,

which require the particles to have a mass median geometric diameter in the range of 1-

5 μM (Adjei and Garren, 1990). Formulations not fulfilling this criterion are either

deposited in the oropharyngeal cavity, or are cleared by lungs’ clearance mechanisms

(phagocytosis by alveolar macrophages, mucociliary clearance), causing a reduced drug

residence time in the lungs and thus reduced therapeutic efficacy (Edwards et al., 1997).

In a seminal paper Edwards et al. proposed “Large Porous Microparticulate Delivery

Systems” i.e. microparticles with size >10μM and mass density of <0.4 g/cm3, could

facilitate respirability and deep lung deposition of the formulation, and could also provide

a mean to escape lungs’ natural clearance mechanisms. Porous microspheres have

hollow spaces and channels to release the drug in a controlled fashion. Edwards et al.

proposed that these formulations, despite being of large geometric diameter, will still

have Aerodynamic diameter in the respirable range of 1-5 μM due to low mass density

(Edwards et al., 1997).

Where; da = aerodynamic diameter, d = particle’s geometric diameter, ρ = mass density

3 of the particle, and ρa = reference density (1 g/cm ).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

As discussed earlier, PLGA microparticles have been thoroughly investigated for their

efficacy in delivering various therapeutic agents (both large and small molecular weight)

via pulmonary route (Wang et al., 1999; Tsapis et al., 2003; Jiang et al., 2005).

Microparticulate delivery systems can be designed to release the drug at a controlled

rate over prolonged period of time. Unfortunately, there are no data so far to establish

the efficacy of PLGA microparticles for sustained delivery of anti-PAH medications,

despite PAH being such a deadly disease of the pulmonary circulation.

Several factors led us to believe that PLGA microparticles will be an ideal delivery

system to achieve our goals to develop a controlled release inhalable delivery system

that will provide prolonged pulmonary vasodilation in PAH induced rats and ultimately in

PAH patients. Since PAH is a disease of distal pulmonary arteries and any medication

delivered via the parenteral route will result in systemic side-effects. However, inhalable

delivery system will deliver the drug locally into the diseased areas of the lungs and thus

will eliminate the possibilities of developing systemic side-effects. All currently approved

and investigational anti-PAH drugs suffer from a common shortcoming of very short

biological half-life−5-10 minutes in case of PGE1−which calls for a delivery system that can deliver the drug into the lungs over a prolonged period of time and achieve sustained pulmonary vasodilation. Respirable PLGA microparticles deposit in deep lungs and can deliver the drug right into the diseased area of the pulmonary circulation.

Moreover, PLGA core of microparticles degrades in a time dependent manner and continuously release the drug over a prolonged period of time. Further, PGE1 is extensively metabolized in the lungs (∼80%) thus making it pulmonary-selective.

Encapsulation of PGE1 in PLGA microspheres will result in protection of unreleased

PGE1 from metabolic enzymes present in the lungs and will thus contribute to the sustained pulmonary vasodilatory effects of the delivery system.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

The Monocrotaline-Induced Animal Model for PAH

Monocrotaline (MCT) is a toxic crystalline 11-membered macrocyclic pyrrolizidine

alkaloid of plant origin, which is found in Crotolaria spectabilis and other plants of

Crotolaria genus. Toxic manifestations of MCT exposure include hepatic and pulmonary

lesions. In the lungs, vascular lesions are prominent that causes swelling of capillary

endothelial cells, lesions of the arterial media, and thrombi accumulation with large

number of platelets resulting in increased pulmonary arterial pressure and right

ventricular hypertrophy. Various studies have documented the similarities observed

between lesions in rats induced by MCT and that in the lungs of patients of PAH and

chronic pulmonary vascular disease (Hilliker et al., 1982). A single subcutaneous

injection of MCT (60 mg/kg) in rats results in lung injury characterized by pulmonary

vascular remodeling, pulmonary hypertension and compensatory right heart hypertrophy

over 3-4 weeks (Todd et al., 1985). Several investigators have studied suitability of

MCT-induced rodent model of PAH and have demonstrated that MCT treatment in rats

induces right ventricular hypertrophy, increases the cross-sectional area of small

pulmonary arteries (100-200 μM) and reduces the luminal diameter that eventually leads

to reduction in lung function. MCT treatment results in muscularization of small

pulmonary arterioles thus causes increased MPAP and right ventricular hypertrophy (Lai

and Law, 2004). Prolonged exposure to hypoxia is also reported to induce PAH like

symptoms in rodents. While hypoxia causes an immediate increase in PAP followed by

vascular remodeling, MCT treatment causes injury and induces structural changes in the

pulmonary circulation resulting in an increase in PAP (van Suylen et al., 1998). In this

dissertation, we intend to investigate both preventive and protective roles of PGE1 when

administered as inhalable controlled release formulations in-vivo. MCT-induced rodent

model of PAH meets all the requirements of an ideal animal model for the project.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Hypothesis and Specific Aims

To date, there are no reports regarding controlled release formulation of any anti-PAH

drug that can provide sustained pulmonary vasodilation of distal pulmonary arteries.

Because of pulmonary selectivity, PGE1 would be an excellent candidate for localized

delivery to lungs to produce its vasodilatory effects. However, little is known about the

feasibility of PGE1 as a therapeutic agent when encapsulated in particulate carriers.

Therefore, the overall objective of this dissertation is to develop a safe and efficacious

inhalable particulate delivery system for PGE1 which can (i) ameliorate symptoms of

PAH for a prolonged period of time and (ii) reduce the progression of the disease. With

this in mind, we have designed this dissertation to answer the following questions:

(i) Are PLGA microspheres feasible carriers for non-invasive pulmonary delivery of

PGE1?

(ii) How does the incorporation of absorption enhancers, polyethyleneimine (PEI)

and HPβCD in the microparticulate core affect distribution of PGE1 following

pulmonary administration?

(iii) Does PGE1 encapsulated in microspheres release the drug to produce its

pharmacological effect?

(iv) Do the optimized PLGA microspheric formulations of PGE1 provide symptomatic

relief against pulmonary hypertension to the monocrotaline (MCT) induced

chronic pulmonary hypertensive rats?

To answer these questions, we have proposed the following research plans:

1. To establish the feasibility of plain PLGA microspheres as inhalable long acting

carriers for PGE1 (Chapter 2).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

2. To investigate the suitability of core modified large porous PLGA microspheres in

delivering PGE1 to the lungs via pulmonary route (Chapter 3).

3. To determine the efficacy of hydrophilic complexes of PGE1 with hydroxypropyl-

β-cyclodextrin (PGE1-HPβCD) encapsulated in PLGA microspheres in delivering

PGE1 via the non-invasive pulmonary route (Chapter 4).

4. To determine the safety of the optimized microspheric formulations in-vivo and in

in-vitro cell culture model (Chapters 2, 3, and 4).

5. Study the efficacy of optimized PGE1 microspheres in ameliorating symptoms of

PAH in monocrotaline (MCT) induced-PAH rats (Chapter 5).

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Table 1.1: Updated Clinical Classification of Pulmonary Hypertension (Dana Point, CA, 2008) (modified from Simonneau et al., 2009)

1. Pulmonary Arterial Hypertension (PAH) 1.1. Idiopathic PAH 1.2. Heritable 1.2.1. BMPR2 1.2.2. ALK1, endoglin (with or without heritable hemorrhagic telangiectasia) 1.2.3. Unknown 1.3. Drug and Toxin induced 1.4. Associated with 1.4.1. Connective tissue diseases 1.4.2. HIV infection 1.4.3. Portal hypertension 1.4.4. Congenital heart diseases 1.4.5. Schistosomiasis 1.4.6. Chronic hemolytic anemia 1.5. Persistent pulmonary hypertension of the newborn 1’. Pulmonary veno-occlusive disease (PVOD) and/or pulmonary capillary hemanglomatosis (PCH) 2. Pulmonary hypertension owing to left heart disease 2.1. Systolic dysfunction 2.2. Diastolic dysfunction 2.3. Valvular disease 3. Pulmonary hypertension owing to lung diseases and/or hypoxia 3.1. Chronic obstructive pulmonary disease (COPD) 3.2. Interstitial lung disease 3.3. Other pulmonary diseases with mixed restrictive and obstructive pattern 3.4. Sleep-disordered breathing 3.5. Alveolar hypoventilation disorders 3.6. Chronic exposure to high altitude 3.7. Developmental abnormalities 4. Chronic thromboembolic pulmonary hypertension (CTEPH) 5. Pulmonary hypertension with unclear multifactorial mechanisms 5.1. Hematologic disorders: myeloproliferative disorders, splenectomy 5.2. Systemic disorders: sarcoidosis, pulmonary langerhans cell histiolysis: lymphangloleiomyomatosis, neurofibromatosis, vasculitis 5.3. Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders 5.4. Others: tumoral obstruction, fibrosing mediastinitis, chronic renal failure on dialysis ALK1⎯Activin receptor like kinase type 1, BMPR2⎯Bone morphogenetic protein receptor type 2, HIV⎯Human immunodeficiency virus

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Table 1.2: Advantages and Limitations of Prostacyclin Analogues for PAH Treatment (modified from Gomberg-Maitland and Olschewski, 2008)

Name Route Half-Life Advantages Disadvantages FDA Approval Epoprostenol I.V. 6 minutes Easy to titrate - Requires permanent I.V. Class III (Flolan®) Longest experience catheter Class IV - Risk of line infection - Risk of syncope or cardiovascular collapse - Need for ice packs - Mixing every 24 hours Treprostinil S.C. 4.6 hours Smaller pump - Pain at the site of infusion Class II (Remodulin®) No mixing Class III I.V. 4.4 hours Cassette changed every 48 - Risk associated with the Class IV hours permanent I.V. catheter No need for ice packs Less risk of cardiovascular collapse Iloprost Inhaled 20-30 No need for IV catheter - 6-9 inhalations per day Class III (Ventavis®) minutes Local delivery limits side- - More risk of syncope Class IV effects I.V. Same as epoprostenol but - Same as epoprostenol less experience Beraprost Oral 35-40 Oral delivery - Gastrointestinal No minutes intolerance - Unclear efficacy

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Maintenance of Vascular Tone

Fig. 1.1: Factors responsible for maintaining vascular tone in the pulmonary circulation. EDRF, endothelium-derived relaxing factor (modified from Gupta and Ahsan, 2010).

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Pathophysiology of PAH

Fig. 1.2: Schematic representation of different vascular abnormalities associated with pulmonary hypertension and compared with normal circulation throughout the pulmonary circulation. EC, endothelial cell; SMC, smooth muscle cell (modified from Rabinovitch, 2008).

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Cellular Mechanisms of PAH Progression

Fig. 1.3: Diagrammatic illustration of different cell layers involved in the pathophysiology of pulmonary hypertension. The figure depicts different factors and phenotypic responses involved in histological progression of the pulmonary circulation from normal to pathogenically activated in pulmonary arterial hypertension (modified from Chan and Loscalzo, 2008).

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Treatment Strategies for PAH

Fig. 1.4: Targets for current and emerging therapies in pulmonary arterial hypertension (modified from Humbert et al., 2004).

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Prostaglandin E1 (PGE1) and Its Biosynthesis

Fig. 1.5: (A) Structure of Prostaglandin E1 (PGE1); and (B) Biosynthesis of PGE1 from dihomo-γ-linolenic acid (DGLA; 20:3, ω-6)

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Non-invasive Delivery of Anti-PAH Medications

Fig. 1.6: Inhaled route of administration of prostacyclin analogues. Black arrows indicate the areas where locally deposited drug can penetrate the airway wall and directly diffuse into the pulmonary arterial wall. Terminal arterioles, carrying most of the resistance, are completely surrounded by alveolar surfaces. Cap – pulmonary capillaries (modified from Gomberg-Maitland and Olschewski, 2008).

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Polyethyleneimine, Hydroxypropyl-β-cyclodextrin, PLGA

A Ter tiar y Am ines, 25% Secondary Am ines, 50%

NH (CH CH N) (CH CH NH) 2 2 2 x 2 2 y

CH 2 CH 2 NH 2

P rim ary A m in es, 25%

Fig. 1.7: Chemical structures of (A) polyethyleneimine; (B) Hydroxypropyl-β-Cyclodextrin (HPβCD); and (C) Poly (lactide-co-glycolic) acid (PLGA).

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CHAPTER 2

FEASIBILITY STUDY OF AEROSOLIZED PROSTAGLANDIN E1 MICROSPHERES

AS A NON-INVASIVE THERAPY FOR PULMONARY ARTERIAL HYPERTENSION

INTRODUCTION

Pulmonary arterial hypertension (PAH) is a chronic and debilitating disorder of the pulmonary circulation that affects about 50,000-100,000 people in the United States

(Gaine and Rubin, 1998). PAH is characterized by mean pulmonary arterial pressure

(MPAP) above 25 mm Hg at rest or 30 mm Hg after exercise. The primary cause of PAH progression is an imbalance of neurochemical mediators⎯prostacyclins, nitric oxide, endothelin-1⎯that are required to maintain the vascular tone of pulmonary arteries. This imbalance causes vasoconstriction, pulmonary vascular remodeling, and increased pulmonary vascular resistance that result in occlusion and narrowing of the pulmonary arteries and eventually right ventricular enlargement (Martin et al., 2006). Development of PAH is associated with a reduction in prostacyclin levels in the pulmonary circulation

(Humbert et al., 2004). Various prostacyclin I2 (PGI2) analogues, including epoprostenol,

iloprost and treprostinil, are currently available for PAH treatment. However, because of

their very short biological half-lives, prostacyclin analogues are required to be

administered by continuous intravenous or subcutaneous infusion. Use of indwelling

catheters to administer the drugs results in a variety of side effects, including infection

and cardiovascular collapse (Gomberg-Maitland and Preston, 2005). To overcome these

limitations, an inhalable prostacyclin analogue (iloprost, Ventavis®) has been developed and is now commercially available in the United States (Olschewski et al., 2003). An inhalable form of treprostinil has recently been tested in humans (Sandifer et al., 2005;

Voswinckel et al., 2006). However, the limitations of very short biological half-life and

Modified from Gupta et al. (2010), Journal of Pharmaceutical Sciences, 99(4):1774-89.

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metabolic instability continue to be major limitations for PAH therapy. Inhaled iloprost is required to be taken 9-12 times a day, which often leads to patient noncompliance.

Prostaglandin E1 (alprostadil, PGE1) is one of the prostacyclins that has been

investigated for PAH therapy (Kunimoto et al., 1997; Shen et al., 2005). PGE1 acts as a

selective pulmonary vasodilator when administered as an aerosol into the lungs and

thereby eliminates the complications associated with systemic vasodilation (Sood et al.,

2004). In fact, intravenous PGE1 has been reported to be used for treatment of PAH,

acute respiratory distress syndrome, hypoxemic respiratory failure, and in lung

transplantation (Meyer et al., 1998; Della Rocca et al., 2008), However, it has a half-life

of 5-10 minutes because 70-90% of the drug metabolizes in the lungs in a single pass.

Moreover, because of its extensive peripheral distribution, intravenous PGE1 is associated with severe side-effects such as systemic hypotension and low cardiac output (Meyer et al., 1998). The limitations of PGE1-based PAH therapy can be

overcome by delivering the drug as long-acting inhalable particles. Approaches that

have been used to increase the residence time of PGE1 in the lungs include: (i) chemical

modification (Hollo, 2007), (ii) formulation in a β-cyclodextrin complex (Gu et al., 2005),

and (iii) encapsulation in particulate carriers such as lipids, microspheres (Igarashi et al.,

2001; Shen et al., 2005), liposomes (Feld et al., 1994) and other polymeric carriers (Pan

et al., 2007). Further, PGE1 is reported to be continually released upon encapsulation in

PLGA particles (Huang et al., 2008; Ishihara et al., 2008). Despite all these studies,

there is no formulation of PGE1 which is capable of showing long term efficacy in PAH treatment.

Recently, we showed that PLGA microspheres can be used to achieve prolonged- release of low molecular weight heparins after pulmonary administration (Rawat et al.,

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2008). Incorporation of PGE1 into microspheric carriers is likely to offer the following

advantages: (i) prevent inactivation by phase-I metabolic enzymes in the lungs, (ii)

release the drug over a longer period of time and produce a prolonged therapeutic

effect, and (iii) minimize the distribution of PGE1 in the body by localized delivery of the

drug in the lungs. However, the pulmonary route suffers from some important limitations

such as poor deposition of particles that are outside the respirable size range (1-5 μm)

and loss of drug due to oropharyngeal deposition. The problem of poor deposition in the

respiratory tract can be addressed by encapsulating the drug in particles with mass

densities <0.4 g/cm3 and geometric diameter >5 μm, as proposed by Edwards et al.

(Edwards et al., 1997). This approach will facilitate respirability, enhance deep-lung deposition and produce prolonged release by avoiding uptake by alveolar macrophages.

This study therefore tests the hypothesis that PGE1-loaded PLGA-based microspheres, when administered via the pulmonary route, release the drug over a prolonged period of time and prevent enzymatic degradation of PGE1 in the lungs.

MATERIALS AND METHODS

Materials

PLGA 50:50 with inherent viscosity 0.55-0.75 dL/g (average molecular weight = 43.5 kDa) and PLGA 85:15 with inherent viscosity 0.55-0.75 dL/g (average molecular weight

= 85.2 kDa) were purchased from Lactel Absorbable Polymers (Pelham, AL).

Prostaglandin E1 (PGE1) was purchased from Cayman Chemical Company (Ann Arbor,

MI). Polyvinyl alcohol (PVA) and dichloromethane (DCM) were from Sigma-Aldrich Inc.

(St. Louis, MO) and VWR International (West Chester, PA), respectively.

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Preparation of PGE1-Loaded PLGA Microparticles

PGE1-loaded PLGA microparticles were prepared by the water-in-oil-in-water (W/O/W) double emulsion/solvent evaporation method as previously established in our laboratory

(Rawat et al., 2008; Thomas et al., 2009). Briefly, the primary emulsification was performed by probe sonication with a Branson Sonifier 450 (Branson Ultrasonics

Corporation, Danbury, CT). An aliquot of saline (internal aqueous phase, IAP) was emulsified with 4.0 mL of DCM (organic phase, OP) containing 200 mg of PLGA polymer. The drug (PGE1), being hydrophobic, was dissolved in the organic phase before emulsification. Probe sonication was performed for 1 min (20W, 6 cycles of 10 sec each at 40% duty cycle). The ratio of IAP:OP was 1:10 for all formulations. The secondary emulsification was performed by homogenizing the water-in-oil (W/O) emulsion with 20.0 mL of PVA (1, 2 and 5% w/v) solution (external aqueous phase,

EAP) at 10,000 rpm for 10 min with an Ultra-Turrex T-25 basic (IKA, Wilmington, DE).

The formulations were kept overnight at room temperature under constant stirring to remove the solvent. The next day, the formulations were washed 3 times by centrifugation at 50,000xg for 15 min at 40C and then freeze-dried for 24-48 h to obtain a

free-flowing powder. Each formulation was prepared in triplicate and a total of 6

formulations were prepared: 3 with PLGA 50:50 and 3 with PLGA 85:15 (Table 2.1).

Particle Characterization

The particles were characterized for morphology, size, zeta potential and aerodynamic diameter. The particle morphology was investigated by scanning electron microscopy

(SEM). The samples for SEM were prepared by sprinkling a small amount of freeze- dried formulation (1-2 mg) onto double-sided adhesive tape attached to an aluminum stub and then sputter-coated with gold under argon (Emitech K550X, Kent, UK). The

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samples were then examined by SEM and photomicrographs of the formulations were taken by using a Hitachi S-3400N (Freehold, NJ) scanning electron microscope. Particle size distribution and volume-based mean diameter was analyzed by using a Microtrac®

S3500 (North Largo, FL) particle size analyzer. Samples for particles size analysis were prepared by dispersing the freeze-dried formulations in a 0.2% w/v solution of Tween 80.

Polydispersity indices (PDIs) were calculated as a ratio of mean-volume averaged particle size to mean-number averaged particle size. The experiemnts for particle sizing were performed in triplicate and data are presented as the mean±SEM.

The density of microspheric formulations was estimated from the tapped density measurements as described previously (Vanbever et al., 1999). An aliquot of 100 mg of microspheres was transferred to a 10 (±0.05) mL graduated cylinder and the initial volume was recorded. Tapped density of the formulations (ρ) was calculated as the ratio between sample weight (g) and the volume (mL) occupied after 50 taps. Theoretical mass mean aerodynamic diameter (MMADt) of the formulations was also calculated from tapped density measurements by using the following equation (Vanbever et al.,

1999):

1/2 MMADt = d(ρ/ρ0 X)

Where d is the geometric mean diameter determined from particle size analysis, ρ is the

3 tapped density, ρ0 is the reference density of 1 g/cm , and X is the shape factor, which is

1 for a sphere. The actual aerodynamic diameter of the dry powder formulations was also determined by using an eight-stage Marc-II Anderson Cascade Impactor (Westech

Instruments Inc., Marietta, GA). The dry powder formulations were fired into the cascade impactor at a flow rate of 28.3 L/min. The amount of formulation deposited at each stage of the impactor was determined by weighing the glass fiber filter papers that were put on

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the plates for each stage. All studies were performed in triplicate and the data thus obtained were plotted on a log graph to calculate the mass median aerodynamic diameter.

Encapsulation Efficiency

The encapsulation efficiency of the formulations was determined by quantitating the amount of unentrapped PGE1 in the EAP, recovered after centrifugation and washing of

the microspheres. Briefly, the samples recovered from the EAP were diluted 105 times and the free drug was assayed by using a commercially available PGE1-ELISA kit

(Correlate-EIA, Assay Designs, Ann Arbor, MI). A higher degree of dilution was required

due to the high sensitivity of the commercial kit, which can reliably measure 4.88-5000

pg/ml of PGE1. All assays were preformed in triplicate and results are presented as the mean±SEM.

In Vitro Release Studies

For studying in vitro release profiles, 10 mg freeze-dried microparticles were suspended in 1.0 mL of phosphate-buffered saline (PBS) containing 0.02% Tween 80 in a microcentrifuge tube (2.0 mL) and incubated in an isotemp incubator at 37±1ºC with gentle stirring. At predetermined time points ranging from 0-120 h, samples were removed from the incubator and centrifuged at 16,000 ×g for 15 min at 4ºC. Two

hundred microliters of supernatant were collected and assayed for PGE1 content as

described above. The volume of supernatant collected at each time point was replaced

with an equal volume of fresh PBS with 0.02% Tween 80. The data are presented as

percent cumulative drug release±SEM (N = 3).

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In Vivo Absorption Studies

Adult male Sprague-Dawley rats (Charles River Laboratories, Charlotte, NC) weighing between 250 and 300 g were used for in vivo absorption studies (n = 6-8). The absorption and circulation time of the drug was monitored by measuring plasma PGE1 content. Before beginning the experiments, the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Anesthesia was maintained with additional intramuscular injections of ketamine (20 mg/kg) and xylazine (100 mg/kg) on an as needed basis during the course of experiments. The freeze-dried microparticles of PGE1 were administered intratracheally by using a microsprayer specially designed for aerosol inhalation in small animals (Penn-Century, Philadelphia, PA). The microparticles were administered as a dispersion in PBS buffer and the dose administered was 80 μg PGE1 for each kg of body weight. For intravenous

administration, 80 μg/kg PGE1 in PBS buffer was administered into the animals as a

single 100-μL injection into the jugular vein. Blood samples were collected at

predetermined time points (0-24 h) from the tail and stored in citrated centrifuge tubes

containing 10 μg/mL indomethacin as a PGE1 synthase inhibitor. The plasma was separated by centrifuging the blood samples at 3000 rpm for 10 min and stored at -200C until analysis.

Enzyme Immunoassay for Estimation of PGE1

The amount of PGE1 in the blood was determined by a commercially available ELISA kit

TM (Correlate PGE1-EIA, Assay Designs, Ann Arbor, MI). The assays were performed

according to the manufacturer’s protocol. A standard curve was prepared by using the

samples provided with the kit and compared with the standard curve prepared for the

drug used to prepare the formulations. The ELISA kit used for estimation of PGE1

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concentration had a quantification range of 4.88-5000 pg/ml. The kit was standardized in our laboratory to further confirm the quantitation. For analysis, the plasma samples were diluted 20 times with the assay buffer to minimize matrix interference. Plasma samples from untreated rats were used as negative controls to estimate the influence of endogenous PGE1.

Metabolic Degradation Studies of PGE1 Microparticles in Rat Lung Homogenate

The metabolic degradation of the microparticulate formulations was studied in lung homogenates. Lung homogenates of adult male Sprague-Dawley rats (300-350 g) were prepared according to a protocol described by Nakano et al (Nakano et al., 1973).

Briefly, the lungs were surgically removed and homogenized with 4 volumes of cold homogenization buffer (Bücher medium; 20 mM KH2PO4, 72 mM K2HPO4, 27.6 mM nicotinamide, and 3.6 mM MgCl2, pH 7.4) using a motor-driven Potter-Elvehjem glass homogenizer. The homogenate was centrifuged at 10,000 ×g, 40C, for 20 min to remove

the cell debris, and the supernatant was collected and used for the stability studies.

Protein concentration of the rat lung homogenate was determined by a BCATM Protein

Assay Kit (Pierce, Rockford, IL). For metabolic degradation studies, 80 ng/ml PGE1 or 5 mg of MCP-4 containing about 50 μg of PGE1 were incubated for 8 h in 1.0 ml lung

homogenate in the presence of 2 mM NAD+. The reaction was terminated at predetermined time points by adding 200 μL of 0.1 N HCl to the incubation mixture.

Samples were collected periodically and assayed for PGE1 content by using a commercial ELISA kit. For metabolic degradation of MCP-4 formulations, we also determined the amount of drug that was not released from the particles after 8 h of incubation. To determine the amount of unreleased drug, the particles in the homogenate were digested and extracted with 5 volumes of DCM by vortexing for 30

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seconds followed by centrifugation at 3500 rpm for 5 min. Following extraction, the aqueous phase and proteins were discarded and the organic layer (DCM with polymer and drug) was transferred to a clean tube and evaporated with an air stream by using N-

Evap (Organomation Associates Inc, Berlin, MA). After evaporation of the organic solvent, PGE1 was extracted by vortexing the residue in 1.0 ml of PBS (pH 7.2) for 30

seconds. PGE1 thus obtained in PBS solution was assayed by ELISA and this amount of

PGE1 was considered as the amount of PGE1 that was not released upon incubation in

lung homogenates.

Cytotoxicity Studies

For cytotoxicity studies, Calu-3 cells were seeded at a density of 50,000 cells per well in flat-bottom 96-well microtiter tissue culture plates and allowed to grow in MEM medium

[+10% fetal bovine serum + 1% 200 mM L-glutamine + 1% penicillin/streptomycin] to

90% confluency. Immediately prior to the start of the experiment, the medium was removed from the wells, the cells were washed twice with sterile saline, 20 μl of test

formulations were added, and the plates were incubated at 37oC for 4 h. The test samples contained microspheric formulations at 0.5, 1.0, 2.0, 5.0 and 10.0 mg/ml as a solution with normal saline. A 0.1% w/v solution of sodium dodecyl sulfate (SDS) was used as positive control for cytotoxicity. Cell viability was measured by MTT assay as described earlier (Scudiero et al., 1988). Briefly, after 4 h of incubation with the test formulations, the formulations were removed from the wells, the bottom of each well was washed with saline, 20 μl of MTT solution (5 mg/ml) was added to each well, and the cells were again incubated at 37oC for 4 h. After 4 h, the solution in each well was removed carefully, and 100 μl of 100% DMSO was added to each well, and the plates were incubated for 1 h. Absorbance was measured at 570 nm on a microtiter plate

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

reader (TECAN U.S. Inc, Research Triangle Park, NC). Each assay was performed on

16 samples, and cell viability was expressed as the percentage of MTT released by cells exposed to the test formulations or 0.1% SDS compared to cells incubated with saline alone.

Bronchoalveolar Lavage Studies

Bronchoalveolar lavage (BAL) studies were performed to determine the safety profile of the optimized formulations as per our previously published procedure (Thomas et al.,

2008). For BAL studies, adult male Sprague-Dawley rats weighing 300-350 g were

divided into 5 groups (n = 4). The rats were anesthetized with an intraperitoneal injection

of pentobarbital sodium (60 mg/kg). Three groups of animals received the following

optimized formulations: MCP-1, MCP-4, and MCP-6. One group of animals received

saline as a negative control. The remaining group received lipopolysaccharide solution

(LPS, 0.1 μg/mL) as a positive control. After 12 h of administration of the formulations, all

animals were weighed and the lungs were surgically removed after re-anesthetizing the

rats. Briefly, the respiratory apparatus was exposed by a mid-level incision in the

thoracic cavity and the lungs were carefully removed after exsanguination of the animal

by severing the abdominal aorta. Lung wet weights were recorded and then the lungs

were lavaged by instilling 5-mL aliquots of normal saline into the trachea. The instilled

saline was left in the lungs for 30 s, withdrawn, re-instilled for 30 s, and finally withdrawn

and collected in centrifuge tubes. The collected fluid was centrifuged at 500 ×g for 10

min and then stored at -20 0C until further analysis. The BAL fluid collected was assayed

for the presence of injury markers, lactate dehydrogenase (LDH) and alkaline

phosphatase (ALP), with commercially available LDH and ALP kits (Pointe Scientific,

Canton, MI) and the enzyme levels are reported as IU/L. The lung wet weights are

reported as g/100 g body weight.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

All animal studies were conducted in accordance with NIH guidelines for the Care and

Use of Laboratory Animals under a protocol approved by the Texas Tech University

Health Sciences Center (TTUHSC) Animal Care and Use Committee (AM-10012).

Pharmacokinetic and Statistical Analyses

The pharmacokinetic parameters were determined by using standard non- compartmental analysis (Kinetica®, Version 4.0, Innaphase Corp. Philadelphia, PA) of in

vivo absorption data. The data were analyzed for area under the plasma concentration

versus time curve (AUC0-24 h), Cmax, Tmax, and t1/2. The results obtained were compared by one-way ANOVA followed by Newman-Keuls post-hoc analysis (GraphPad Prism version 3.03, GraphPad software, San Diego, CA). Values showing p<0.05 were considered significant.

RESULTS AND DISCUSSION

Particle Characterization

Scanning electron microscopy (SEM) was performed to study the particle morphology of the formulations. As shown in Fig. 2.1, the particles in all the formulations are spherical with smooth surfaces. The particle morphology changed significantly with increasing

PVA concentration in the EAP (1%, 2%, and 5%). SEM images of formulations with a

PVA concentration of 2% or more (Figs. 2.1B, C, E, and F) showed the presence of spherical particles. Moreover, with increasing PVA concentration, the surface of the microspheres became smoother, and fewer empty and fragmented cores were observed. These observations are in accord with published data (Jeong et al., 2003;

Cirpanli et al., 2005). PVA has been reported to act as an emulsion stabilizer for preparation of PLGA microspheres and to affect the hydrophobicity and digestibility of

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the microparticle surface (Lee et al., 1999). PVA provides enhanced stability to the surface of PLGA nanoparticles by strongly adsorbing on the surface (Murakami et al.,

1999). Though the microspheric formulations prepared with double-emulsion solvent- evaporation do not show any surface pores, internal pores should be present owing to the presence of the aqueous phase. The use of saline (0.9% NaCl) as the IAP is likely to make the particles porous and bulkier. In fact, it has been demonstrated that increased osmotic pressure in the IAP renders particles more porous by promoting inward diffusion of water from the EAP toward the IAP during solvent evaporation (Srinivasan et al.,

2005). During lyophilization, water is removed from inside the particles, leaving void spaces in the core.

Particle Size, Tapped Density and Aerodynamic Diameter

As discussed earlier, successful deep-lung deposition of inhaled formulations is a function of particle density and aerodynamic diameter of the formulations. A geometric diameter of 1-5 μm and a particle density of ∼ 1.0 g/cm3 are optimal for efficient deposition of particles in the lungs. However, Edwards et al, (Edwards et al., 1997) in a seminal paper, proposed that particles with a geometric diameter >5 μm and a mass density <0.4 g/cm3 could be used to prolong the release and extend the residence time

of inhaled formulations (Edwards et al., 1997). We have determined the particle size,

tapped density and mass median aerodynamic diameter (MMAD) to investigate the

suitability of particles for pulmonary delivery. As shown in Fig. 2.2A, the formulations

were moderately polydispersed, with a polydispersity index (PDI) ranging from 2.03 to

3.52 (Table 2.1). For both polymer types, PLGA 50:50 and PLGA 85:15, particle size

decreased significantly as the PVA concentration in the EAP increased (1%, 2%, and

5%). For the PLGA 50:50 particles, the geometric diameter decreased from 7.55±2.65

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

μm (MCP-1) to 2.54±0.82 μm (MCP-3), and for the PLGA 85:15 particles, the geometric diameter decreased from 10.66±3.71 μm (MCP-4) to 2.36±0.47 μm (MCP-6) (Fig. 2.2A).

These data, in conjunction with previous reports (Lee et al., 1999; Yang et al., 2001), illustrate the reduction in particle size with increasing PVA concentration in the EAP.

Higher PVA concentrations in the water phase provide a hydrophilic environment and stabilize the emulsion droplets, thus causing a reduction in particle size (Jeong et al.,

2003).

In addition to particle size, the tapped density and theoretical and actual mass median aerodynamic diameters (MMADt and MMADa) were also determined. As can be seen in

Fig. 2.2B, all formulations have comparable tapped densities, ranging from 0.16 g/ml to

0.19 g/ml, thus making the particles lighter than conventional particles. The lower tapped densities observed for all the formulations can be attributed to the use of saline (0.9%

NaCl) instead of water as the IAP, as discussed in the previous section. Mass median aerodynamic diameter is an important determinant of particle distribution and lung deposition pattern. Theoretical MMAD was calculated from volume-based mean diameter and tapped density, and MMADa was determined by using an eight-stage

Marc-II Anderson Cascade Impactor, a widely used technique to simulate particle behavior following inhalation from therapeutic inhalational devices (Mitchell and Nagel,

2003). The theoretical and actual MMAD data presented in Fig. 2.2C show a pattern similar to that observed for the geometric diameter. Further, MMADt closely resembles the experimental MMAD (MMADa). Both MMADt and MMADa decreased with increasing

PVA concentration in the EAP. MMADt decreased from 3.29±1.16 to 1.26±0.41 μm for

PLGA 50:50 particles, and from 4.77±2.03 to 0.95±0.23 μm for PLGA 85:15 particles.

Similarly, MMADa decreased from 4.0±1.74 to 1.85±0.31 μm for PLGA 50:50 particles, and from 4.25±0.9 μm to 1.23±0.4 μm for PLGA 85:15 particles. More importantly, both

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the MMADt and MMADa values of all the formulations are <6 μm, which is considered ideal for efficient deposition of particles in the lungs (Srinivasan et al., 2005). However,

MCP-3 and MCP-6 showed MMADa values of 1.85±0.31 μm and 1.23±0.4 μm, respectively, making them more susceptible to degradation by macrophages (Edwards et al., 1997). On the whole, the data obtained from physical characterization of the

microparticulate formulations suggest that varying the polymer composition and PVA

concentration in the EAP plays an important role in determining the physical and

aerodynamic properties of the dry powder microparticulate formulations.

Entrapment Efficiency

The entrapment efficiency of the microspheres was determined indirectly by assaying the amount of drug recovered in the supernatant after washing the microspheres. The amount of drug entrapped increased with increasing PVA concentration in all formulations (Fig. 2.2D). Moreover, formulations prepared with PLGA 85:15 polymer showed better entrapment efficiency than those prepared with PLGA 50:50 polymer.

MCP-1 (1% PVA in EAP) showed an entrapment efficiency of 20.13±2.26%, which increased to 55.6±1.91% for MCP-2 (2% PVA) and to 65.6±2.76% for MCP-3 (5% PVA).

Similarly, particles prepared with PLGA 85:15 (MCP-4) showed an entrapment efficiency of 59.44±3.61%, which increased substantially to 65.51±2.62% in MCP-5 when 2% PVA was used. However, MCP-5 and MCP-6 (64.6±3.39%) showed comparable entrapment efficiencies. As discussed in the preceding section, the concentration of PVA plays an important role in determining various physical characteristics of microspheres, including particle size, entrapment efficiency, and in vitro release. Moreover, PVA acts as an O/W emulsion stabilizer (Lee et al., 1999; Cirpanli et al., 2005), thus improving the entrapment efficiency further. However, at higher concentrations (5%), PVA has been

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

reported to produce a reduction in drug loading and entrapment efficiency (Jeong et al.,

2003). as is evident in the case of MCP-6. The reason PLGA 85:15 microspheres showed better entrapment than the PLGA 50:50 microspheres may be because of differences in the lactide:glycolide (L/G) ratio between the two polymers. PLGA 85:15, with a greater proportion of lactic acid, tends to be more hydrophobic, thus facilitating its interaction with hydrophobic PGE1 and resulting in greater drug entrapment in the formulation.

In Vitro Release Profiles

The amount of PGE1 released from the microspheric formulations was determined by

using a commercially available ELISA kit, and the release kinetics was calculated. PGE1 released at the zero time point was considered to be surface-associated drug, and the

PGE1 released during the first 30 min of the experiment was considered to be the initial burst release. The in vitro release profile data shown in Fig. 2.3 suggest a release pattern that is biphasic for all the formulations. An initial burst release phase was followed by a slow, release of PGE1. The biphasic release pattern consists of (i) an initial

zero-order burst release phase for first 30 minutes, followed by (ii) a first order release

pattern thereafter. Burst release phenomena can be explained on the basis of

development of a very high concentration gradient across the particle surface, due to the

release of PGE1 adsorbed onto or close to the microspheres’ surface but not entrapped in the core. MCP-1 microspheres prepared with PLGA 50:50 and 1% PVA in the EAP showed a cumulative drug release of 34.95±4.95% (Fig. 2.3A). The amount of drug released decreased considerably with increasing PVA concentration in the EAP

(20.4±2.38% for MCP-2, and 11.8±0.91% for MCP-3). MCP-1 showed a surface- associated burst release of 7.61%; that is, a large amount of drug entrapped in MCP-1 was actually associated with the surface of the particles. Other formulations, however,

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

showed lower burst releases (3.91±0.13% for MCP-2, and 3.06±0.28% for MCP-3).

Similarly, Fig. 2.3B and 2.3C show the in vitro release profiles of microspheres prepared with PLGA 85:15. For this polymer, MCP-5 (2% PVA) and MCP-6 (5% PVA) showed cumulative drug releases of 21.32±0.35% and 20.52±0.96%, respectively, whereas

MCP-4 (1% PVA) showed a cumulative drug release of 10.49±0.16% despite having less PVA in the EAP. All PLGA 85:15 formulations released a very small amount of drug in the burst release phase (MCP-4 = 2.65±0.26%, MCP-5 = 3.03±0.27%, and MCP-6 =

3.63±0.43%).

Drug release from microspheres occurs by two mechanisms: (i) diffusion through the microspheric matrix, and (ii) hydrolytic degradation of the polymer (Ronneberger et al.,

1996). As reported by Takada et al. (Heya et al., 1991; Takada et al., 2003), the amount of drug released from microspheric formulations is dependent upon the lactic/glycolic ratio and also upon the molecular weight of the polymer used. As shown in Fig. 2.3A and

2.3B, microspheres prepared with PLGA 85:15 released a smaller amount of drug in the burst phase when compared to the PLGA 50:50 particles, which can be attributed to the larger molecular weight of PLGA 85:15 (85.2 kDa) compared to PLGA 50:50 (43.5 kDa).

Also, the cumulative release from MCP-1(PLGA 50:50) was faster than from MCP-4

(PLGA 85:15); thus, MCP-4 has better sustained-release properties. Moreover, as noted above, the PVA concentration in the EAP also affects drug release from microspheric formulations: drug release from microspheres decreases with increasing PVA concentration (Cirpanli et al., 2005), which is consistent with the data obtained for the

PLGA 50:50 microspheres. However, in the case of the PLGA 85:15 microspheres,

MCP-4 showed a smaller release than MCP-5 and MCP-6, which may be due to a reduced amount of surface-associated drug and increased hydrophobic interactions between the polymer and PGE1 (log P = 3.3) in MCP-4. The rate of release was

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

independent of the entrapment efficiency, thus yielding a constant release after the burst phase. However, no significant changes in release were observed past 20 h, perhaps because of differences in the degradation profiles of the polymers used in preparing the microparticles; the average degradation time for PLGA 50:50 is around 10 weeks, whereas that for PLGA 85:15 is 30 weeks (Shive and Anderson, 1997). A fraction of

PGE1 present in the core of the microparticles may not have been released over the 5- day in vitro release study. Though data obtained from in vitro release studies give an indication about the release pattern of formulations in vivo, caution should be exercised when extrapolating such data to drug release in the lungs, because the compositions of in vitro release media differ significantly from that of lung fluid.

In Vivo Pulmonary Absorption Studies of PGE1 from Microspheric Formulations

Pulmonary absorption of PGE1 encapsulated in microspheric formulations (MCP-1, 4

and 6) was studied in a rodent model and the amount of PGE1 absorbed into the

systemic circulation was determined by an ELISA. To compare the efficacy of the

microspheric formulations in increasing the residence time of the drug in the body, plain

drug was also administered via the intravenous and pulmonary routes. As is shown in

Table 2.2 and Fig. 2.4A, administration of PGE1 via the intravenous route resulted in an ultra-rapid onset of absorption, with a Cmax of 80.09±9.37 ng/ml and a half-life (t1/2) of

1.5±0.2 min. On the other hand, drug levels following pulmonary administration of plain

PGE1 did not achieve a Cmax as high as that produced by intravenous administration

(Cmax = 56.73±6.2 ng/ml), but showed an increase in the half-life of the drug

(t1/2=3.49±0.41 min). The lower Cmax achieved following pulmonary administration can be explained on the basis of extensive inactivation of PGE1 by metabolizing enzymes

present in the lungs. Administration of microspheric formulations containing an

equivalent dose of the drug demonstrated a considerable increase in the residence time

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

of the drug in the body for all the formulations, although the Cmax was lower compared to

that of plain drug administered via either the intravenous or pulmonary route (Figs. 2.4B

and 2.4B inset).

As shown in Table 2.1, the microspheric formulations tested in vitro were prepared by

using both polymers with varying lactide/glycolide ratios or different concentrations of

PVA in the EAP. MCP-1 microspheres, prepared with PLGA 50:50, showed a Cmax of

13.18±1.34 ng/ml and a t1/2 of 40.86±4.65 min (Fig. 2.4B). In contrast, MCP-4 microspheres, prepared with PLGA 85:15, showed a Cmax of 8.48±2.45 ng/ml and a t1/2 of

582.39±70.3 min (about 10 h). Of all the formulations tested, MCP-1 showed the highest burst release (7.61±2.49%; Fig. 2.3C). For this reason, more drug was available for immediate metabolism, which resulted in a larger Cmax and a shorter t1/2 compared to

MCP-4. Because PLGA 85:15 used in MCP-4 is more hydrophobic than PLGA 50:50

used in MCP-1, the former formulation had a greater entrapment and produced a slower

release (Figs. 2.2D, 2.3A and B). MCP-6 (PLGA 85:15, 5% PVA) administered via the

pulmonary route showed a Cmax of 4.94±1.23 ng/ml and a t1/2 of 20.92±3.2 min. As discussed earlier, release of a drug from microspheres is highly influenced by the concentration of PVA in the EAP during preparation of the microspheres (Lee et al.,

1999). Increasing the PVA coating prevents degradation of the inner PLGA core by hydrolytic enzymes, thus slowing the release of drug from the particles. Further, MCP-6 was more susceptible to phagocytosis by alveolar macrophages due to its smaller geometric diameter, as shown in Fig. 2.2A. For all the formulations tested, the time required to achieve maximum concentration (Tmax) was longer than that observed for

intravenous and pulmonary administration of plain PGE1. Also, the elimination phase of all three formulations was longer than that for intravenous and pulmonary administration

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

of plain PGE1. MCP-4 showed a significant increase in relative bioavailability when compared with plain drug administered via the pulmonary route (p<0.05) (Table 2.2).

The increase in half-life and bioavailability of PGE1 administered in microspheric

formulations can be attributed to several factors. First, drug administered in microspheric

formulations is released slowly compared to plain drug. Second, the prolonged-release

effect of the drug formulations can be attributed to the inability of alveolar macrophages

to digest the particles because of their larger size (MCP-1 and MCP-4) (Edwards et al.,

1997). A further increase in actual size may also occur due to accumulation of moisture

(Labiris and Dolovich, 2003). However, the assumption that larger particles are too big to be phagocytosed may not hold true if the size of the particles decreases because of disintegration and dissolution in the respiratory fluid. But PLGA particles are unlikely to disintegrate or dissolve immediately in the lung fluid because the average degradation time of PLGA particle varies from 5 to 10 weeks depending on the lactic to glycolic acid ratio (Shive and Anderson, 1997). Because of the long degradation time, we believe that any shrinkage due to dissolution or disintegration is unlikely to have a significant impact on particle characteristics in vivo. In addition to particle size, the composition and molecular weight of the PLGA used is also a major determinant of the biodegradability of the polymer and hence the amount of drug released from the formulations (Takada et al., 2003). However, the fate of PLGA after degradation and drug release is unknown and further studies are required to investigate its metabolism after pulmonary administration.

Metabolic Degradation Studies of PGE1 Microparticles in Rat Lung Homogenate

The physiological stability of MCP-4, the formulation that showed the best in vivo performance, was determined by studying the degradation of microparticles and subsequent drug release in rat lung homogenate. In rat lungs, PGE1 is mainly

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

metabolized by the microsomal enzyme NAD+-dependent 15-hydroxy prostaglandin dehydrogenase. This oxidation reaction is responsible for the ultra-rapid lung metabolism and thus high pulmonary clearance (70-90%) of PGE1. The protein

concentration of the homogenate was found to be between 10 and 15 mg/mL. Plain

PGE1 incubated in lung homogenate was metabolized quickly and the drug

concentration was reduced from 74.38±12.21 ng/mL at time zero to 3.96±0.57 ng/mL after 60 min, and the drug was undetectable at the 120-min time point (Fig. 2.5A).

However, as shown in Fig. 2.5B, when PGE1-entrapped particles (MCP-4) were

incubated in the lung homogenate, the concentration of drug in the homogenates was

increased from 273.5±65.78 ng/mL at time zero to 1063.01±82.44 ng/mL at 8 h (Fig.

2.5B). The increase in PGE1 concentration with time can be attributed to continuous

release of the drug from MCP-4. In agreement with the increase in PGE1 concentration

in lung homogenates, a decrease in the concentration of PGE1 in the particles was

observed. The concentration of unreleased PGE1 decreased from 43.8±1.44 μg/mL at the zero time point to 5.7±0.52 μg/mL after 8 h of incubation. This pattern suggests a

time-dependent degradation of the polymeric core of the particulate formulation (MCP-

4). The data obtained from the metabolic degradation studies are consistent with the in

vivo absorption data that suggest an increase in the half-life of PGE1 upon pulmonary

administration of drug-entrapped particles (Table 2.2).

MTT Cytotoxicity Studies

Cytotoxicity studies were performed to evaluate the effect of formulation ingredients on the lung epithelial cell line Calu-3. Cells grown in 96-well micro-titer plates were incubated with different concentrations of optimized formulations (MCP-1, 4 and 6), and the viability of the cells was determined by MTT assay. MTT [3-(4,5-Dimethyl-2- thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide], a tetrazolium salt, is cleaved by

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

mitochondrial dehydrogenase present in living cells, forming a purple/dark blue product,

“formazan”. Because damaged cells show reduced or no production of the dehydrogenase, formazan production will also be reduced. As shown in Fig. 2.6A, Calu-

3 cells incubated with sterile saline showed high levels of cell viability (70%), whereas only 16% of cells were viable following treatment with 0.1% SDS solution (p=0.05). A concentration-dependent increase in cell death was observed for all the optimized formulations. The cell viability for MCP-1 was comparable to that of saline; the percent survival decreased from 75.1±4.2% to 43.86±1.79% as the concentration of the formulation was raised from 0.5 to 10.0 mg/ml. MCP-4 and MCP-6, on the other hand, actually had little or no effect on cell viability, with a percent survival ranging from 100% to 90.84±1.89% (MCP-4) and 102.56±3.02% to 66.95±3.39% (MCP-6) with increasing concentrations of the formulations. The increased cell viability is in accordance with some recent reports underlining the importance of PLGA scaffolds for better cell adhesion. Also, PLGA 85:15 was shown to provide the best cell adherence when compared with PLGA polymers with different L/G ratios (Gabler et al., 2007; Ellis and

Chaudhuri, 2008). Overall, cytotoxicity of the optimized formulations was comparable to that of the saline control.

Bronchoalveolar Lavage Studies

Bronchoalveolar lavage (BAL) studies are performed to determine the extent of injury caused by any product inhaled into the lungs. These studies have been frequently employed for in vivo monitoring of pulmonary injury in experimental animals. In the present study, lipopolysaccharide (LPS), an immuno-stimulant obtained from E. coli, was used as a positive control. Changes in lung wet weight were determined as per previously reported procedures (Hussain et al., 2006), and the levels of expression of two injury markers (LDH and ALP) were determined in BAL fluid following pulmonary

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

administration of the formulations. An increase in lung weight is observed in the event of lung injury, due to the accumulation of extracellular fluid by the epithelial cell lining of the respiratory wall (Xu et al., 2004). Corrected lung weight was recorded 12 h after pulmonary administration of saline, LPS or optimized formulations (MCP-1 and 4). As can be seen in Fig. 2.6B, there was a significant difference in lung wet weights for MCP-

1 and MCP-4 (0.42 and 0.38 g/100 g body weight, respectively) versus LPS-treated rats

(0.49 g/100 g), whereas no significant differences were observed for MCP-1 and MCP-4 versus the lung wet weight of saline-instilled rats.

The acellular content of the BAL fluid was examined by analyzing the enzymatic activities of LDH and ALP. Lactate dehydrogenase is a cytoplasmic enzyme and its presence in the BAL fluid is indicative of cell damage and lysis. As shown in Fig. 2.6C, there was no statistically significant difference between the LDH levels for saline and

MCP-1 or saline and MCP-4 treatments 12 h after pulmonary administration. However,

LDH levels following administration of 0.1 μg/ml LPS were significantly higher (p<0.05)

than those for animals treated with saline or optimized formulations (4-fold increase in

LDH levels; 583.5±72.83 IU/L as compared to 157.32±17.95 IU/L with saline). Alkaline phosphatase is a membrane-bound lysosomal enzyme whose presence in BAL fluid indicates tissue damage—specifically alveolar type II cell proliferation in response to type I cell damage (Hussain and Ahsan, 2005). Fig. 2.6D depicts the ALP levels in BAL fluid 12 h after pulmonary administration of saline, MCP-1, MCP-4, and LPS. In the case of MCP-1, the ALP levels were increased to 93.09±7.7 IU/L as compared to 54.4±12.12

IU/L, but were still within the normal range (35-123 IU/L). BAL fluid from MCP-4-treated rats did not show a significant difference in ALP level (50.52±11.12 IU/L) when compared to saline-treated rats. However, similar to LDH levels, LPS-treated rats showed enormously increased ALP levels when compared with saline-treated rats

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

(220.5±40.52 IU/L, 4-fold increase compared to saline) (p<0.05). The data presented in

Figs. 2.6B-D establish the short-term safety of inhalable microspheric formulations of

PGE1. However, safety profiles for these formulations after long-term administration are yet to be established. Besides the levels of various injury markers and cytokines, histological changes occurring in the lungs after pulmonary administration can give better insight into the safety of the PGE1 microspheres delivered by the pulmonary route.

In summary, PLGA microspheres can be successfully used for encapsulating PGE1. In terms of particle size, aerodynamic diameter, in vitro release, entrapment efficiency and safety profiles, the microspheric formulations were optimal for pulmonary delivery of

PGE1. The microspheres showed good metabolic stability and produced prolonged release of PGE1 after pulmonary administration. PLGA-based microspheres of PGE1 could be a viable approach for non-invasive therapy of pulmonary arterial hypertension.

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Labiris NR and Dolovich MB (2003) Pulmonary drug delivery. Part I: physiological factors affecting therapeutic effectiveness of aerosolized medications. Br J Clin Pharmacol 56:588-599.

Lee SC, Oh JT, Jang MH and Chung SI (1999) Quantitative analysis of polyvinyl alcohol on the surface of poly(D, L-lactide-co-glycolide) microparticles prepared by solvent evaporation method: effect of particle size and PVA concentration. J Control Release 59:123-132.

Martin KB, Klinger JR and Rounds SI (2006) Pulmonary arterial hypertension: new insights and new hope. Respirology 11:6-17.

Meyer J, Theilmeier G, Van Aken H, Bone HG, Busse H, Waurick R, Hinder F and Booke M (1998) Inhaled prostaglandin E1 for treatment of acute lung injury in severe multiple organ failure. Anesth Analg 86:753-758. Mitchell JP and Nagel MW (2003) Cascade impactors for the size characterization of aerosols from medical inhalers: their uses and limitations. J Aerosol Med 16:341-377.

Murakami H, Kobayashi M, Takeuchi H and Kawashima Y (1999) Preparation of poly(DL-lactide-co-glycolide) nanoparticles by modified spontaneous emulsification solvent diffusion method. Int J Pharm 187:143-152.

Nakano J, Prancan AV and Morsy NH (1973) Metabolism of prostaglandin E1 in stomach, jejunum chyle and plasma of the dog and the rat. Jpn J Pharmacol 23:355- 361.

Pan H, Kopeckova P, Liu J, Wang D, Miller SC and Kopecek J (2007) Stability in plasmas of various species of HPMA copolymer-PGE1 conjugates. Pharm Res 24:2270- 2280.

Rawat A, Majumder QH and Ahsan F (2008) Inhalable large porous microspheres of low molecular weight heparin: in vitro and in vivo evaluation. J Control Release 128:224-232.

Ronneberger B, Kao WJ, Anderson JM and Kissel T (1996) In vivo biocompatibility study of ABA triblock copolymers consisting of poly(L-lactic-co-glycolic acid) A blocks attached to central poly(oxyethylene) B blocks. J Biomed Mater Res 30:31-40.

Sandifer BL, Brigham KL, Lawrence EC, Mottola D, Cuppels C and Parker RE (2005) Potent effects of aerosol compared with intravenous treprostinil on the pulmonary circulation. J Appl Physiol 99:2363-2368.

Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D and Boyd MR (1988) Evaluation of a soluble tetrazolium/formazan assay for cell

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growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48:4827-4833.

Shen J, He B and Wang B (2005) Effects of lipo-prostaglandin E1 on pulmonary hemodynamics and clinical outcomes in patients with pulmonary arterial hypertension. Chest 128:714-719.

Shive MS and Anderson JM (1997) Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv Drug Deliv Rev 28:5-24. Sood BG, Delaney-Black V, Aranda JV and Shankaran S (2004) Aerosolized PGE1: a selective pulmonary vasodilator in neonatal hypoxemic respiratory failure results of a Phase I/II open label clinical trial. Pediatr Res 56:579-585.

Srinivasan C, Katare YK, Muthukumaran T and Panda AK (2005) Effect of additives on encapsulation efficiency, stability and bioactivity of entrapped lysozyme from biodegradable polymer particles. J Microencapsul 22:127-138.

Takada S, Yamagata Y, Misaki M, Taira K and Kurokawa T (2003) Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique. J Control Release 88:229-242.

Thomas C, Gupta V and Ahsan F (2009) Influence of surface charge of PLGA particles of recombinant hepatitis B surface antigen in enhancing systemic and mucosal immune responses. Int J Pharm.

Thomas C, Rawat A, Bai S and Ahsan F (2008) Feasibility study of inhaled hepatitis B vaccine formulated with tetradecylmaltoside. J Pharm Sci 97:1213-1223.

Vanbever R, Mintzes JD, Wang J, Nice J, Chen D, Batycky R, Langer R and Edwards DA (1999) Formulation and physical characterization of large porous particles for inhalation. Pharm Res 16:1735-1742.

Voswinckel R, Ghofrani HA, Grimminger F, Seeger W and Olschewski H (2006) Inhaled treprostinil [corrected] for treatment of chronic pulmonary arterial hypertension. Ann Intern Med 144:149-150.

Xu H, Verbeken E, Vanhooren HM, Nemery B and Hoet PH (2004) Pulmonary toxicity of polyvinyl chloride particles after a single intratracheal instillation in rats. Time course and comparison with silica. Toxicol Appl Pharmacol 194:111-121.

Yang YY, Chung TS and Ng NP (2001) Morphology, drug distribution, and in vitro release profiles of biodegradable polymeric microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. Biomaterials 22:231-241.

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Table 2.1: Composition and polydispersity index (PDI) of the microspheric formulations

Formulation Polymer IAP OP EAP PDI MCP-1 Saline 1% w/v PVA 2.03±0.31 PLGA Di-chloro- MCP-2 Saline 2% w/v PVA 2.88±0.35 50:50 methane MCP-3 Saline 5% w/v PVA 2.18±0.24 MCP-4 Saline 1% w/v PVA 2.02±0.19 PLGA Di-chloro- MCP-5 Saline 2% w/v PVA 3.52±0.46 85:15 methane MCP-6 Saline 5% w/v PVA 2.02±0.34

IAP = Internal Aqueous Phase OP = Organic Phase EAP = External Aqueous Phase PDI = Polydispersity Index

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Table 2.2: Pharmacokinetic parameters of PGE1-loaded PLGA microspheric formulations. Data represent mean ± SEM (n=6-8)

AUC t Absolute Formulation C (ng/ml) 0-24 1/2 max (ng/ml.min) (minute) Bioavailability (F) Plain PGE I.V. 1 80.09±9.37 723.53±86.4 1.5±0.2 - Plain PGE Pulmonary 1 56.73±6.2 594.16±66.1 3.49±0.41 0.821±0.15 MCP-1 13.18±1.34 171.87±21.2 40.86±4.65 0.238±0.08 MCP-4 8.48±2.15 741.08±90.1 582.39±70.3 1.024±0.15* MCP-6 4.94±1.23 216.1±24.14 20.92±3.2 0.30±0.06

*Significantly different from plain PGE1 pulmonary administration (p<0.05)

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SEM Images of PLGA Microparticles

A B

C D

E F

Fig. 2.1: SEM of PGE1-loaded PLGA microspheres. (A) MCP-1, (B) MCP-2, (C) MCP-3, (D) MCP-4, (E) MCP-5, and (F) MCP-6. See Table 2.1 for composition of the different formulations.

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Physical Characterization of Microparticulate Formulations

A B

* *

16 0.22 14 0.20 12 0.18 10 8 0.16

6 0.14 4 0.12 2 (g/ml) Density Tapped

Mean Volume Diameter (um) Diameter Volume Mean 0 0.10 1 2 3 4 5 6 -1 -2 -3 -4 -5 -6 P- P- P- P- P- P- CP CP CP CP CP CP C C C C C C M M M M M M M M M M M M

C D * *

Theoretical MMAD 7 Actual MMAD 70 6 60 5 50

M) μ 4 40

3 30

MMAD ( MMAD 2 20

1 Entrapped Drug % 10 0 0 1 2 3 4 5 6 -1 -2 -3 -4 -5 -6 P- P- P- P- P- P- CP CP CP CP CP CP MC MC MC MC MC MC M M M M M M

Fig. 2.2: Physical characteristics of PGE1-loaded PLGA microspheres. (A) Volume- based mean diameter, (B) Tapped density, (C) Theoretical mass median aerodynamic diameter (MMADt), and (D) Entrapment efficiency. Data represent mean ± standard error of the mean (n=3).

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In-vitro Release Profiles

A B

45 24 MCP-1 MCP-4 MCP-2 22 40 MCP-5 MCP-3 20 MCP-6 35 18 30 16 14 25 12 20 10 15 8 6 10 4 5 % Cumulative Release%

% Cumulative Release Cumulative % 2 0 0 0 20 40 60 80 100 120 0 20406080100120 Time (Hours) Time (Hours)

C 11 10 % Surface Associated Drug % Burst Release 9 Release Rate (% Release/hr) 8 7 6

5 4 3 % Drug Release 2 1 0 1 2 3 4 5 6 P- P- P- P- P- P- MC MC MC MC MC MC

Fig. 2.3: Physical characteristics of PGE1-loaded PLGA microspheres. (A) In vitro release profiles of PGE1 from microspheres prepared with PLGA 50:50, (B) In vitro release profiles of PGE1 from microspheres prepared with PLGA 85:15, and (C) In vitro release pattern showing surface-associated release, burst release, and percent drug released per hour. Data represent mean ± standard error of the mean (n=3).

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In-vivo Absorption Studies

A 80 Saline 70 PGE1 I.V. PGE1 Pulmonary 60

20 Concentration (ng/ml) 1 10 PGE 0 0 2 4 6 8 10 12 14 16 18 Time (minutes) B 14 MCP-1 MCP-4 14 12 MCP-6 MCP-1 MCP-4 10 12 MCP-6 8

10 6

Concentration (ng/ml) 4 1 8 2

PGE

0 6 0 102030405060 Time (min) 4 Concentration (ng/ml) 1 2 PGE 0 0 10 20 30 300 600 900 1200 1500 TIme (min)

Fig. 2.4: In vivo absorption studies: (A) Changes in plasma levels of PGE1 after administration of plain PGE1 (80 µg/kg) via the intravenous and pulmonary routes compared with PGE1 levels after pulmonary administration of saline (n=6), and (B) In vivo performances of the formulations (MCP-1, -4, and -6) following pulmonary administration at a dose of 80 µg/kg. ((n=6-8). Data represent the mean ± standard error of the mean.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Metabolic Degradation Studies

Concentration (ng/mg Protein) 1 0 PGE 0 20406080100120 Time (min) B

Fig. 2.5: Metabolic degradation of PGE1 in rat lung homogenate after addition of (A) plain PGE1 (80 ng/mL) and (B) MCP-4 (5 mg). Data represent mean ± standard error of mean (n=3).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

In-vitro and In-vivo Safety Studies

A B

140 0.5 10 mg/ml 5 mg/ml * 120 2 mg/ml 0.4 1 mg/ml 100 0.5 mg/ml

0.3 80

60 0.2

40 % Cell Viability Cell % 0.1 20 *

Weight) (g/100g Weight Wet Lung 0 0.0

e S 1 4 6 e 1 4 ) in D P- P- P- in P- P- l al S C C C al C C g/m S 1% M M M S M M c 0. 1 m 0. S C D LP

300 700

600 * 250 *

500 200

400 150 300 100 200 50 ALP Activity (IU/L) LDH Activity (IU/L) Activity LDH 100

0 0 e 1 4 ) e 1 4 l) in - - ml lin P- P- /m al CP CP g/ Sa C C cg S M M c M M m 1 m .1 0. (0 ( S PS LP L

Fig. 2.6: (A) Effects of microspheric formulations (MCP-1, 4, and 6) on the viability of Calu-3 cells. The test samples contained 0.5, 1.0, 2.0, 5.0, or 10.0 mg/ml of microspheres (n=16). (B⎯D) Safety studies with bronchoalveolar lavage (BAL) fluid analysis 12 h after administration of the formulations. (B) Corrected wet lung weights. (C) Levels of lactate dehydrogenase (LDH), and (D) alkaline phosphatase (ALP). Data represent mean ± standard error of the mean (n=4).

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

CHAPTER 3

PEI-MODIFIED PLGA MICROSPHERES OF PGE1 FOR NONINVASIVE TREATMENT

OF PULMONARY ARTERIAL HYPERTENSION

INTRODUCTION

The lungs are affected by an array of disorders, including infection, inflammation, obstruction, fibrosis, and vascular diseases such as thrombosis and arterial hypertension. Many lung disorders are currently treated by therapeutic agents that are required to be administered by systemic routes such as the parenteral and oral routes

(Shulman, 2000; Rubin et al., 2002; Ewert et al., 2007; Taniguchi et al., 2010). Because of this systemic administration, the body is exposed to drugs that may harm other vital organs such as the heart and kidney. Such off-target effects in the treatment of lung diseases can be minimized by administering the drugs directly to the lungs. Indeed, the pharmacotherapy of certain pulmonary disorders, including asthma and pulmonary arterial hypertension (PAH), currently involves the use of nebulizers and inhalers for localized delivery of drugs to the lungs (Olschewski et al., 2002; Papi et al., 2007).

However, these formulations or delivery systems suffer from a wide range of limitations that include multiple inhalations a day, short duration of action, metabolic instability in the lungs, and drug loss due to premature deposition in the oropharyngeal tract

(Lipworth, 1995; Chrystyn, 2000; Lee and Rubin, 2005). Short duration of action and metabolic instability often stem from the fact that currently marketed inhalable formulations consist of drug dissolved in a mixture of solvents and propellants or plain drug formulated with respirable lactose (Labiris and Dolovich, 2003b; Labiris and

Dolovich, 2003a). These shortcomings can be addressed in two ways: chemical modification of the drug, or reformulation of the drug in controlled-release polymeric

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carriers. However, the latter approach is preferred because chemical modification often leads to reduction in pharmacological activity. Chemical modification of heparin, for example, has resulted in reduced anti-coagulant activity (Lee et al., 2001; Park et al.,

2010).

In fact, polymeric particulate carriers have been used for many years to prolong the duration of action and improve the stability of numerous drugs (Shive and Anderson,

1997; Lemoine and Preat, 1998; Ertl et al., 1999). Of the various polymeric carriers, poly

(lactic-co-glycolic acid) (PLGA)-based particles have been extensively investigated for the delivery of drugs via the pulmonary route (Kawashima et al., 1999; Suarez et al.,

2001; Mohamed and van der Walle, 2006; Ohashi et al., 2009; Hirota et al., 2010).

Moreover, there has been intense interest in the use of large porous PLGA particles for prolongation of the duration of action of inhaled drugs since the publication of Edwards’ seminal paper in 1997. In that paper, Edwards and colleagues proposed that large porous particles with mass densities <0.4 g/cm3 and mean volume diameter >5 μm provide deep lung deposition of the inhaled drug and help bypass clearance mechanisms in the lungs, thereby providing enhanced respirability and prolonged residence time in the lungs (Edwards et al., 1997). In agreement with this study, we and others have shown that porous particles with a density of <1 g/cc release drugs for a longer period of time compared to nonporous, high-density particles (Rawat et al., 2008).

PAH is a progressive disease that results from remodeling of the pulmonary vasculature

(Gaine and Rubin, 1998; Martin et al., 2006). It is a debilitating disease affecting 50,000-

100,000 Americans, with 300-500 new cases diagnosed each year. The current therapeutic strategies for PAH involve the use of short-acting inhalable or injectable formulations of anti-PAH drugs. Unfortunately, the current pharmacotherapeutic

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

approaches for PAH are plagued with many disadvantages, including a requirement of 9 to 12 inhalations a day (Ventavis®, Iloprost inhalation solution), or intravenous or subcutaneous injections (Ricachinevsky and Amantea, 2006). In a very recent study, we showed that PLGA microparticles can be used to overcome the limitations of short duration of action and metabolic instability of an investigational anti-PAH drug, prostaglandin E1 (PGE1) (Kunimoto et al., 1997; Shen et al., 2005; Gupta and Ahsan,

2010; Gupta et al., 2010). In fact, PGE1 is a potent pulmonary vasodilator with a very

short biological half-life, 3-5 minutes; however, it produces systemic side effects when

administered intravenously (Meyer et al., 1998). Our published study suggests that it is

feasible to develop a long-acting inhalable formulation of PLGA-based microparticles of

PGE1 that releases the drug for a period of 8 hours and provides better metabolic stability when compared with plain drug administered via the pulmonary route. However, reduced drug loading and poor drug deposition patterns remain problematic for PLGA- based inhalable formulations of this anti-PAH drug.

Recently, Rawat et al. showed that incorporation of a porosigen such as polyethyleneimine-25 kDa (PEI-25 kDa) in the aqueous core of PLGA microparticles results in highly porous particles for pulmonary delivery of low molecular weight heparins

(Rawat et al., 2008). PEI, a hydrophilic polycation, is known to produce large porous particles with a uniform distribution of pores owing to the osmotic pressure gradient produced between the microparticle core and the external aqueous phase. In addition to facilitating formation of porous particles through the osmotic pressure gradient, PEI is likely to form an electrostatic complex with PGE1 that results in improved solubility and

enhanced entrapment of the drug in microparticulate systems. Further, PEI-25 kDa, at

therapeutically safe doses, has been reported to work as an absorption enhancer for

pulmonary delivery of low molecular weight heparins (Yang et al., 2006). This study

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therefore tests the hypothesis that PEI-modified large porous microparticles of PGE1 have high drug-loading capacity, an extended drug-release profile, and favorable respirability for deep lung deposition.

MATERIALS AND METHODS

Materials

Poly (lactic-co-glycolic acid) (PLGA) 85:15 with an inherent viscosity of 0.55-0.75 dl/g

(average molecular weight = 85.2 kDa) and prostaglandin E1 (PGE1) were obtained from

Lactel Absorbable Polymers (Pelham, AL) and Spectrum Chemicals (Gardena, CA),

respectively. Polyethyleneimine (PEI) 25 kDa, poly vinyl alcohol (PVA), and

dichloromethane (DCM) were purchased from Sigma-Aldrich, Inc. (St Louis, MO). Kits to

assay PGE1 and protein were acquired from Assay Designs, Inc. (Ann Arbor, MI) and

Pierce Biotechnology (Rockford, IL), respectively. Alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) kits were procured from Pointe Scientific, Inc. (Canton,

MI). All other chemicals were of analytical grade and used without any modification.

Preparation of Core-Modified Large Porous PLGA Microparticles

Core-modified large porous PGE1-loaded PLGA microparticles were prepared by a

water-in-oil-in-water (W/O/W) double emulsion/solvent evaporation method after slight

modification of a published protocol (Rawat et al., 2008; Gupta et al., 2010). Briefly, 250

mg of PLGA was initially dissolved in 5 mL of DCM (organic phase, OP). The internal

aqueous phase (IAP) was prepared by adding 5 mg of PGE1 to 500 μL of deionized water after solubilizing the drug in a minimal quantity of absolute alcohol. PEI 25 kDa, used as a core-modifying agent, was added to IAP at varying concentrations (1.25, 2.5, and 5% w/v; Table 3.1). The IAP and OP were emulsified (W/O) by probe sonication for

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

1 minute using a Branson® Sonifier 450 (Branson Ultrasonics Corporation, Danbury,

CT). The primary emulsion was then homogenized with 25 mL of PVA solution (external

aqueous phase, EAP) at two different concentrations (1% and 2%; Table 3.1) at 8,000

rpm for 10 minutes with an Ultra-Turrex T-25 Basic (IKA, Wilmington, DE). The resultant

W/O/W emulsion was stirred overnight at room temperature to facilitate the removal of

the OP. Next day, the formulations were washed by centrifugation with deionized water

to remove excess PVA and lyophilized to obtain free flowing powdered microparticles

(FreeZone 2.5, Labconco Corporation; Kansas City, MO).

Physical Characterization

Particle Morphology: Particle morphology was examined by using a scanning electron microscope (Hitachi® S-3400N; Hitachi High Technologies America, Inc, Pleasanton,

CA). The samples were prepared by sprinkling a small amount of powdered formulation onto a double-sided adhesive tape attached to an aluminum stub. The particles placed on the stub were sputter coated with gold under argon (Emitech K550X; Quorum

Technologies Ltd, Kent, UK) and were viewed under the electron microscope.

Photomicrographs of the formulations were taken at varying magnifications.

Particle Size: Particle size distribution and mean volume diameters of all the formulations were determined by Tri-laser diffraction technology using a Microtrac®

S3500 (Microtrac, Inc; Largo, FL). Samples for wet-fluid sampling were prepared by dispersing 2 mg of microparticulate formulations in 0.2% w/v solution of Tween-80.

Polydispersity indices (PDIs) were calculated as a ratio of volume-averaged mean particle size to number-averaged mean diameter. All the formulations were measured in triplicates and the data are presented as mean±SD.

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Tapped Density Measurements: The density of microspheric formulations was estimated from tapped density measurements as described previously (Vanbever et al., 1999). An aliquot of microspheres was transferred to a 10 (±0.05) mL graduated cylinder and the initial volume was recorded. The tapped density of the formulations (ρ) was calculated as the ratio between sample weight (g) and the volume (mL) occupied after 100 taps.

Mass Median Aerodynamic Diameter Determination: The mass median aerodynamic diameter (MMAD) was studied in an 8-stage Mark-II non-viable Anderson Cascade

Impactor (ACI) (Westech Instruments, Marietta, GA). Briefly, freeze dried microparticulate formulations (10 mg) were filled in size 3, hard gelatin capsules and fired into the cascade impactor via the induction port using a Rotahaler (Spiriva®

Handihaler®, Boehringer Ingelheim, Inc., Ridgefield, CT). The impaction was performed

at a flow rate of 28.3 L/min for 2 minutes. To prevent bounce or re-entrainment of the

particles, glass fiber filter papers were placed in an inverted position on collection plates

of all stages 0-7 (Vanbever et al., 1999). The amount of particles deposited at each

stage was determined gravimetrically by calculating the differences in the weights of the

glass fiber filter papers. Data obtained were plotted on semilog graph paper and MMAD

was determined at 50% of the particle distribution profile. The fine particle fractions

(FPF) of the formulations were determined using the following equation:

< 4.7 3 100

Entrapment Efficiency: The amount of PGE1 entrapped in the microparticulate formulations was determined by quantifying the amount of PGE1 in the EAP, i.e. unentrapped PGE1 recovered after the first washing of the microparticles. The samples

4 5 recovered were diluted 10 -10 times and were assayed for the unentrapped PGE1 using

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

an ELISA kit (Assay Designs, Ann Arbor, MI) according to the protocol provided by the manufacturer. The kit can reproducibly quantify PGE1 in the range between 4.88 and

5,000 pg/mL. All measurements were performed in triplicates and data are presented as mean±SD.

Surface Area and Pore Size Determination: For determination of surface area and the extent of pore formation, nitrogen physiosorption isotherms were obtained at 77K using a Nova 3200e Surface Area Analyzer (Quantachrome Instrument Corporation, Boynton

Beach, FL). For physiosorption isotherm determination, the samples were first degassed at room temperature for 24 hours to remove any of the dissolved components from the surface so as to yield a higher surface area. After completion of degassing, samples were placed on the surface area analyzer in a sample cell with a dewar flask filled with liquid nitrogen underneath the sample cell. After generation of the isotherm, surface areas of all the microparticulate formulations were evaluated by using Brunauer-Emmett-

Teller (BET) and Barrett-Joyner-Halenda (BJH) adsorption isotherms. The average pore radius and cumulative pore volumes were calculated from the desorption curve of the isotherm (Gill et al., 2009). The analysis was performed in triplicates for 24 hours with a

150 s equilibrium interval.

In-vitro Release Studies in Simulated Interstitial Lung Fluid

In-vitro drug-release studies were performed in a simulated interstitial lung fluid (SILF) prepared based on the “Moss Formula” (Moss, 1979) (Table 3.2). Briefly, SILF was prepared as per the recipe described in Table 3.2 (Moss, 1979) and the pH of the fluid was maintained at 7.0-7.2 throughout the experiment. An aliquot of formulations (10 mg) was dispersed in 1 mL of SILF and incubated at 37±10C under moderate shaking (∼200 rpm). Samples were withdrawn at predetermined time intervals and replaced with an

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

equivalent amount of fluid to maintain the pH between 7.0-7.2. Collected samples were centrifuged at 16,000 g at 40C for 15 minutes and the supernatant was collected. The

drug content in the supernatant was quantified by ELISA. All measurements were

performed in triplicates and data are presented as mean±SD.

In-vivo Absorption Studies

Adult male Sprague-Dawley rats (Charles River Laboratories, Charlotte, NC) weighing between 250 g and 300 g were used for studying pulmonary absorption (n = 6-8). Briefly, rats were first anesthetized by an intramuscular (IM) injection of ketamine and xylazine cocktail (90 mg/kg + 10 mg/kg). The optimized formulations (PEI-1, and PEI-4) were administered via the intratracheal route utilizing a microsprayer (Penn-Century,

Philadelphia, PA). Microparticulate formulations were administered dispersed in normal saline at a dose of 120 μg/kg. Three control groups received the following formulations:

(i) normal saline via the pulmonary route, (ii) intravenous bolus dose of 80 μg/kg PGE1 via the jugular vein, and (iii) pulmonary dose of 80 μg/kg PGE1. Following administration, blood samples were collected for 24 hours in citrated microcentrifuge tubes containing

10 μg/mL indomethacin as a PGE1 synthase inhibitor. The plasma was separated by centrifuging the blood (5,000 rpm for 5 minutes) and stored in separate microcentrifuge

0 tubes at -20 C until further analysis. The plasma levels of PGE1 were determined by using the ELISA kit as described above. The plasma samples from untreated rats were used as negative controls to take into the account the influence of endogenous PGE1 on

the analysis.

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Metabolic Degradation Studies in Rat Lung Homogenates

To determine the stability of the optimized formulations (PEI-1 and PEI-4) under physiological conditions and to assess their controlled release properties, metabolic degradation studies were performed in rat lung homogenates according to a protocol established in our laboratory with slight modification (Gupta et al., 2010). Briefly, lungs from healthy adult male Sprague-Dawley rats (300-350 g) were surgically removed and homogenized in 4 volumes of cold homogenization buffer (Bücher medium – 20 mM

KH2PO4, 72 mM K2HPO4, 27.6 mM nicotinamide, and 3.6 mM MgCl2, pH 7.4) (Nakano et

al., 1973) using a Potter-Elvehjem glass homogenizer. Supernatant collected after

centrifugation was used for further studies. For metabolic degradation studies, lung

homogenates were incubated with (i) plain PGE1 (10 μg/mL), (ii) PEI-1, or (iii) PEI-4

+ 0 containing 10 μg PGE1 each for 8 hours in the presence of 2 mM NAD at 37 C in an

oscillating water bath. The reaction was terminated at predetermined time points by

adding 200 μL of 0.1N HCl to the incubation mixture. The amount of drug released in the

homogenate samples was determined by PGE1-ELISA. To determine the amount of drug

remaining in the formulations, particles in the homogenate were digested and extracted

with 5 volumes of DCM. Following the extraction, the organic layer containing DCM with

polymer and drug was transferred to a clean test-tube and evaporated in a N2 stream by

using N-Evap (Organomation Associates Inc, Berlin, MA). PGE1 was then extracted by vortexing the residue in the test-tube with 1.0 mL of phosphate-buffered saline (PBS, pH

7.2) for at least 30 seconds. The PBS solution thus obtained was assayed for PGE1 content by ELISA. This amount of PGE1 was considered to be the amount of PGE1 not

released upon incubation in lung homogenates.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Bronchoalveolar Lavage Studies

Bronchoalveolar lavage (BAL) fluid studies were performed to establish the safety profile of the formulations (PEI-1 and PEI-4) according to our previously published procedure

(Hussain and Ahsan, 2005; Thomas et al., 2008; Gupta et al., 2010). Briefly, four groups of adult male Sprague-Dawley rats (300-400 g) (n = 4) were anesthetized with a cocktail of ketamine and xylazine, and were treated with (i) saline as a negative control, (ii) PEI-1 equivalent to 120 μg/kg PGE1, (iii) PEI-4 equivalent to 120 μg/kg PGE1 and (iv) 0.1% w/v

solution of sodium dodecyl sulfate (SDS) as a positive control. The lungs were surgically

removed 12 hours post administration from anesthetized rats by exposing the respiratory

system by a mid-level incision in the thoracic cavity and severing the abdominal aorta.

Excised lungs were carefully cleaned from adjoining tissues/organs and were weighed to

investigate the possibility of edema formation. Lungs were lavaged with 5 mL normal

saline instilled through the trachea. Saline was left into the lungs for 30 s, withdrawn, re-

instilled for 30 s, and finally withdrawn and collected in microcentrifuge tubes. The BAL

fluid samples thus collected were centrifuged at 500g for 10 minutes, and the

supernatant was collected and stored at -200C until further analysis (24-48 hours). BAL

fluid samples were analyzed for (i) total protein concentration, (ii) ALP and (iii) LDH.

All animal studies were performed in accordance with NIH Guidelines for the Care and

Use of Laboratory Animals under a protocol approved by Texas Tech University Health

Sciences Center (TTUHSC) Animal Care and Use Committee (AM-10012).

Cell Viability Studies

For determining the extent of cytotoxic effects of the optimized formulations on lung epithelial cell lines, cell viability studies were performed by propidium iodide exclusion

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

fluorescence assay using the Calu-3 cell line (Nieminen et al., 1992). Briefly, Calu-3 cells

(ATCC, Manassas, VA) were seeded at a density of 20,000 cells/well in a 96-well black microtiter plate (Corning, Inc., Corning, NY) in MEM medium. The cells were allowed to

0 attach overnight in a 5% CO2 incubator at 37 C. All experiments from here onwards

were performed in Krebs-Ringer-N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid

(HEPES) buffer (KRH) containing 115 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM KH2PO4,

1.2 mM MgSO4, and 25 mM NaHEPES (pH 7.4) (Qian et al., 1997).

Immediately prior to the experiment, media was removed from the wells, cells were

washed with KRH buffer twice and 20 μL of test formulations were added at

concentrations of 0.5, 1.0, 2.0, and 5.0 mg/mL. SDS at a concentration of 0.1% was

used as a positive control. Following a 4 h incubation at 37°C, test samples were

removed, cells were washed with KRH buffer twice and background fluorescence for

each well was recorded after adding 100 μL buffer. The cells were then treated with

propidium iodide solution (100 μL of 5 μM), incubated at 370C for 30 minutes and

measured for fluorescence (F). Digitonin at a final concentration of 375 μM was added to

permeabilize the cells and subsequently label them with propidium iodide. Following

addition of digitonin, the fluorescence measurement was repeated every 5 minutes for

15 minutes, which was considered as the final fluorescence (Fmax) and used to calculate

cell viability using the following equation (Sarafian et al., 1994; Sarafian et al., 2002):

% 100 100

All fluorescence measurements were performed in a SynergyMX microplate reader

(Biotek, Winnoski, VT) using an excitation wavelength (Ex) of 546 nm and an emission wavelength (Em) of 620 nm at a band pass of 20 nm.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Pharmacokinetic and Data Analysis

Pharmacokinetic parameters for in-vivo absorption studies were determined by using

standard non-compartmental extravascular analysis (Kinetica®, version 5.0, Thermo

Fisher Scientific, Waltham, MA). Data were analyzed for area under the concentration time curve (AUC0-24h), Cmax, Tmax, and t1/2. All data were analyzed by one-way ANOVA followed by appropriate post hoc analysis (GraphPad Prism version 5.0, GraphPad

Software, La Jolla, CA). Values showing p<0.05 were considered significantly different.

RESULTS AND DISCUSSION

Physical Characterization:

A series of experiments was performed to study the micromeritics−i.e., morphology, size, density, porosity and pore size−of the particles to be used for depositing PGE1 in the

respiratory tract.

First, the morphology of the particles was examined under a scanning electron

microscope. All formulations were comprised of spherical particles with a porous surface

(Fig. 3.1). The degree of porosity increased with increasing PEI concentration

(5.0%>2.5%>1.25%), which agrees with our previous study that showed that

incorporation of PEI in the IAP resulted in very large particles with highly porous

surfaces (Gupta et al., 2010). PEI, an osmotically active polycation, increases the

porosity by pulling water into the particle core during emulsification. Subsequent removal

of this water by lyophilization results in the formation of holes or pores on the particle

surface. This assumption agrees with other published studies suggesting that surface

porosity results from increased osmotic pressure in the aqueous core (Pistel and Kissel,

2000; Ravivarapu et al., 2000). The concentration of PVA (1% and 2%) used in the EAP

also significantly influenced particle morphology. Formulations with 1% PVA (Figs. 3.1A,

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

B, and C) showed spherical particles with a somewhat dented or bumpy surface, whereas formulations with 2% PVA in the EAP (Figs. 3.1D, 3.1E, and 3.1F) showed uniformly distributed spherical particles with even surfaces. These observations are in agreement with published data that showed that PVA works as an emulsion stabilizer and can affect the hydrophobicity and digestibility of PLGA microparticles by being adsorbed on the particle surface (Jeong et al., 2003).

The particle size data presented in Fig. 3.2A suggest that the particle size of the various formulations varied depending on PEI concentration in the IAP and PVA concentration in the EAP. The particle size increased significantly when the PEI concentration was increased from 10.72±0.04 μM (PEI-1) to 22.53±0.255 μM (PEI-3)

and from 7.01±0.009 μM (PEI-4) to 12.26±0.115 μM (PEI-6). The increase in particle

size with increasing PEI concentration is because PEI leads to increased porosity

(Rawat et al., 2008). However, a reduction in particle size was observed when the PVA

concentration was increased from 1% to 2%. This reduction is because PVA stabilizes

the emulsion droplets by providing a hydrophilic environment (Jeong et al., 2003). The

polydispersity index values presented in Table 3.1 show that the formulations were

moderately polydispersed, with polydispersity indices ranging from 0.99 to 3.73.

The tapped density of particulate formulations can provide important information

concerning flowability, particle porosity and particle size distribution. Therefore, tapped

density was measured, and a decrease in tapped densities was observed with

increasing concentrations of PEI, suggesting that larger but lighter particles were

formed. However, no significant differences in tapped density were observed when the

PVA concentration was increased from 1% to 2%.The increase in tapped density with

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the increase in PEI concentration was due to the increased porosity of the particles, as observed in SEM photomicrographs.

The mass median aerodynamic diameter (MMAD), the most important particle parameter that determines the flight of the formulation over the route of inhalation, was determined by using an 8-stage non-viable Anderson Cascade Impactor. All formulations showed an aerodynamic diameter between 2.5 μm and 3.5 μm. While no direct

correlation between PEI concentration and MMAD was observed, a slight increase in

MMAD was observed with increasing PVA concentration despite the smaller particle size

(Fig. 3.2A). Importantly, MMAD was between 1-6 µm, which is considered optimal for

efficient deposition in the respiratory tract. In fact, for effective deposition of a particulate

drug-delivery carrier, a geometric diameter of 1-5 μm with a particle density of ∼1 g/cm3 is desirable (Adjei and Garren, 1990). However, Edwards et al. suggested that particles larger than 5 µm with reduced density (<0.4 g/cm3) can enhance deep lung deposition

and provide enhanced bioavailability by prolonging the release period of the drug

(Edwards et al., 1997).

The fine particle fraction (FPF) of all the formulations was determined from the fraction

of particles that was deposited at stage 3 or lower in the cascade impactor. All

formulations showed a FPF in the range of 50.98% to 63.16% (Fig. 3.2B). Similar to the

MMAD data, FPF also showed little or no correlation with PEI concentration, but

decreased with the increase in PVA concentration.

Surface area, pore volume, and pore radii were determined using a Nova 3200e

surface area analyzer (Gill et al., 2009). The surface area increased as a function of PEI

and PVA concentrations. In fact, the surface area data generated from two isotherms,

BET and BJH, showed very similar patterns (Fig. 3.3A). These data agree with the

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

particle size data presented in Fig. 3.2, which show that the presence of PEI produces a lighter, more porous particle in a concentration-dependent manner. Further, the data also agree with the assumption that increasing the PVA concentration produces less porous, smaller particles. Interestingly, the increase in surface area was directly proportional to the increase in particle size, which contradicts an established fact that surface area is inversely related to particle size. Indeed, mathematically, the external surface area of a non-porous particle decreases with increasing particle size. However, in the case of porous particles, the internal surface area takes into account the porous nature of the particles in addition to the external surface area. For this reason, there was a net increase in the specific surface area with the increase in porosity of the particles, as pointed out by Jeyanthi et al (Jeyanthi et al., 1997). Similar to the surface area data, an increase in the pore volume was observed with the increase in PEI or PVA concentration (Fig. 3.3B). The pore volume data corroborate the porous character and extent of porosity observed by SEM. The pore radii of the formulations were 27-28 Å, and no significant differences in pore radii were observed among the various formulations (Fig. 3.3C).

Following the micromeritic analysis, the entrapment efficiencies for all the formulations were evaluated by analyzing drug content in the supernatant recovered after washing the microparticles (Fig. 3.4A). All PEI-modified formulations showed excellent entrapment efficiency compared to their plain counterparts. In the first set of formulations

(1% PVA), the entrapment efficiency was 83.26±3.04% when PEI was used at a concentration of 1.25%. However, the entrapment efficiency increased from 97% to 99% when the PEI concentration was increased from 1.25% to 2.5% or 5%. Similar increases in drug loading were observed when the PVA concentration was 2%, although no major differences were observed with the change in PEI concentration. In fact, PEI-

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

modified particles showed a considerable increase in drug loading compared to our previously published study on PLGA particles of PGE1, in which drug entrapment

efficiency was between 59.44% and 65.51% (Gupta et al., 2010). Such a dramatic

increase in drug loading in PEI-modified particles can be explained on the basis of

following two assumptions: First, PEI is a positively charged polymer which forms an

electrostatic complex with negatively charged PGE1, resulting in retention of a larger

amount of drug in the particle core. Second, both PEI and PVA increase entrapment by

enhancing the stability of the primary emulsion (Lee et al., 1999; Cirpanli et al., 2005).

Overall, the micromeritic analysis of the particles shows that PEI as a porosigen and

PVA as an emulsifier play important roles in controlling the size and porosity of the

formulations and that the particles meet the standard for an inhalable formulation.

Similar to the influence of PEI and PVA on the physical properties of the particles, these

formulation additives play a major role in the loading of drug in PLGA particles.

In-vitro Release Studies in Simulated Interstitial Lung Fluid:

The release of PGE1 from PLGA particles was studied in simulated interstitial lung fluid

(SILF). Drug released from all formulations, except PEI-1, followed a two-step process: an initial burst release phase followed by a nearly zero-order release (Fig. 3.4B). There was no further release after 10 hours of the study. Formulations containing 1.25% PEI produced a cumulative release of 55.36±0.06% over a 24-hour period. The amount of drug released from 1.25% PEI-modified particles was 2- to 5-fold higher than that from plain PLGA particles reported in our earlier paper on PLGA particles of PGE1 (Gupta et al., 2010). Such a large improvement in drug release was because of the porous nature of the particles, as was observed for porous PLGA particles of low molecular weight heparin (Rawat et al., 2008). In the case of porous particles, a larger amount of release medium comes in contact with the particle surface and facilitates drug release. However,

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the amount of drug released was dramatically reduced when the PEI concentration was increased. For example, the cumulative drug release for the formulations containing

2.5% and 5% PEI was 35.02±2.31% and 16.59±2.17%, respectively. Similar to the first set of formulations containing 1% PVA, in the second set of formulations containing 2%

PVA, a significant increase in drug release was observed for the 1.25% PEI-modified microspheres, whereas the formulations containing 2.5% or 5% PEI showed lower amounts of drug release. The observed decrease in cumulative drug release with increasing PEI concentration may stem from the fact that PEI, when used at a higher concentration, forms a strong electrostatic complex with PGE1 that prevents dissociation of the drug from PEI. Further, because of its hydrophobic nature, PVA has been reported to significantly reduce the release of drugs at higher concentrations (Jeong et al., 2003).

The in-vitro release profiles were further analyzed to calculate the following release parameters: (i) surface-associated drug release, i.e., the amount of drug released at 0 minutes; (ii) burst release, the amount of drug released in the first 30 minutes; (iii) and the amount of drug released per hour. With increasing PEI concentration there was an increase in the amount of surface-associated release (Fig. 3.4C). The percentages of surface-associated drug released from the first three PEI-modified formulations containing 1% PVA were 1.85±0.73%, 2.86±0.01% and 6.28±0.31%, respectively.

Formulation containing 2% PVA showed a similar pattern. Interestingly, PEI-2 (1% PVA, and 2.5% PEI) showed the greatest increase in burst release, followed by PEI-3 and

PEI-1, which suggests the presence of greater amounts of drug on the particle surface or in the distal core of the particles. This behavior may have been due to rapid degradation of the particle core during the in-vitro release studies, thereby causing a higher burst release.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

The release profiles performed in SILF suggest that PEI-1 and PEI-4 microspheres are likely to produce a prolonged-release effect after inhalation. It is worth noting that SILF is a solution of electrolytes that mimics the electrolytes present in the surfactant-rich fluid released by Type II alveolar cells, which fills the space between alveolar cells and maintains the surface tension of water in the lungs (Daniels and Orgeig, 2003). In terms of electrolyte composition, SILF is virtually identical to actual human lung fluid, although it lacks the proteins present in actual lung fluids. However, in SILF the total ionic contribution from protein is compensated for by citrate ions (Moss, 1979). Because SILF provides an environment that is close to that of the mucosal fluids in the respiratory system, in-vitro release studies in this medium is likely to give a more accurate estimate of the drug that would be released upon pulmonary or intratracheal administration of the formulations. Importantly, there are very few studies that have used SILF for studying the release properties of an inhaled formulation (Davies and Feddah, 2003; Cook et al.,

2005). This is the first study to document the release profile of an anti-PAH drug in a simulated lung fluid.

In-vivo Absorption Studies:

The formulations that showed prolonged released properties in vitro were tested for in-

vivo pulmonary absorption in adult male Sprague-Dawley rats. As shown in our earlier

study (Fig. 3.5A, reproduced from (Gupta et al., 2010)), administration of plain PGE1 (80

μg/kg body weight) via the intravenous route resulted in a rapid rise of PGE1 in the

blood, with a Cmax of 80.09±9.37 ng/mL and a half-life of 1.5±0.2 minutes. Pulmonary administration of plain PGE1 showed a Cmax of 56.73±6.2 ng/mL and a half-life of

3.49±0.49 minutes with a relative bioavailability (F) of 0.821±0.15. A rapid rise in drug concentration in the blood suggests that intact PGE1 traversed the pulmonary epithelial

membrane and reached the systemic circulation in biologically active form (Gupta et al.,

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

2010). Administration of two microspheric formulations, PEI-1 and PEI-4, at a dose of

120 μg/kg resulted in a significant increase in the half-life of PGE1 compared to the plain

drug administered via the pulmonary or IV route. With the increase in half-life of the drug

a concomitant reduction in Cmax was observed. In fact, compared to plain PGE1 administered via the pulmonary route, a 2.5- to 4-fold reduction in Cmax and a 100-110 times extension in half-life were observed when either of the two microspheric formulations was administered intratracheally (Table 3.3, Fig. 3.5B). The Cmax for PEI-1 microspheres was slightly higher than that for PEI-4, perhaps because the former had a more pronounced surface-associated drug release compared to the latter, as observed in Figs. 3.4B and 3.4C. Importantly, the data from the in-vitro release study provide a good estimate of in-vivo absorption after pulmonary administration.

Several factors may have contributed to the reduced Cmax and extended half-life of the two formulations. First, because of their porous nature and large geometric diameter, phagocytosis of the particles by alveolar macrophages was delayed or prevented, which allowed for prolonged release of the drug (Edwards et al., 1997). Second, the average biodegradation time for PLGA is about 5-10 weeks, which may also have contributed to the prolonged release of the drug after pulmonary administration. Third, it is also possible that the PVA coating on the microparticle surface prevents polymer degradation by hydrolysis and thus facilitates continuous drug release over a prolonged period (Lee et al., 1999). Fourth, the glass transition temperature of the polymer used also plays an important role in drug release upon polymeric degradation. The glass transition temperature of PLGA 85:15 used in this study is ∼450C, thus making it more prone to degradation under physiological conditions (Park and Jonnalagadda, 2006). Overall, both large porous formulations showed a significantly extended half-life and excellent bioavailability as compared to our published report with nonporous PLGA microparticles.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Metabolic Degradation Studies in Rat Lung Homogenates:

To determine whether the formulations that showed increased blood levels in the pulmonary absorption studies underwent metabolic degradation in the respiratory epithelium, we investigated the metabolic degradation profiles of PEI-1 and PEI-4 in rat lung homogenates. As discussed elsewhere, lung is a major site of metabolism of PGE1 and 70-80% of the drug is metabolized by oxidation, which is catalyzed by a microsomal enzyme, NAD+-dependent 15-hydroxy prostaglandin dehydrogenase (Nakano et al.,

1973). This metabolic pathway is primarily responsible for rapid clearance of PGE1 from the lungs. The metabolic degradation study showed that plain PGE1 (10 μg/mL) underwent rapid degradation in lung homogenate, with little or no drug being detectable after 2 hour’s incubation. The drug concentration decreased from 9.415±1.005 μg/mL at

time 0 to 0.505±0.05 μg/mL at 120 minutes. However, when the equivalent amount of

PGE1 encapsulated in PLGA microspheres was incubated in lung homogenates, the drug concentration in the homogenate continually increased over the period of the experiment. For PEI-1, the amount of PGE1 released increased from 78.9±5.42 ng/mL at

time 0 minutes to 141.9±15.42 ng/mL at 8 hours; for PEI-4, the amount of PGE1 increased from 32.83±4.21 ng/mL at time 0 to 142.42±20.741 ng/mL at 8 hours (Fig.

3.6A). The presence of PGE1 in the homogenates is an indication of continual release of the drug from the formulations as a result of time-dependent degradation of the polymeric core of the microparticles.

To further confirm the drug degradation in the lung homogenate, we also determined the amount of drug that remained unreleased from the microspheric formulations by extracting PGE1 from the microparticles incubated with the homogenates. As shown in

Fig. 3.6B, with the increase in drug release, a concomitant decrease in the amount of

drug in the polymeric microparticles was observed. The amount of drug in PEI-1

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

microspheres decreased from 3.96±0.45 μg/mL to 0.115±0.0142 μg/mL after 8 hours of incubation. A similar pattern was observed for PEI-4 microspheres: the drug concentration decreased from 12.87±1.3 μg/mL to 1.91±0.21 μg/mL after 8 hours (Fig.

3.6B). On the whole, in concurrence with the pulmonary absorption profiles, data on

metabolic degradation suggest that polymeric microparticles continuously release PGE1 over an extended period and protect the unreleased drug against metabolic degradation.

Safety Studies:

The safety of the formulations was studied in two sets of experiments: analysis of bronchoalveolar lavage and a cell viability study using a propidium iodide assay.

The acute safety of the formulations was studied by analyzing the bronchoalveolar lavage fluid collected from rats treated with two optimized formulations, PEI-1 and PEI-4, and a positive control 0.1% w/v solution of SDS. In addition to determining the levels of injury markers, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in the BAL fluid, total protein content and wet lung weight were also recorded to investigate accumulation of extracellular fluid in the epithelial cell lining of the respiratory wall upon drug inhalation. The presence of LDH, a cytoplasmic enzyme, in BAL fluid is representative of cell damage and lysis (Henderson et al., 1978). ALP is a membrane- bound lysosomal enzyme, and its presence in the BAL fluid represents alveolar type II cell proliferation in response to type I cell damage (Hussain and Ahsan, 2005). Further, total protein content of the BAL fluid is an important biomarker of permeability of the alveolar capillary barrier and inflammation (Beck et al., 1982).

The wet lung weights of rats treated with PEI-1 were significantly higher than those of saline-treated animals, but lower than those of 0.1% SDS-treated rats. The increase in wet lung weights caused by PEI-4 was much smaller than that produced by the PEI-1

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

formulations (Fig. 3.7A). Further, compared to the saline-treated animals, no formulations produced a remarkable increase in total protein content or elevation in LDH and ALP levels. Both formulations were safe compared to 0.1% SDS. However, these data should be treated with caution because of the acute nature of the study. A long- term study using PEI-1 and PEI-4 would give better insight into the pathological changes that may occur upon repeated administration of the formulations via the lungs.

The cell viability study was performed using a propidium iodide exclusion

fluorescence assay in Calu-3 cells, a lung epithelial cell line. Propidium iodide is a

fluorescent dye and an intercalating agent that binds to double-stranded nucleic acids of

non-viable cells by intercalating between bases. It is a membrane-impermeable

molecule and excluded from viable cells (Pavlik et al., 1985; Nieminen et al., 1992).

When bound to nucleic acids, propidium iodide causes a red shift and about a 20-to 30-

fold increase in fluorescence compared to background. The cell viability data presented

in Fig. 3.7C show high levels of cell viability when incubated with saline. In fact, saline-

treated cells were 100% viable, whereas SDS-treated cells showed a viability of

37.79±7.03%. However, neither formulation produced a major reduction in cell viability.

In the case of PEI-1, cell viability was reduced from 92.49±7.61% to 89.13±6.85% when

the concentration of the formulation was increased from 0.5 mg/mL to 5 mg/mL. A

similar reduction in cell viability was observed with PEI-4-treated cells at concentrations

between 0.5 and 5 mg/mL. It is important to note that, although PEI is a polycation, it did

not produce much cytotoxicity, perhaps because its interaction with PGE1 lowered its

charge density.

In summary, this study is first to investigate in-vitro release profiles of polymeric PLGA

microspheres of PGE1 in a simulated lung fluid. PEI-modified microspheres can enhance

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

the drug payload and produce large porous particles. The resulting microspheres meet micromeritical standards of inhaled particles. The microspheres provided an extended biological half-life in addition to offering protection against metabolic degradation, suggesting that these formulations can overcome important limitations of existing therapies for pulmonary hypertension, a pulmonary vascular disorder. Currently, studies are underway in our laboratory to investigate the efficacy of these formulations in producing sustained vasodilation of pulmonary arteries in a rodent animal model of pulmonary hypertension.

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Taniguchi H, Ebina M, Kondoh Y, Ogura T, Azuma A, Suga M, Taguchi Y, Takahashi H, Nakata K, Sato A, Takeuchi M, Raghu G, Kudoh S and Nukiwa T (2010) Pirfenidone in idiopathic pulmonary fibrosis. Eur Respir J 35:821-829.

Thomas C, Rawat A, Bai S and Ahsan F (2008) Feasibility study of inhaled hepatitis B vaccine formulated with tetradecylmaltoside. J Pharm Sci 97:1213-1223.

Vanbever R, Mintzes JD, Wang J, Nice J, Chen D, Batycky R, Langer R and Edwards DA (1999) Formulation and physical characterization of large porous particles for inhalation. Pharm Res 16:1735-1742.

Texas Tech University Health Sciences Center, Vivek Gupta, December 2010

Table 3.1: Composition and Polydispersity Indices (PDI) of Different Microspheric Formulations

Formulations IAP:OP:EAP IAP EAP PDI Tapped Composition Composition Density (w/v) (w/v) (g/cm3) Plain-1 ⎯ 3.73±0.059 0.36±0.12 PEI-1 1.25% PEI 1.41±0.005 0.21±0.03 1% PVA PEI-2 2.5% PEI 0.93±0.012 0.15±0.02 PEI-3 5% PEI 0.95±0.012 0.1±0.003 0.5:5:25 Plain-2 ⎯ 2.14±0.058 0.45±0.11 PEI-4 1.25% PEI 1.42±0.001 0.25±0.02 2% PVA PEI-5 2.5% PEI 0.92±0.003 0.2±0.014 PEI-6 5% PEI 0.99±0.0250.146±0.012 IAP, Internal Aqueous Phase; OP, Organic Phase; EAP, External Aqueous Phase; PEI, Polyethyleneimine 25 kDa; PVA, Polyvinyl Alcohol; PDI, Polydispersity Index

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Table 3.2: Compositions of Actual Lung Fluids and Simulated Lung Fluids (modified from (Daniels and Orgeig, 2003)).

Ion Actual Lung Fluid Simulated Lung Fluid (mEq) (mEq) Calcium, Ca++ 5.0 5.0 Magnesium, Mg++ 2.0 2.0 Potassium, K+ 4.0 4.0 Sodium, Na+ 145.0 145.0 Total Cations 156.0 156.0 - Bicarbonate, HCO3 31.0 31.0 Chloride, Cl- 114.0 114.0 3- Citrate, H5C6O7 - 1.0 - Acetate,H3C2O2 7.0 7.0 2- Phosphate, HPO4 2.0 2.0 2- Sulfate, SO4 1.0 1.0 Protein 1.0 - Total Anions 156.0 156.0 pH 7.3-7.4 7.3-7.4

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Table 3.3: Pharmacokinetic parameters of PGE1-loaded PLGA microspheric formulations. Data represent mean ± standard deviation (n=6-8).

Formulation Cmax (ng/mL) t1/2 (min) AUC0-24 Relative (ng/mL*min) Bioavailability (F)

Plain PGE1 I.V.* 80.09±19.37 1.5±0.2 723.53±86.4 --

Plain PGE1 56.73±6.2 3.49±0.41 594.16±66.1 0.821±0.15 Pulmonary* PEI-1 20.12±3.81 362.296±50.56 1038.11±180.1 0.957±0.13 PEI-4 14.51±2.91 390.66±55.71 973.52±130.2 0.897±0.17

AUC, Area under the concentration time curve; I.V., Intravenous; PEI, Polyethyleneimine 25 kDa *Adapted from Gupta et al., 2010

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SEM Images of PEI Modified Large Porous PLGA Microparticles

A B

C D

E F

Fig. 3.1: SEM of PGE1-loaded large porous PLGA microspheres. (A) PEI-1, (B) PEI-2, (C) PEI-3, (D) PEI-4, (E) PEI-5, and (F) PEI-6. See Table 3.1 for compositions of the different formulations.

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Physical Characterization of PLGA Microparticles

A Mean Volume Diameter 25 Mass Median Aerodynamic Diameter

M) 20 μ

Particle Diameter ( 5

-1 I-1 I-2 I-3 -2 I-4 I-5 I-6 ain E E E ain E E E Pl P P P Pl P P P

1% PVA 2% PVA B

Fine Particle Fraction (%)

-1 I-1 I-2 I-3 -2 I-4 I-5 I-6 ain E E E ain E E E Pl P P P Pl P P P

1% PVA 2% PVA

Fig. 3.2: Micromeritic profile of PGE1-loaded large porous PLGA microspheres. (A) Volume-based mean diameter and actual mass median aerodynamic diameter (MMAD); (B) % Fine particle fraction. Data represent mean ± standard deviation (n=3).

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Physiosorption-based Surface Area Characteristics

A Plain-1

45 * PEI-1 PEI-2

40 ** PEI-3 Plain-2 35 ** * PEI-4 /g) 2 30 PEI-5 PEI-6 25

10 Surface Area (m Surface Area 5 0

BET Isotherm BJH Isotherm

B C

0.07 * 35

0.06 30

0.05 25

/g) ** 3

0.04 20

0.03 15

0.02 10

5 Pore Volume (cm Volume Pore 0.01 Pore Radius (Angstrom)

0.00 0 -1 -1 -2 -3 -2 -4 -5 -6 -1 I-1 I-2 I-3 -2 I-4 I-5 I-6 in EI EI EI in EI EI EI ain E E E ain E E E la P P P la P P P Pl P P P Pl P P P P P 1% PVA 2% PVA 1% PVA 2% PVA

Fig. 3.3: Physiosorption-based surface characteristics of PGE1-loaded large porous PLGA microspheres. (A) Surface area as determined by BET and BJH adsorption isotherms, (B) Pore volume, and (C) Pore radii. Data represent mean ± standard error of mean (n=3). *Significant differences among the group (1% PVA) (p<0.05); **Significant differences among the group (2% PVA) (p<0.05).

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Entrapment Efficiency and In-vitro Release Pattern

A C 32 % Surface Associated Drug % Burst Release 30 Release Rate (% Drug Release/hr) 99.06±0.03 97.73±0.74 96.69±1.21 95.48±0.46 100 94.72±0.08 28 83.26±3.04 80 67.22±4.98

61.19±4.91 10 60

% DrugRelease 5

% DrugEntrapped % 20

0 0 1 1 2 3 2 4 5 6 n- I- I- I- n- I- I- I- -1 I-1 I-2 I-3 -2 I-4 I-5 I-6 ai PE PE PE ai PE PE PE ain E E E ain E E E Pl Pl Pl P P P Pl P P P

1% PVA 2% PVA 1% PVA 2% PVA

B Plain-1 60 PEI-1 PEI-2

50 PEI-3 Plain-2 PEI-4 40 PEI-5

PEI-6 30

% Cumulative Release Cumulative % 10

0 5 10 15 20 25 Time (Hrs)

Fig. 3.4: (A) Entrapment efficiencies of PGE1-loaded large porous PLGA microspheres, (B) In-vitro release profiles of PGE1 from PEI-modified PLGA microspheres, and (C) In- vitro release patterns showing surface-associated release, burst release, and percent drug released per hour. Data represent mean ± standard deviation (n=3).

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In-vivo Absorption Profile

A 80 Saline PGE Intravenous 70 1 PGE Pulmonary 1 60

20 Concentration (ng/mL)

1 10

PGE 0 024681012141618

Time (min) B 25 25 PEI-1 20 PEI-4 15 20 10 Concentration (ng/mL)

1 5 15 PGE 0 0 102030405060 10 Time (min)

Concentration (ng/mL)

0 0 100 200 300 400 1350 1500

Time (minutes)

Fig. 3.5: In-vivo absorption studies. (A) Changes in plasma levels of PGE1 after administration of plain PGE1 (80 µg/kg) via the intravenous and pulmonary routes (n=6, modified from (Gupta et al., 2010)), and (B) In-vivo performances of the formulations (PEI-1, and PEI-4) after pulmonary administration at a dose of 120 µg/kg of PGE1. Data represent the mean ± standard deviation (n=6-8).

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Metabolic Degradation Studies of Large Porous Microspheres

A PGE released from PEI-1 1 ( PGE1 Concentration PGE released from PEI-4 1.0 14 1 Plain PGE 1 12 0.8

10 0.6

g/mg Protein) μ 8 0.4 6 μ

g/mg Protein) g/mg 4 0.2

2 0.0 Concentration (

1 0 02468 PGE Time (Hours)

B 1.2 PGE entrapped in PEI-1 1 PGE entrapped in PEI-4 1.0 1

0.8 g/mg Protein)

μ 0.6

0.4

0.2 Concentration ( 1

0.0 PGE 012345678 Time (Hours)

Fig. 3.6: Metabolic degradation of PGE1 in rat lung homogenate. (A) Concentration of PGE1 upon incubation of 10 μg/mL plain PGE1, PEI-1, and PEI-4 containing 10 μg/mL PGE1. (B) The amount of drug extracted from PEI-1, and PEI-4 microspheres incubated in lung homogenates. Data represent mean ± standard deviation (n=3).

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In-vivo and In-vitro Toxicity Studies

0.70 (mg/mL) Concentration Protein Wet Lung Weight * 8 Protein Concentration 0.65 7 ** 6 0.60 5 0.55 4

0.50 3 2 0.45 1 0.40 0 Saline PEI-1 PEI-4 0.1% SDS Wet Lung Weight (g/100 weight)g body

0.5 mg/mL B Saline C 1 mg/mL 800 120 * PEI-1 2 mg/mL PEI-4 700 5 mg/mL 0.1% SDS 100 600 80 500 * 400 60 300 40

200 Viability Cell % Enzyme Activity (IU/L) Activity Enzyme 20 100 0 0 e 4 ALP LDH I- lin EI-1 E SDS P P Sa % 0.1

Fig. 3.7: Acute in-vivo toxicity studies of optimized formulations PEI-1 and PEI-4 in bronchoalveolar lavage (BAL) fluid after 12 hours of administration. (A) Corrected wet lung weights (g/100 g body weight) and total protein concentration (mg/mL). *Means are significantly different from (p<0.05) (protein concentration), and **Means are significantly different (p<0.05) (wet lung weight). (B) Levels of lactate dehydrogenase (LDH) (IU/L), and alkaline phosphatase (ALP) (IU/L). *Means are significantly different (p<0.05). Data represent mean ± standard deviation (n=4). (C) Acute cytotoxicity studies to determine in-vitro safety profiles of the optimized formulations, PEI-1, and PEI-4, by propidium iodide fluorescence exclusion assay in the Calu-3 respiratory epithelial cell line at 0.5, 1.0, 2.0, and 5.0 mg/mL of microspheric formulations. Data represent mean ± standard deviation (n=16).

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CHAPTER 4

INHALABLE PLGA MICROPARTICLES ENCAPSULATING PROSTAGLANDIN E1-

HYDROXYPROPYL-β-CYCLODEXTRIN (PGE1-HPβCD) COMPLEX FOR

PULMONARY ARTERIAL HYPERTENSION (PAH) TREATMENT

INTRODUCTION

Physiological absorption of a drug across biological membranes is governed primarily by its physicochemical properties, especially its aqueous solubility and chemical stability.

Many potential therapeutic drug candidates fail because of their extreme hydrophobic nature, which results in low absorption and poor bioavailability. The hydrophobicity of a molecule is a major determinate of its drug-like properties, influencing adsorption, distribution, selective protein binding, as well as metabolism. A physiologically effective drug needs to have an intermediate distribution coefficient that is neither too hydrophobic nor hydrophilic.

A variety of techniques and delivery systems aimed at increasing the aqueous solubility of hydrophilic molecules have been investigated. Cyclodextrin complexation is among the few techniques which have attracted the attention of the scientific community (Irie and Uekama, 1997; Hirayama and Uekama, 1999; Stella and He, 2008). Cyclodextrins are cyclic oligosaccharides derived from amylose sugar and are composed of a varying number of α-1-4- glucose units linked together in a ring structure. Glucose chains in the cyclodextrin molecule form a toroid-like structure, with a characteristic arrangement of primary and secondary hydroxyl groups that results in the formation of a cone-like cavity that is less hydrophilic than the aqueous environment. This cone-like cavity provides an environment for hydrophobic drug molecules, forming noncovalent inclusion complexes,

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thereby modifying the physicochemical properties of the drug molecule (Gould and

Scott, 2005; Jug and Becirevic-Lacan, 2008). Of the cyclodextrins commonly used as formulation vehicles, β-cyclodextrin (β-CD) has seven glucose units and exhibits an excellent absorption profile, but is also associated with hepatotoxicity and nephrotoxicity.

This led to development of 2-hydropropyl-β-cyclodextrin (HPβCD), a hydroxylalkyl derivative with improved water solubility, which is relatively non-toxic irrespective of the route of administration (Carpenter et al., 1995).

Prostaglandin E1 (PGE1; Alprostadil) is a compound with vasodilatory, anti-inflammatory,

anti-aggregatory, and anti-proliferative properties (Kloeze, 1969; Wallace, 1992; Sood et

al., 2004). PGE1 is approved by the U.S. Food and Drug Administration (FDA) for the treatment of erectile dysfunction in men and patent ductus arteriosus in newborns. It has been investigated in the treatment of various cardiopulmonary disorders, especially pulmonary arterial hypertension (Nakazawa et al., 1998; Sakuma et al., 1999; Igarashi et al., 2001; Della Rocca et al., 2008). However, the clinical utility of PGE1 has been limited by its chemical instability and extremely hydrophobic nature (log P = 3.3), which results in low drug absorption and poor bioavailability. Several reports have suggested using complexes of cyclodextrin and PGE1 to increase the aqueous solubility of PGE1 (Wiese

et al., 1991; Yamamoto et al., 1992; Uekama et al., 2001; Gu et al., 2005). Gu et al.

reported that a PGE1-HPβCD inclusion complex resulted in increased aqueous solubility

of PGE1 and increased absorption following nasal administration in Wistar rats (Gu et al.,

2005).

It has been reported that PGE1, when administered via the pulmonary route, works as a selective pulmonary vasodilator (Meyer et al., 1998). However, the main limitation of pulmonary delivery of PGE1 is its very short half-life (3 to 4 minutes) as a result of first-

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pass metabolism in the lungs. We have shown recently that PLGA microparticles of

PGE1 release the drug over a prolonged period of time, while keeping the drug stable in

lungs’ metabolic environment (Gupta et al., 2010). However, PGE1-encapsulated PLGA microparticles exhibit poor deep-lung deposition due to unfavorable aerodynamic properties. Moreover, the extreme hydrophobicity of PGE1 limits its passage through the

air-blood barrier. Among the physicochemical properties altered by cyclodextrin inclusion

complexes are the release properties of drug-encapsulated polymeric drug delivery

systems (Bibby et al., 2000; De Rosa et al., 2005). Many investigators have emphasized

the effectiveness of cyclodextrins in enhancing drug absorption via tight junctions

(Marttin et al., 1998; Yang et al., 2004). Considering the suitability of HPβCD in (i)

forming an inclusion complex with PGE1, (ii) enhancing the drug absorption, and (iii) modulating the drug release profile of polymeric drug delivery systems, we hypothesized that encapsulation of PGE1-HPβCD complex in PLGA microparticles would result in a

microparticulate drug delivery system with favorable aerodynamic properties, better drug

release, and increased absorption of drug through the air-blood barrier.

This study tests the efficacy of PGE1-HPβCD-loaded, inhalable PLGA microparticles in providing sustained release of PGE1 over a prolonged period of time while

demonstrating improved release in vitro and a better drug absorption profile in vivo.

MATERIALS AND METHODS

Materials

PGE1 (Alprostadil) was purchased from Spectrum Chemicals (Gardena, CA), and (2- hydroxypropyl)-β-cyclodextrin (HPβCD; Mw ≈ 1,380) was purchased from Sigma-Aldrich,

Inc. (St. Louis, MO). PLGA 85:15 polymer for the preparation of microparticulate

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formulations (inherent viscosity 0.55 to 0.75 dl/g; average Mw ≈ 85 kDa) was purchased from Lactel Absorbable Polymers (Pelham, AL). Kits for various assays were purchased from the following vendors: PGE1 ELISA kit from Assay Designs, Inc. (Ann Arbor, MI);

BCA protein assay kit from Pierce Biotechnology (Rockford, IL); alkaline phosphatase

(ALP) and Lactate dehydrogenase (LDH) kits from Pointe Scientific, Inc. (Canton, MI).

Polyvinyl alcohol (PVA), dichloromethane (DCM), and all other chemicals were of analytical/HPLC grade and were purchased from different commercial resources. Adult male Sprague-Dawley (SD) rats were procured from Charles River Laboratories

(Wilmington, MA).

Preparation of PGE1-HPβCD Complex

PGE1-HPβCD complex was prepared in molar ratios of 1:4 and 1:10 by slightly modifying the freeze-drying procedure reported previously by Gu et al. (Gu et al., 2005).

Molar ratios of 1:4 and 1:10 have been reported to be suitable for PGE1 complex

formulations with various cyclodextrins (Wiese et al., 1991; Yamamoto et al., 1992).

Briefly, PGE1 was dissolved in a minimal amount of absolute ethanol, and the

corresponding quantity of HPβCD was dissolved in double-distilled deionized water. The

resulting solutions were mixed together, vortexed for 60 seconds, and freeze dried for 48

hours using a FreeZone 2.5 lyophilizer (Labconco Coporation, Kansas City, MO). The

freeze dried complex of PGE1 and HPβCD was used for the preparation of polymeric

microparticles and for in vivo absorption studies.

Preparation of PLGA Microspheres Encapsulating PGE1-HPβCD Complex

Polymeric microparticles of PGE1-HPβCD complex were prepared by water-in-oil-in- water (W/O/W) double emulsion/solvent evaporation, with slight modification to the

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previously described procedure (Gupta et al., 2010). Briefly, PLGA 85:15 was dissolved in DCM (organic phase, OP) and emulsified (primary W/O emulsion) with deionized water containing 5 mg of PGE1 alone (Plain) or PGE1-HPβCD complex equivalent to 5

mg active PGE1 (internal aqueous phase, IAP) using a Branson Sonifier 450 (Branson

Ultrasonics Corporation, Danbury, CT). The primary W/O emulsion was emulsified again

with an aqueous PVA solution (secondary emulsion) by homogenization at 10,000 rpm

for 10 minutes (Ultra-Turrex T-25 basic, Ika, Wilmington, DE). The prepared formulations

were stirred overnight (∼50-100 rpm) for removal of the OP. The following day, the formulations were washed three times by centrifugation at 50,000 g for 15 minutes at

4°C (Avanti J-25I, Beckman Coulter, Inc. Brea, CA) to remove excess PVA. The washed formulations were lyophilized (FreeZone 2.5, Labconco Corporation) for 48 hours to obtain nine unique, free flowing, powdered microparticulate formulations: Plain-1, Plain-

2, Plain-3, CD-1, CD-2, CD-3, CD-4, CD-5, and CD-6. Formulation properties are listed in Table 4.1.

Physical Characterization

The prepared microparticulate formulations were characterized for morphology, size, tapped density, mass median aerodynamic diameter, surface properties, drug loading, and entrapment efficiency.

Particle Morphology. To understand the morphology of the prepared particulate

formulations, scanning electron microscopy (SEM) experiments were performed using a

Hitachi S-3400N SE microscope (Hitachi High Technologies America, Inc, Pleasanton,

CA). Briefly, a small amount of powdered formulation was sprinkled onto double-sided

adhesive tape attached to an aluminum stub and was sputter coated with gold under

argon (Emitech K550X; Quorum Technologies Ltd, Kent, UK) and viewed under SE

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microscope. Photographs were taken at varying magnifications so as to reveal surface characteristics of the formulations.

Particle Size. Mean volume diameter and particle size distribution for all the formulations were studied using Tri-laser diffraction technology (Microtrac S3500; Microtrac, Inc;

Largo, FL). Briefly, samples were prepared by dispersing 2 mg of powdered formulations in 0.2% w/v tween-80 solution, which was poured into the wet sample port of the instrument for analysis. Mean volume diameter (Mv) was recorded. In addition,

polydispersity indices (PDIs) of all the formulations were also determined using the

following equation:

(1)

Tapped Density, Carr’s Index and Theoretical Mass Median Aerodynamic Diameter.

Tapped density was measured to determine the bulkiness of the formulations and to

calculate theoretical mass median aerodynamic diameters (MMADt) as described previously (Rawat et al., 2008). Briefly, 100 mg of particles was placed in a graduated cylinder (10 ± 0.5 mL). The initial volume (Vinitial) was recorded, and the cylinder was

tapped 200 times onto the work station from a consistent height. The final volume (VFinal)

was recorded and was used to calculate tapped density (ρ) using the following equation

(Ungaro et al., 2009):

ρ (2)

Bulk density (ρi) was calculated using the following equation:

ρ (3)

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Thus, calculated tapped and bulk densities were used to determine the flow properties of the powdered formulations by calculating the compressibility index (Carr’s Index) using the following equation:

ρ ′ 100 (4) ρ

Theoretical mass median aerodynamic diameter (MMADt) was calculated using the equation:

/ (5)

3 where d = geometric mean diameter; ρ = tapped density (g/cm ); ρ0 = reference density

for a sphere (1 g/cm3); and X = shape factor (1 for sphere).

Actual Mass Median Aerodynamic Diameter (MMADa). Actual mass median

aerodynamic diameter (MMADa) was determined using an 8-stage Mark-II nonviable

Anderson Cascade Impactor (ACI) (Westech Instruments, Marietta, GA). Briefly, a size 3

hard gelatin capsule was filled with 10 mg powdered formulation and fired into the

impactor at a constant flow rate of 28.3 L/min using a Spiriva Handihaler (Boehringer

Ingelheim Pharmaceuticals). The amount of the formulation deposited at each stage was

determined gravimetrically by weighing glass fiber filters placed at each stage (0-7) on

inverted collection plates (to prevent bounce or re-entrainment of the particles). Data

were plotted on semi-log graph, and MMADa was determined at 50% of total particle distribution. In addition to MMADa, the fine particle fraction (FPF) for each formulation was determined as the ratio of cumulative weight of particles < 4.7 μM (stage 3 or lower) to total weight of particles recovered from the cascade impactor (Yang et al., 2009).

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Entrapment Efficiency. The quantity of PGE1 entrapped in the microparticulate formulations was determined indirectly by determining the amount of PGE1 present in

the supernatant recovered after the first washing of the microparticles. Briefly, the

4 5 supernatant was diluted 10 to 10 times and was analyzed using a PGE1 ELISA kit

(Assay Designs, Ann Arbor, MI), according to the manufacturer’s protocol. Sample dilution was necessary due to the high sensitivity of the ELISA kit (limit of quantitation =

4.88 to 5,000 pg/mL) and to minimize the matrix interference. All measurements were performed in triplicate and data represent mean ± SD.

Determination of Surface Characteristics by Physioabsorption Isotherms. To determine the surface porosity and pore characteristics, we used nitrogen physiosorption isotherms obtained at 77K using a Nova 3200e surface area analyzer (Quantachrome Instrument

Corporation, Boynton Beach, FL). Briefly, the microparticulate samples were degassed for 24 hours at room temperature to remove any dissolved components that could interfere with the analysis. The samples were run on a surface area analyzer in a sample cell placed in a liquid nitrogen-filled dewer to maintain equilibrium temperature. Each sample was run for 24 hours with a 150-second equilibrium interval to obtain Brunauer-

Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) isotherms. Average surface areas (m2/g) for all formulations were determined by both BET and BJH isotherms, whereas average pore volumes (cm3/g) and pore radii (Å) were determined using the desorption branch of the BJH isotherm (Gill et al., 2009).

In-vitro Release Studies in PBS and SILF

In vitro release studies were performed in simulated interstitial lung fluid (SILF; pH 7.2) prepared at 37°C, prepared according to the Moss Formula (Table 4.3) (Moss, 1979).

Briefly, 10 mg of the formulation was dispersed in 1 mL of release medium (PBS or

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SILF), and incubated at 37 ± 1°C with gentle shaking (∼150 to 200 rpm). At predetermined time intervals ranging from 0 to 48 hours, samples were centrifuged at

16,000 × g for 15 minutes at 4°C, and 200 μL of the supernatant was collected for PGE1 analysis. Collected sample volume was replaced with fresh release media. Data obtained were plotted against time (h) as percent cumulative drug release. Data represent mean ± SD of three simultaneous measurements.

In-vivo Absorption Studies

To determine the effect of HPβCD complexation and encapsulation into PLGA microparticles on the circulation half-life (t1/2) of PGE1, in vivo absorption studies were

performed in adult male Sprague-Dawley (SD) rats (250-300 g). All in vivo studies were

performed under anesthesia, induced by an intramuscular injection of a ketamine /

xylazine cocktail (90 mg/kg ketamine, 10 mg/kg xylazine). The anesthesia was

maintained as needed during the experiment.

The rats (n = 42) were divided into seven groups and were dosed with the equivalent of

120 μg/kg of PGE1 via intravenous (IV) or intratracheal (IT) administration as follows: (i)

saline, IT; (ii) PGE1, IV; (iii) PGE1, IT; (iv) PGE1-HPβCD complex (1:4), IT; (v) PGE1-

HPβCD complex (1:10), IT; (vi) microparticulate formulation CD-3, IT; and (vii)

microparticulate formulation CD-5, IT. Intravenous drug was delivered via the penile

vein, and intratracheal delivery employed a Microsprayer (PennCentury, Philadelphia,

PA) designed specifically for aerosol administration to small rodents. Blood samples

were collected in citrated microcentrifuge tubes at predetermined time points (0 to 24

hours). While collecting the blood samples, 10 μg/mL indomethacin was added to each

tube to inhibit prostaglandin synthase. Plasma was collected from blood samples by

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centrifuging at 2,300 × g for 5 minutes, and was stored at -20°C until analysis. Plasma

PGE1 concentrations were determined using ELISA as described above.

Metabolic Degradation Studies in Rat Lung Homogenates

Metabolic degradation studies were performed to determine the stability of optimized microparticulate formulations under physiological conditions. These studies were performed according to the protocol previously established in our lab (Gupta et al., 2010) with slight modifications. Briefly, rat lungs were removed surgically and homogenized in

4 volumes of cold buffer (Bücher medium) using a Potter-Elvehjem glass homogenizer.

The homogenate was centrifuged at 10,000 × g for 20 minutes at 4°C, and the supernatant was used for further studies. For degradation studies, PGE1 (10 μg/mL) or

optimized formulations (CD-3 or CD-5) at a concentration of 10 μg/mL PGE1 were incubated with lung homogenates at 37°C in the presence of 2mM NAD+. At predetermined time points (0-8 hours), the reaction was terminated by adding 200 μL

0.1N HCl to the incubation mixture, and the PGE1 concentration was determined by

ELISA. To determine amount of PGE1 remaining in the microparticles following

incubation, PGE1 was extracted from the microparticles by the procedure previously published (Gupta et al., 2010), and quantified by ELISA, providing a measure of the controlled release properties for the PGE1-HPβCD complex polymeric microparticles.

Bronchoalveolar Lavage (BAL) Fluid Analysis

To establish the safety profile of the optimized formulations, various lung injury and toxicity markers were analyzed in the bronchoalveolar lavage (BAL) fluid, according to a published procedure (Hussain et al., 2006; Thomas et al., 2008). Briefly, male SD rats

(350 to 400 g; n = 16) were divided into 4 groups and administered (i) saline, (ii) 0.1%

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sodium dodecyl sulfate (SDS), (iii) CD-3 equivalent to 120 μg/kg dose of PGE1, and (iv)

CD-5 equivalent to 120 μg/kg PGE1 via the IT route. Lungs were removed surgically from anesthetized rats 12 hours after administration, by exposing the respiratory system with a mid-level incision in the thoracic cavity. Lungs were cleaned of surrounding tissues/organs, weighed (g/100 g body weight) to assess the possibility of edema formation. To collect BAL fluid, 5 mL of normal saline was instilled into the lungs through trachea, left for 30 seconds, withdrawn and re-instilled for 30 seconds, and finally withdrawn. The collected BAL samples were centrifuged at 500 × g for 10 minutes. The supernatant was used to determine the levels of the injury markers ALP and LDH (IU/L;

Pointe Scientific, Canton, MI), as well as total protein concentration (mg/mL; Pierce

Biotechnology, Rockford, IL).

All animal studies were performed in accordance with NIH Guidelines for the care and use of Laboratory Animals under a protocol approved by TTUHSC Animal Care and Use

Committee (AM-10012).

Cell Viability Studies

To access the in vitro safety profile, acute cytotoxicity studies were performed on Calu-3 lung epithelial cells using a propidium iodide exclusion fluorescence assay (Nieminen et al., 1992). Briefly, Calu-3 cells were seeded at 20 000 cells/well in a 96-well, black microtiter plate using EMEM cell culture media (+ 10% fetal bovine serum + 1% 200 mM

L-glutamine + 1% penicillin/streptomycin solution) and allowed to attach overnight at

37°C in a 5% CO2 incubator. Immediately prior to the experiment the medium was

removed, and cells were washed twice with KRH (Krebs-Ringer-HEPES) Buffer (Qian et

al., 1997) and were treated for 4 hours at 37°C with the following: (i) 20 μL saline; (ii) 20

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μL 0.1% SDS (positive control); 0.5, 1.0, 2.0 or 5.0 mL of test formulations (iii) CD-3, and

(iv) CD-5.

After 4 hours the cells were washed twice with KRH buffer, and initial fluorescence

(Blank) was measured with 100 μL of KRH buffer in each well. Propidium iodide (PI, 5

μM) was added to each well, and the plate was incubated at 37°C for 30 minutes. After

30 minutes, fluorescence (F) was measured and 375 μM of digitonin was added to permeabilize the cells and label all the nuclei with PI.

Fluorescence was measured every 5 minutes up to 20 minutes. Fluorescence at 20 minutes (Fmax) was used for all the calculations using following equation (Sarafian et al.,

2002):

% 100 100

Fluorescence was measured at an excitation wavelength (Ex) of 546 nm and emission

wavelength (Em) of 620 nm at a band pass of 20 nM, using a SynergyMX microplate

reader (Biotek, Winnoski, VT).

Pharmacokinetic and Statistical Analyses

Pharmacokinetic parameters were determined by Kinetica Version 5.0 (Thermo

Scientific, Waltham, MA), using standard non-compartmental extravascular analysis.

Data were analyzed for Cmax, Tmax, t1/2, and area under concentration-time plot (AUC0-24 h). Data analysis was performed by using one-way ANOVA followed by appropriate post

hoc analysis (GraphPad Prism 5.0, GraphPad Software, La Jolla, CA). Values of p <

0.05 were considered statistically significant.

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RESULTS AND DISCUSSION

The main aim of this study was to determine the effect of HPβCD incorporation on the preparation and properties of PGE1-loaded PLGA microparticles, in vivo and in vitro.

Physical Characterization

Physical characterization studies were performed to determine the effect of process variables and additives on the morphology and aerodynamic behavior of microparticulate formulations, in order to determine the suitability of microparticulate formulations for pulmonary administration.

Particle Morphology. Scanning electron microscopy experiments were performed to determine the morphology of PGE1-HPβCD complex encapsulated microparticulate formulations. As can be seen in Figs. 4.1A – 4.1F, all the formulations had a relatively smooth outer surface and were spherical in shape. Despite presence of few surface pores in all the formulations, SEM images suggest that the incorporation of HPβCD as

an excipient did not significantly affect particle morphology. As has been suggested in

many reports, HPβCD works as an osmotic agent creating high osmotic pressure in the

internal aqueous phase during particle hardening (Zannou et al., 2001), which causes an

influx of water and generally results in the development of a porous particle surface

(Pistel and Kissel, 2000). Although relatively few surface pores are visible in the images,

these formulations are more likely to produce internal particle pores not visible from the

surface. We recently reported on the development of bulky and internally porous PLGA

microparticles of PGE1 by incorporation of 0.9% NaCl in the internal aqueous phase

(Gupta et al., 2010). At the same time, others have reported the development of internal

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pores in microparticles with increased osmotic pressure in the IAP rendering influx of water from the EAP (Srinivasan et al., 2005).

Particle Size. As shown in Fig. 4.2A, the mean volume diameter for all formulations was dependent on the concentrations of HPβCD and PVA. Particle size increased significantly with increasing concentration of HPβCD (Plain-2 = 8.54 ± 0.055 μm; CD-4 =

10.09 ± 0.11 μm; and CD-3 = 14.54 ± 0.03 μm), an indication of the bulking and porosigenic properties of HPβCD. In addition, particle size decreased as the concentrations of PVA was increased from 0.5% to 2% (Fig. 4.2A). This decrease in particle size can be explained by the emulsion-stabilizing properties provided by the hydrophilic environment of PVA (Lee et al., 1999). All the formulations were moderately dispersed, with PDI ranging from 0.99 to 1.73.

Tapped Density and Carr’s Index. Tapped density measurements provide important information regarding the flowability, porosity, and size distribution of the particulate formulations, as well as information regarding interparticulate forces (cohesive and adhesive) working in the system (Rawat et al., 2008). As described in Table 4.2, the tapped densities of the prepared formulations decreased with increasing HPβCD concentration (Plain-1 = 0.33 ± 0.04; CD-1 = 0.185 ± 0.03; and CD-2 = 0.188 ± 0.04). A similar pattern was seen with the other formulations. Tapped densities also decreased with increasing PVA concentration (Table 4.2). A decrease in tapped density is suggestive of an increase in the bulk and porosity of particles with HPβCD incorporation.

Carr’s index provides an estimate of the flow properties of the particulate formulations.

Carr's index defines particle flowability: excellent (5 to 12%); good (12–18%); fair (18–

25%); poor, cohesive (25–32%); very poor (32–38%); extremely poor (>40%). As

described in Table 4.2, formulations CD-1 to CD-5 were in range of fair to poor

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flowability, with CD-3 (22.2 ± 2.92%) and CD-5 (23.3 ± 3.16%) exhibiting superior flow properties. Plain PLGA particles exhibited Carr’s index values > 40% and thus had extremely poor flow properties.

Theoretical and Actual Aerodynamic Diameter (MMADt and MMADa). Mass median aerodynamic diameter is the most important parameter determining particle deposition and distribution following pulmonary administration. As can be seen in Fig. 4.2B, MMADt for all the formulations was below < 10 μm, confirming their good flow properties (Ungaro et al., 2009).

Actual aerodynamic diameters (MMADa) for all the formulations were determined by a nonviable 8-stage Anderson cascade impactor. As shown in Fig. 4.2B, MMADa for all the formulations was between 0.7 ± 0.12 μm (CD-1) and 4.7 ± 0.294 μm (CD-4) for all the PGE1-HPβCD inclusion complex formulations. Plain particles, however, showed

MMADa values in the range of 3.19 ± 0.162 μm (Plain-3) to 5.49 ± 0.19 μm (Plain-1),

which underlines their poor suitability for pulmonary administration. Among all CD

formulations, CD-3 (2.2 ± 0.41 μm) and CD-5 (2.7 ± 0.29 μm) had the most favorable

aerodynamic properties. The fine particle fraction (FPF) for all formulations was

determined from the fraction of particles deposited at stage 3 or lower. As shown in

Table 4.2, FPF values for all the CD formulations were higher than those for the Plain

formulations, indicating an overall improvement in aerodynamic properties with HPβCD

complex formation. However, the FPF data did not follow any specific pattern.

Large porous particles > 5 μm in diameter and with densities < 0.4 g/cm3 (MMADa

between 1-5 μm) have been reported to exhibit desirable aerodynamic and deep lung

deposition properties. Porous particles enhance bioavailability and prolong drug release

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(Edwards et al., 1997). The microparticulate formulations developed in this project exhibit aerodynamic properties optimal for pulmonary delivery.

Entrapment Efficiency. Formulation entrapment efficiencies were determined indirectly by measuring drug content in the supernatant recovered after washing the formulations.

As can be seen in Fig. 4.2C, drug entrapment was comparable for all formulations, but did decrease somewhat with increasing HPβCD concentration (40.47 ± 2.39% for CD-2 and 34.05 ± 3.01 for CD-1, compared to 46.77 ± 10.24% for plain particles). The same pattern was seen with other formulations as well, with higher entrapment efficiencies of

52.02±7.69% and 55.79±4.38% with CD-5 and CD-6, respectively. At the same time, increased PVA concentration was critical in increasing drug entrapment: CD-2 = 40.47 ±

2.39% (0.5% PVA); CD-4 = 50.74 ± 2.51% (1% PVA); and CD-6 = 55.79 ± 4.38% (2%

PVA). This result is congruent with previous reports suggesting PVA works as an emulsion stabilizer (Lee et al., 1999; Jeong et al., 2003).

A decrease in entrapment efficiency with the incorporation of HPβCD in microparticles can be attributed to the bulk introduced by HPβCD in the internal aqueous core. As can be seen in Table 4.1, a very high molar ratio of PGE1:HPβCD were used to increase the

hydrophilicity of PGE1, which resulted in reduced drug entrapment due to (i) the

increased water solubility of the PGE1-HPβCD inclusion complex, which makes it more prone to leach out of microparticulate core, and (ii) increased bulk due to HPβCD incorporation. At the same time, similar pattern was observed in the calculated percent drug loading (Table 4.2).

Surface Characteristics Determination. Surface characteristics of the prepared formulations were determined by the physiosorption-isotherm-based method. As shown in Fig. 4.3A, the specific surface area of all formulations increased as a function of

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HPβCD concentration, which agrees with our particle size data. BET and BJH isotherms used for surface area determination show a similar pattern. Various reports have suggested that HPβCD, when incorporated in the internal aqueous phase, helps in the development of porous particles (Ungaro et al., 2006). Due to the porous nature of the particles, the internal surface area is significantly higher than in nonporous particles, resulting in a porosity-dependent increase in specific surface area (Jeyanthi et al., 1997).

As with surface area, pore volume also increased in an HPβCD concentration- dependent manner, with plain PLGA particles being the least porous of them all (Fig.

4.3B). At the same time, both specific area and pore volume decreased with increasing

PVA concentration, which is in agreement with the particle size data presented in Fig.

4.2. All the formulations exhibited comparable pore sizes in the range of 26-30 Å, and no significant differences were observed with any of the formulation variables. As depicted in Fig. 4.1, there were not many surface pores visible in these formulations. Instead, internal pores contributed to the low density, porous nature of the current formulations, as is evident from the surface characteristics.

Overall, the physical data reveal HPβCD to be a bulky, porosity-inducing agent with PVA acting as an emulsion stabilizer, both of which modify the physical properties of the microparticles in a concentration-dependent manner.

In vitro Release Studies in SILF

The release profiles of all formulations were studied in SILF, first described by Moss as a solution of electrolytes mimicking the concentrations of ions present in surfactant-rich human lung fluid secreted by Type II alveolar cells (Moss, 1979). This fluid is important in maintaining water surface tension in the lungs by filling the space between alveolar cells. In SILF, citrate ions are used to compensate for the ionic contribution from protein

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in human alveolar fluid. SILF was used as the release medium for in vitro release studies to provide a good estimate of drug released following in vivo pulmonary administration of the microparticulate formulations.

Fig. 4.4 illustrates that the in vitro drug release profiles for the formulations were significantly different from one another. Plain PLGA formulations without HPβCD showed very low percent cumulative release, which subsequently decreased with increase in

PVA concentration ranging from 23.41 ± 2.95 (Plain-1, 0.5% PVA) to 12.41 ± 1.42%

(Plain-3, 2% PVA) in agreement with previous reports. Incorporation of HPβCD resulted in a significant, concentration-dependent increase in percent cumulative release from the formulations over a period of 48 hours (Fig. 4.4A, B, and C). A total of 62.48 ± 6.73% of drug was released from CD-4 (1:4 molar ratio of PGE1:HPβCD) over 48 hours, and this increased to 68.95 ± 8.77% with CD-3 (1:10 molar ratio) (Fig. 4.4B). A similar pattern was seen within each pair of formulations having the same PVA concentration. Increase in drug release following HPβCD inclusion can be attributed to the increased

hydrophilicity of PGE1, which arises from its incorporation into a hydrophilic toroid cavity of HPβCD that results from the spatial arrangement of primary and secondary hydroxyl groups. As a result of the increase in aqueous solubility, the PGE1-HPβCD inclusion

complex tends to leach out of the microparticulate core into the external aqueous

environment. In addition, all the formulations showed a biphasic drug release, i.e., an

initial burst release phase followed by a nearly zero-order drug release phase.

Drug release profiles were analyzed further to calculate (i) surface-associated drug

release (drug released at 0 minutes) and (ii) burst release (drug release in first 30

minutes). As shown in Fig. 4.4D, a surface-associated drug release ranging from 5-10%

was apparent for all formulations incorporating cyclodextrin , except CD-4 (19.87 ±

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1.32%) and CD-6 (13.81 ± 0.91%), which suggests the presence of more drug on the surface of CD-4 and CD-6 particles. Similarly, all the formulations showed favorable burst release profiles (ranging from 7-26%) (Fig. 4.4D).

The data obtained from the in vitro release studies are vital for characterizing the formulations, because they were performed under conditions that mimic physiological conditions, providing a useful estimate of the correlation between in vitro release and in vivo absorption.

Overall, formulations CD-3 and CD-5 showed optimum flowability, aerodynamic properties, entrapment efficiencies, and favorable drug release with desired burst release properties. These formulations would be expected, therefore, to produce deep lung deposition and prolonged release of drug following intratracheal administration.

In vivo Absorption Studies

The absorption profiles of the optimized formulations (CD-3 and CD-5) were studied in adult male SD rats following intratracheal administration at a physiological dose of 120

μg/kg body weight. As is depicted in Fig. 4.5A, administration of PGE1 via the IV or IT route resulted in a rapid increase in PGE1 concentration in the circulation, with a Cmax of

134.22 ± 14.52 ng/mL (IV) and 96.85 ± 5.33 ng/mL (IT), respectively. However, the drug disappeared from the circulation very quickly, exhibiting t1/2 of 3.5 ± 0.28 minutes (IV)

and 6.49 ± 0.41 minutes (IT), respectively. As depicted in Table 4.4, intratracheal

administration of PGE1 resulted in a relative bioavailability of 0.67 ± 0.078, indicating a rapid absorption of the drug in biologically active form into blood via the air-blood barrier.

When PGE1-HPβCD complexes (molar ratio 1:4 and 1:10) were administered at equivalent dose via intratracheal route, we observed a significant decrease in Cmax (1:4

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complex = 75.28 ± 28.1 ng/mL; 1:10 complex = 50.26 ± 18.47 ng/mL), but a significant increase in t1/2 for PGE1 (1:4 complex = 30.95 ± 3.26 minutes; 1:10 complex = 18.57 ±

2.27 minutes). A significant increase in bioavailability was also observed (1:4 complex =

0.91 ± 0.12; 1:10 complex = 0.86 ± 0.095) relative to that seen with intratracheal

administration of plain PGE1 (0.67 ± 0.078) (Table 4.4 and Fig. 4.5B).

When PGE1-HPβCD-encapsulated optimized formulations were administered via the

pulmonary route, PGE1 was measurable in the circulation for up to 8 hours (CD-3) or 12 hours (CD-5) after administration (Fig. 4.5C). Tmax values (CD-3 = 60 minutes; CD-5 = 30 minutes) were increased and Cmax values (CD-3 = 14.26 ± 2.13 ng/mL; CD-5 = 22.05 ±

2.4 ng/mL) were slightly lower for optimized microparticulate formulations relative to

those for PGE1 alone or for either PGE1-HPβCD complex (Fig 4.5A-C). This was

accompanied by a simultaneous and significant increase in t1/2 for the microparticulate

relative to other formulations (CD-3 = 249.47 ± 32.56 minutes; CD-5 = 136.23 ± 15.63

minutes). At the same time, the relative bioavailabilities of the microparticulate

formulations (CD-3 = 0.76 ± 0.15; CD-5 = 0.79 ± 0.14) were comparable to those of the

cyclodextrin inclusion complexes (Table 4.4).

As can be seen, the data obtained with the plain PGE1 administration at a dose of 120

μg/kg is significantly different from the data published earlier at a dose of 80 μg/kg

(Gupta et al., 2010), which indicates toward presence of dose-dependent

pharmacokinetics of PGE1. Earlier reports published with human studies indicate that pharmacokinetics of PGE1 is not dose-dependent, but are dose proportional (Cawello et al., 1995; Cawello et al., 1997). However not many studies have been performed with rats, and we certainly cannot correlate clinical data with preclinical animal studies.

Henceforth, PGE1 pharmacokinetics needs to be studied in more detail. The data

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indicate formation of a PGE1-HPβCD inclusion complex results in increased drug

bioavailability, which can be attributed to the ability HPβCD to increase the hydrophilicity

of PGE1 (extremely hydrophobic) thus enhancing its distribution in blood. There may be

several factors that contributed to increased t1/2 and reduced Cmax of PGE1 in

microparticulate formulations. Lungs have alveolar macrophages to clear foreign

particles from the respiratory system via phagocytosis. Due to large particle size and low

density, the CD-3 and CD-5 formulations were able to elude macrophages, thus

prolonging release (Edwards et al., 1997). In addition, time-dependent biodegradation of

the PLGA polymeric core also contributes to the prolonged-release property of the

formulations (Shive and Anderson, 1997). However, HPβCD in the microspheric core is

responsible for increasing the hydrophilicity of the drug, facilitating its absorption through

the air-blood barrier. As we discussed recently (Gupta et al., 2010), prolonged release

can be achieved with plain PLGA microparticles of PGE1, but the Cmax achieved is

significantly lower than that from microparticles encapsulating drug-cyclodextrin inclusion

complexes. This phenomenon can be attributed to increased hydrophilicity of PGE1- cyclodextrin complex, which results in increased absorption of available PGE1 into the circulation. Overall, both the optimized formulations showed a significantly extended half-life and excellent bioavailability.

Metabolic Degradation Studies in Rat Lung Homogenates

Metabolic degradation studies in rat lung homogenates were used to establish the stability of optimized formulations in a physiological environment. PGE1 undergoes 70 to

80% oxidation as a result of first pass metabolism in the lungs. PGE1 is metabolized by

+ NAD -dependent 15-hydroxy prostaglandin dehydrogenase into 13,14-dihydro-PGE1, a stable PGE1 metabolite with anti-aggregatory properties. This accounts for its rapid

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clearance from the lungs and circulation (Nakano et al., 1973; Ney et al., 1991). When incubated at a concentration of 10 μg/mL with rat lung homogenates, PGE1 is rapidly cleared/degraded with almost no drug detectable after 2 hours of incubation at 37°C (8.5

± 1.28 μg/mL at time zero; 0.211 ± 0.02 μg/mL after 120 minutes; Fig 4.6A). However,

when optimized PLGA microspheres encapsulating PGE1-HPβCD inclusion complex were incubated at an equivalent dose, PGE1 in the homogenate remained elevated for more than 8 hours, indicating a time-dependent release of PGE1 from the core of polymeric microspheres. The mean PGE1 level from CD-3 was 3.58 ± 0.03 μg/mL at time

zero, decreasing slightly to 2.81 ± 0.13 μg/mL after 8 hours. PGE1 from CD-5 increased from 2.86 ± 0.14 μg/mL at time zero to 4.79 ± 0.8 μg/mL after 8 hours (Fig. 4.6B).

The polymeric core of the microspheres is believed to degrade in a time-dependent

manner to provide continuous availability of PGE1 in the homogenate. To further confirm

this hypothesis, we measured PGE1 from microspheres incubated with lung

homogenate. As shown in Fig. 4.6C, unreleased PGE1 from microspheres decreased

over the course of experiment, indicating a constant rate of release of PGE1 from microspheres. For CD-3, the PGE1 concentration in microspheres decreased from 6.42 ±

0.64 μg/mL at time zero to 0.69 ± 0.17 μg/mL at 8 hours. Similarly, PGE1 concentration

for CD-5 decreased from 5.61±1.28 μg/mL at time zero to 0.93 ± 0.14 μg/mL at 8 hours

(Fig. 4.6C). On the whole, the rat lung homogenate metabolic degradation data suggest

that polymeric microspheres release PGE1 continuously, supporting the view that the

microspheres protect unreleased PGE1 from first pass metabolism in lungs. This finding is consistent with the in vitro release data and in vivo absorption profiles of CD-3 and

CD-5.

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Bronchoalveolar Lavage (BAL) Fluid Analysis

We assessed the acute, in vivo safety of the optimized formulations by analyzing lungs and BAL fluid collected from CD-3 and CD-5-treated rats for presence of edema, protein concentration, and levels the lung injury markers LDH and ALP. Saline- and SDS-treated rats were used as negative and positive controls respectively. An increase in the wet lung weight is an indication of extracellular fluid accumulation in the epithelial cell lining of the respiratory wall. The presence of LDH in BAL fluid is an indication of cell damage and lysis (Henderson et al., 1978), and the presence of ALP represents alveolar type II cell proliferation due to type I cell damage (Hussain and Ahsan, 2005). An increase in total protein concentration is indicative of inflammation and rupture of the alveolar capillary barrier (Beck et al., 1982).

As can be seen in Figs. 4.7A, the wet weights of lungs from CD-3- and CD-5-treated rats were higher than those from saline-treated rats, but were significantly lower those from

SDS-treated rats. Some of the edema can be attributed to stress related to intratracheal administration and anesthesia. No significant changes in the levels of either LDH (Fig.

4.7B), ALP (Fig. 4.7C), or total protein content (Fig. 4.7D) were observed with optimized formulations. In contrast, SDS resulted in significant increase in the levels of all three.

These data suggest that CD-3 and CD-5 may be safe for the noninvasive pulmonary delivery of PGE1. However, long term studies with both CD-3 and CD-5 are required to

provide a better understanding of pathological changes occurring during repeated

administration.

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Cell Viability Studies

Cell viability studies were performed using Calu-3 human airway epithelial cells to determine the effects of microparticulate formulations and excipients on pulmonary epithelial lining following acute exposure. These studies used the propidium iodide fluorescence exclusion assay as described by Nieminen et al. (Nieminen et al., 1992).

Propidium iodide is a fluorescent intercalating dye that binds double stranded nucleic acids from dead cells, and is excluded from viable cells. When bound to nucleic acid, it undergoes a red shift and a 20- to 30-fold increase in fluorescence intensity. As shown in Fig. 4.8, 100% cell viability was observed with saline treatment (negative control), whereas viability decreased to 37.79 ± 4.17% following SDS treatment (positive control).

Neither optimized formulation resulted in any significant loss of cell viability at any concentration over the 4-hour exposure, with cell viability remaining within the 90-100% window (CD-3: 92.29 ± 6.69% to 99.03 ± 2.31%; CD-5: 92.41 ± 4.11% to 100.81 ±

2.74%). HPβCD is a relatively non-toxic derivative of β-CD, with a benign toxicity profile.

PLGA has been approved by FDA as a safe polymer for drug delivery. It has been

suggested that transport of cyclodextrins (height of toroid 0.79 nm) across the alveolar

epithelium is mediated by an abundance of large equivalent pores of 6 nm radius, which

facilitate passage of small hydrophilic molecules < 5 nm in size by passive diffusion without damaging the tight junctions (Matsukawa et al., 1997). Moreover, it was reported recently that HPβCD is one of the safest cyclodextrins for pulmonary drug delivery applications due to its non-toxic interaction with airway epithelial cells (Matilainen et al.,

2008). Hence, PGE1-HPβCD encapsulated PLGA microparticles can be considered

suitable for the pulmonary delivery of PGE1.

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In summary, this study is the first to investigate the suitability of PGE1-HPβCD- encapsulated PLGA microparticles for the pulmonary delivery of PGE1, as a potential medication for pulmonary arterial hypertension (PAH). Our data suggest that it is feasible to encapsulate PGE1-HPβCD complex in PLGA microspheres to produce microspheres

that exhibit favorable particulate properties and meet most micromeritical standards for

noninvasive pulmonary administration. Microspheric formulations provided enhanced

drug release from the particles under physiological conditions, and also provided an

extended biological half-life. At the same time, optimized formulations offer protection

against metabolic degradation of PGE1, and appear to be safe for pulmonary administration on the basis of both in vivo and in vitro studies. Additional studies are currently underway in our laboratory to establish the efficacy of optimized formulations in a rodent model of PAH.

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Ungaro F, d'Emmanuele di Villa Bianca R, Giovino C, Miro A, Sorrentino R, Quaglia F and La Rotonda MI (2009) Insulin-loaded PLGA/cyclodextrin large porous particles with improved aerosolization properties: in vivo deposition and hypoglycaemic activity after delivery to rat lungs. J Control Release 135:25-34.

Ungaro F, De Rosa G, Miro A, Quaglia F and La Rotonda MI (2006) Cyclodextrins in the production of large porous particles: development of dry powders for the sustained release of insulin to the lungs. Eur J Pharm Sci 28:423-432.

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Wallace JL (1992) Prostaglandins, NSAIDs, and cytoprotection. Gastroenterol Clin North Am 21:631-641.

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Table 4.1: Compositions of Various Microparticulate Formulations

PVA Concentration PGE :HPβCD Polydispersity Formulation Polymer IAP:OP:EAP 1 (w/v) Molar Ratio Index (PDI) Plain-1 0.5:5:25 0.5% ⎯ 1.69±0.005 CD-1 0.5:5:25 0.5% 1:10 1.41±0.018 CD-2 0.5:5:25 0.5% 1:4 1.53±0.026 Plain-2 0.5:5:25 1% ⎯ 1.61±0.01 CD-3 PLGA 85:15 0.5:5:25 1% 1:10 1.29±0.001 (0.55-0.75 dl/g) CD-4 0.5:5:25 1% 1:4 1.68±0.011 Plain-3 0.5:5:25 2% ⎯ 0.99±0.16 CD-5 0.5:5:25 2% 1:10 1.536±0.004 CD-6 0.5:5:25 2% 1:4 1.73±0.029

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Table 4.2: Physical Characteristics of PGE1⎯HPβCD Complex Encapsulated Microparticles

Formulation Tapped Density Carr’s Index (%) Drug Loading (%) Fine Particle (g/cm3) Theoretical Actual Fraction (%) Plain-1 0.329±0.039 61.11±2,43 1.016±0.22 35.21±6.71 CD-1 0.185±0.028 30.0±1.27 0.667±0.059 91.82±12.31 CD-2 0.188±0.041 25.0±2.19 0.793±0.047 72.92±10.13 Plain-2 0.40±0.0812 62.5±4.61 1.325±0.07 45.11±4.28 CD-3 0.236±0.02 22.2±2.92 1.96 1.027±0.074 68.33±7.28 CD-4 0.116±0.016 28.9±1.89 0.995±0.049 48.89±5.29 Plain-3 0.435±0.11 64.28±6.19 1.557±0.098 48.24±6.29 CD-5 0.118±0.02 23.3±3.16 1.019±0.15 63.27±7.01 CD-6 0.173±0.018 40.6±4.16 1.093±0.086 54.55±6.02

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Table 4.3: Composition of Simulated Interstitial Lung Fluid (SILF) (Moss Formula)

Chemical Formula Composition (mM)

Magnesium chloride MgCl2.6H2O 1.0 Sodium chloride NaCl 103.0 Potassium chloride KCl 4.0 Sodium phosphate, dibasic Na2HPO4.7H2O 1.5 Sodium sulfate Na2SO4 0.5 Calcium chloride CaCl2.2H2O 2.45 Sodium acetate NaH3C2O2.3H2O 7.0 Sodium bicarbonate NaHCO3 31.0 Sodium citrate Na2H5C6O7.2 0.33

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Table 4.4: Pharmacokinetic parameters of PGE1⎯HPβCD Complex loaded PLGA microspheric formulations. Data represent mean ± standard deviation (n=4-6).

Formulation Cmax (ng/mL) t1/2 (min) AUC0-24 Relative (ng/mL*min) Bioavailability

Plain PGE1 I.V. 134.22±14.52 3.5±0.28 2883.91±286.4 -

Plain PGE1 96.85±25.33 6.49±0.41 1932.22±150.1 0.67±0.078 Pulmonary

PGE1-HPβCD 1:4 75.28±28.1 30.95±3.26 2607.78±200.71 0.91±0.12

PGE1-HPβCD 1:10 50.264±18.47 18.57±2.27 2466.27±289.71 0.86±0.095 CD-3 14.26±2.13 249.47±32.56 2191.78±178.1 0.76±.0.15 CD-5 22.05±2.4 136.23±15.63 2276.9±190.2 0.79±0.14

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SEM Images of PGE1-HPβCD Encapsulated PLGA Microparticles

A B

C D

E F

Fig. 4.1: Scanning electron microscopy (SEM) images of PLGA-HPβCD encapsulated PLGA microparticles: (A) CD-1; (B) CD-2; (C) CD-3; (D) CD-4; (E) CD-5; and (F) CD-6. Please refer to Table 4.1 for composition of different formulations.

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Physical Characterization of PLGA Microparticles

A B

20 M) 9 Theoretical μ 18 Actual M) 8 μ 16 7 14 6 12 5 10 4 8 3 6 2 4

Mean Volume Diameter ( Mean Volume 2 1 0 0 1 1 2 2 3 4 3 5 6 -1 -1 -2 -2 -3 -4 -3 -5 -6 n- D- D- n- D- D- n- D- D- in D D in D D in D D ai C C ai C C ai C C Diameter ( MedianMass Aerodynamic a C C a C C a C C Pl Pl Pl Pl Pl Pl 0.5% PVA 1% PVA 2% PVA 0.5% PVA 1% PVA 2% PVA

20 % Drug Entrapped 10

0 -1 -1 -2 -2 -3 -4 -3 -5 -6 ain CD CD ain CD CD ain CD CD Pl Pl Pl 0.5% PVA 1% PVA 2% PVA

Fig. 4.2: Physical characterization of PLGA-HPβCD encapsulated PLGA microparticles. (A) Volume based mean diameter (μM); (B) Theoretical and actual mass median aerodynamic diameter (MMADt and MMADa); and (C) Entrapment efficiencies of the formulations. Data represent mean ± standard deviation (n = 3).

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Physiosorption-based Surface Area Characteristics

A B Plain-1 CD-1 CD-2 24 0.030 Plain-2

/g) CD-3 2 20 CD-4 0.025 Plain-3 /g)

16 CD-5 3 0.020 CD-6 12 0.015

8 0.010

4 Pore Volume (cm 0.005 Specific Surface (m Area

0 0.000 BET Isotherm BJH Isotherm -1 -1 -2 -2 -3 -4 -3 -5 -6 ain CD CD ain CD CD ain CD CD Pl Pl Pl

0.5% PVA 1% PVA 2% PVA C 35

Pore Radius (Angstroms) Radius Pore 20

-1 -1 -2 -2 -3 -4 -3 -5 -6 ain CD CD ain CD CD ain CD CD Pl Pl Pl 0.5% PVA 1% PVA 2% PVA

Fig. 4.3: Physiosorption based surface characteristics of PLGA-HPβCD encapsulated PLGA microparticles. (A) Surface area as determined by BET and BJH adsorption isotherms; (B) Pore volume (cm3/g); and (C) Pore radius (Angstroms). Data represent mean ± standard deviation (n = 3).

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In-vitro Release Profile of PGE1-HPβCD Encapsulated PLGA Microparticles

A B

80 35 Plain-2 Plain-1 CD-3 70 CD-1 CD-4 30 CD-2 60 25 50

20 40

15 30

10 20

Cumulative Release (%) 10

Cumulative Release (%) 5 0 0 0 1020304050 0 1020304050 Time (hrs) Time (hrs) C D

% Surface Associated Drug Plain-3 30 % Burst Release 80 CD-5 70 CD-6 25

60 20 50

40 15

30 10

20 Drug% Release Cumulative Release(%) 5 10

0 0 0 1020304050 -1 -1 -2 -2 -3 -4 -3 -5 -6 ain CD CD ain CD CD ain CD CD Time (hrs) Pl Pl Pl

Fig. 4.4: In-vitro release profiles of PLGA-HPβCD encapsulated PLGA microparticles in simulated interstitial lung fluid (SILF) up to 48 hours. Release profiles of formulations with (A) 0.5% w/v PVA; (B) 1.0% w/v PVA; and (C) 2.0% w/v PVA. (D) In-vitro release kinetics showing release of surface associated drug and burst release. Data represent mean ± standard deviation (n = 3).

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In-vivo Absorption Profile of PGE1-HPβCD Encapsulated PLGA Microparticles

A B

PGE -HPBCD 1:4 Complex Intravenous PGE 1 160 1 100 PGE -HPBCD 1:10 Complex Pulmonary PGE 1 1 140 80 120

100 60 80

60 40

40 Concentration (ng/mL) 1 20 Concentration (ng/mL) 1 20 PGE

PGE 0 0 0 20 40 60 80 100 120 0 50 100 450 500 550 Time (min) Time (min) C 25 25 CD-3

20 CD-5

20 15

10 15 5

Concentration (ng/mL) 1

10 PGE 0 0 20 40 60 120 Time (min) 5

Concentration (ng/mL)

0 0 100 200 300 400 5001400 1600

Time (minutes)

Fig. 4.5: In-vivo absorption profiles of (A) Plain PGE1 following administration via intravenous and pulmonary route at a dose of (120 μg/kg); (B) PGE1-HPβCD complex (molar ratio 1:4 and 1:10) via pulmonary route (120 μg/kg); and (C) optimized microspheric formulations of PGE1-HPβCD complex (CD-3 and CD-5) following pulmonary administration (120 μg/kg). Data represent mean ± standard deviation (n = 6- 8).

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Metabolic Degradation Studies for PGE1-HPβCD Encapsulated PLGA Microparticles

A B

CD-3 Plain PGE 0.50 CD-5 0.8 1 0.45 0.40 0.6 0.35 g/mg Protein) g/mg Protein)

μ 0.30 μ 0.4 0.25 0.20 0.2 0.15 0.10 Concentration ( Concentration( 1 0.0 1 0.05

02468 02468 PGE PGE Time (Hours) Time (Hours)

CD-3 0.6 CD-5 0.5

0.4 g/mg Protein)g/mg

μ 0.3

0.2

0.1

Concentration ( Concentration 0.0 1 02468 PGE Time (Hours)

Fig. 4.6: Metabolic degradation studies of PGE1 in rat lung homogenate. Concentration of PGE1 in lung homogenate upon incubation of (A) 10 μg/mL plain PGE1; (B) Optimized formulations CD-3 and CD-5 containing 10 μg/mL; and (C) The amount of drug extracted from CD-3 and -5 microspheres incubated in lung homogenates. Data represent mean ± standard deviation (n = 3).

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In-vivo Toxicity Studies in Bronchoalveolar Lavage Studies

A B 0.63 * 500 * 0.60

0.57 400

0.54 300 0.51 200 0.48

0.45 (IU/L) Activity LDH 100

0.42 0 Saline CD-3 CD-5 0.1% SDS Saline CD-3 CD-5 0.1% SDS Wet Lung Weight (g/100 g body weight) (g/100 Weight Lung g body Wet

C D 800 * 8 * 700 7 600 6 500 5 400 4

300 3

200 2 ALP Activity (IU/L) Activity ALP 100 1

0 Protein Concentration (mg/mL) 0 Saline CD-3 CD-5 0.1% SDS Saline CD-3 CD-5 0.1% SDS

Fig. 4.7: Acute in-vivo toxicity studies of CD-3 and -5 in bronchoalveolar lavage (BAL) fluid after 12 hours of administration. (A) Corrected wet lung weights (g/100 g body weight); Levels of (B) LDH activity (IU/L); (C) APL activity (IU/L); and (D) Total protein concentration (mg/mL). *Means are significantly different from each other (p<0.05).

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In-vitro Toxicity Studies in Calu-3 Cell Lines

0.5 mg/mL 1 mg/mL 100 2 mg/mL 5 mg/mL

40 % Cell Viability Cell % 20

0 3 ine D- C CD-5 Sal 0.1% SDS

Fig. 4.8: Acute cytotoxicity studies to determine the safety of CD-3 and -5 microspheric formulations in Calu-3 lung epithelial cell lines. Data represent mean ± standard deviation (n = 8).

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CHAPTER 5

PLGA PARTICLES OF PGE1 AMELIORATES SYMPTOMS OF PAH IN A RAT MODEL

FOLLOWING ACUTE AND CHRONIC ADMINISTRATION VIA THE PULMONARY

ROUTE

INTRODUCTION

We have recently reported that circulation half-life (t1/2) of PGE1 can be tremendously

increased by encapsulating it in polymeric [poly (lactide-co-glycolide) (PLGA)]

microparticles feasible for pulmonary administration (Gupta et al., 2010). As discussed in

chapters 2, 3, and 4, PLGA microparticles serve as an optimized delivery system for

aerosolized PGE1 therapy with favorable aerodynamic properties required for deep lung

deposition of the drug. In addition to providing feasibility for pulmonary administration,

PGE1 loaded PLGA microparticles also provide prolonged release of PGE1 when administered in healthy rodents [t1/2 of 9.5 hrs with plain PLGA particles (Chapter 2) and

6.5 hrs with PEI modified large porous PLGA particles (Chapter 3)].

Despite PGE1 encapsulated inhalable PLGA microparticles exhibiting prolonged and sustained release of PGE1 following pulmonary administration, there is still no evidence available so as to establish the therapeutic potential of these particulate delivery systems in providing sustained and long term relief from PAH symptoms. In addition, it is also not known if PGE1 has some protective effects on PAH i.e. if it reverses the structural remodeling of pulmonary vasculature, the key feature of PAH pathogenesis.

Monocrotaline (MCT) is a toxic crystalline 11-membered macrocyclic pyrrolizidine alkaloid of plant origin, which is found in Crotolaria spectabilis and other plants of

Crotolaria genus. Toxic manifestations of MCT exposure include hepatic and pulmonary

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 lesions. In the lungs, vascular lesions are prominent that causes swelling of capillary endothelial cells, lesions of the arterial media, and thrombi accumulation with large number of platelets resulting in increased pulmonary arterial pressure and right ventricular hypertrophy. Various studies have documented the similarities observed between lesions in rats induced by MCT and that in the lungs of patients of PAH and chronic pulmonary vascular disease (Hilliker et al., 1982). A single subcutaneous injection of MCT (60 mg/kg) in rats results in lung injury characterized by pulmonary vascular remodeling, pulmonary hypertension and compensatory right heart hypertrophy over 3-4 weeks (Todd et al., 1985). Several investigators have studied suitability of

MCT-induced rodent model of PAH and have demonstrated that MCT treatment in rats induces right ventricular hypertrophy, increases the cross-sectional area of small pulmonary arteries (100-200 μM) and reduces the luminal diameter that eventually leads to reduction in lung function. MCT treatment results in muscularization of small pulmonary arterioles thus causes increased MPAP and right ventricular hypertrophy (Lai and Law, 2004). Prolonged exposure to hypoxia is also reported to induce PAH like symptoms in rodents. While hypoxia causes an immediate increase in PAP followed by vascular remodeling, MCT treatment causes injury and induces structural changes in the pulmonary circulation resulting in an increase in PAP (van Suylen et al., 1998). In this dissertation, we intend to investigate both preventive and protective roles of PGE1 when

administered as inhalable controlled release formulations in-vivo. MCT-induced rodent

model of PAH meets all the requirements of an ideal animal model for the project.

In this chapter, we have therefore investigated the efficacy of inhalable PGE1 encapsulated PLGA microparticles in providing symptomatic relief from PAH symptoms in monocrotaline (MCT) induced rodent model of chronic PAH following both acute and chronic treatment via the pulmonary route.

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We have studied the effects of acute single dose treatment and chronic 10 days treatment of above mentioned microparticles on pulmonary and systemic hemodynamics, and right ventricular hypertrophy in MCT induced rat model of PAH, as compared to plain PGE1 administered via I.V. or pulmonary route.

MATERIALS AND METHODS

Materials

PLGA 85:15 (average molecular weight = 85.2 kDa, and inherent viscosity = 0.55 – 0.75 dl/g) for preparation for microparticles was obtained from Lactel Absorbable Polymers

(Pelham, AL). Prostaglandin E1 (PGE1) was obtained from Spectrum Chemicals

(Gardena, CA). To induce PAH, monocrotaline (MCT) was obtained from Sigma-Aldrich,

Inc. (St. Louis, MO). ELISA kit to analyze PGE1 concentration was purchased from

Assay Designs, Inc. (Ann Arbor, MI). All other chemicals were of analytical grade, and

were obtained from various vendors. All the chemicals were used without any

modification.

Microparticle Preparation and Physical Characterization

PGE1 encapsulated PLGA microparticles were prepared by W/O/W double emulsion/solvent evaporation method as described in Chapters 2, and 3. Two optimized formulations [one for plain PLGA particles (MCP-4), and one for PEI modified large porous microparticles (PEI-1)] were prepared and freeze dried according to previously described procedure. After freeze drying, the prepared formulations were tested for in- vitro characterization (mean volume particle size, aerodynamic diameter, entrapment efficiency, in-vitro drug release profile), and in-vivo absorption studies. PGE1

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 concentration after in-vitro release or after in-vivo absorption studies was determined by

ELISA kit as described earlier.

Animals and Experimental Protocols

All experiments were performed on adult male Sprague-Dawley rats (weighing 250-300 g) (Charles River Laboratories, Wilmington, MA). PAH was induced by a subcutaneous injection of MCT (50 mg/kg body weight). MCT solution was prepared by dissolving required amount in 0.1N HCl and then adjusting the pH to 7.4 by 1N NaOH, as described previously (Revermann et al., 2009). All animals were kept at the TTUHSC animal facilities under normal conditions for 28 days with free access to food and water for developing PAH symptoms. All the animal studies were performed in accordance

with NIH Guidelines for the Care and Use of Laboratory Animals under a protocol

approved by Texas Tech University Health Sciences Center (TTUHSC) Animal Care and

Use Committee (AM-10012).

Experimental Design

Experiments were performed to determine efficacy of PGE1 loaded microparticulate formulations in alleviating PAH symptoms following acute and long-term treatment.

Animals were divided into different groups according to the study design as described below:

Acute Treatment Studies: For acute single dose treatment hemodynamic studies, 36

PAH induced animals (MCT28) were used; and were divided into 6 groups of 6 animals each (n = 6): (1) Sham animals (no treatment); (2) single dose of saline via pulmonary route; (3) single I.V. dose of PGE1 (120 μg/kg); (4) single dose of PGE1 (120 μg/kg) via

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pulmonary route; (5) aerosolized MCP-4 equivalent to 120 μg/kg PGE1; and (6)

aerosolized PEI-1 equivalent to 120 μg/kg PGE1.

Chronic Treatment Studies: For chronic treatment studies, the animals were classified into following 6 groups of 6 animals each (n = 6): (1) animals with no treatment (Sham);

(2) MCT injected rats, 28 days postinjection, with no treatment (MCT-28); (3) MCT injected rats treated with aerosolized saline (100 μl) 3 times a day for 10 days; (MCT-

38); (4) MCT injected rats, 28 days postinjection, treated with I.V. PGE1 (120 μg/kg) 3

times a day for 10 days (MCT-IV); (5) MCT injected rats, 28 days postinjection, treated

with aerosolized PGE1 (120 μg/kg) 3 times a day for 10 days (MCT-Pulmonary); and (6)

MCT injected rats, 28 days postinjection, treated with aerosolized PEI-1 equivalent to

120 μg/kg PGE1, 3 times a day for 10 days (MCT-Particle).

I.V. administrations were performed either by penile vein injection (acute studies) or by

sublingual vein injection (chronic treatment studies). Pulmonary administrations were

performed via the intratracheal route utilizing a microsprayer (Penn-Century,

Philadelphia, PA) as described in earlier chapters. All the formulations were prepared

fresh in sterile saline (pH 7.0) for both I.V. and pulmonary administrations.

Hemodynamic Measurements

Hemodynamic measurements were performed to determine mean pulmonary arterial pressure (MPAP) and mean systemic arterial pressure (MSAP) using PowerLab 16/30 with LabChart Pro 7.0 software (ADInstruments, Inc., Colorado Springs, CO). Briefly, animals were anesthetized by an intramuscular injection of a cocktail of Ketamine (90 mg/kg) and xylazine (10 mg/kg). Fluid filled catheters used for blood pressure measurements were connected to the PowerLab setup with Memscap SP844

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 physiological pressure transducers (Memscap AS, Scoppum, Norway) with the help of bridge amplifiers. The ventral neck area and the area dorsally between the scapulae were shaved and were cleaned by scrubbing with betadine solution and ethyl alcohol. A

2-3 cm incision was made over the right ventral neck area so to expose the internal jugular vein. Jugular vein was isolated and kept separated using a suture. The right carotid artery was isolated, and a PE-50 catheter was inserted 3-4 cm into the artery and securely sutured into place for measurements of systemic arterial pressures. For the measurement of pulmonary arterial pressures, a polyvinyl (PV-1) catheter was used with the top end curved at an angle of 60-650 (Stinger et al., 1981). PV-1 catheter was threaded into the pulmonary artery via the right internal jugular vein. A catheter pressure tracing was transduced and was monitored using the PowerLab setup. The location of the tip was identified by the characteristic shapes of the PA pressure waveforms. Once in pulmonary artery, the catheter was then securely sutured into place (Crossno et al.,

2007).

For acute studies, once the animal has been catheterized and the blood pressures have stabilized, formulations to be tested were injected via the desired route (either I.V. via penile vein or pulmonary via intratracheal administration using the PennCentury microsprayer®). Blood pressure measurements were performed soon after surgery as

MCT rats are reported not being able to withstand the stress associated with anesthesia

and surgery. Rats were kept under anesthesia throughout the period of experiment. To

determine the controlled release efficacy of the formulations, blood pressures

measurements were recorded until the PA pressures came back in the vicinity of the

initial starting point. Following the hemodynamic measurements, the animals were

sacrificed by exsanguination and lungs were preserved at -800C for further analysis.

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For chronic studies after the treatment period, rats were catheterized for PA and SA pressure measurements. Blood pressures were recorded for a period of 25-30 minutes, and the animals were sacrificed afterwards by exsanguination so as to obtain the entire heart-lung block for further analysis. Right lung was preserved for measuring levels of various markers of PAH [Proliferating Cell Nuclear Antigen (PCNA), and

Matrixmetalloproteinases (MMPs)]. Left lung was fixed with 10% buffered formalin phosphate solution using inflation fixing method described elsewhere. Following fixation, heart and lungs are removed en bloc. Heart was used to perform right ventricular hypertrophy measurements, and the formalin fixed left lung was used for immunostaining and morphometry studies to determine the extent of muscularization in the arteries.

Right Ventricular Hypertrophy Measurements

For determination of right ventricular hypertrophy, weight ratio of right ventricle vs. left ventricle + septum was determined (RV/LV+S). Briefly, the heart was removed from the animal body following hemodynamic measurements, and was placed the right ventricle on the top of the heart. The two atria were cut off and great vessels were trimmed so as to isolate both the ventricles. Right ventricle was carefully isolated from left ventricle keeping the septum intact with left ventricle. Excess fluid was dried off and the heart and both parts were weighed, so as to calculate RV/LV+S. All the measurements were performed by the same person without the knowledge of the sample being analyzed.

Gelatin Zymography

Gelatin zymography studies were performed to determine the levels of MMP-2 and -9 in the diseased animals, and to determine the effects of microparticulate formulations on

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 these important markers of PAH. For zymography studies, portions of whole lung were homogenized in PBS without protease inhibitors at a concentration of 50 mg/ml using a glass homogenizer. The homogenate thus obtained was centrifuged at 4,000 ×g for 15 minutes at 40C, and the supernatant was collected. Protein concentration was measured by BCA protein assay kit (Pierce Biotechnologies, Rockford, IL). A total of 15 μg protein, mixed with an equal volume of SDS loading buffer (without reducing agent), was loaded on a 10% polyacrylamide-gelatin Novex Zymogram minigel (Invitrogen Corporation,

Carlsbad, CA). Rest of the procedure was performed by using the instructions given by the manufacturer. MMP controls were used to determine the bands of MMP-2 and -9

(R&D Systems, Inc. Minneapolis, MN).

Data Analysis

All data are presented here in terms of mean±SD. Differences among the groups were analyzed by one-way Analysis of Variance (ANOVA) followed by appropriate post hoc analysis (GraphPad Prism version 5.0, GraphPad Software, La Jolla, CA). Values showing p<0.05 were considered significantly different.

RESULTS AND DISCUSSION

Microparticle Preparation and Characterization

As discussed in Materials and Methods section, two optimized microparticulate formulations were prepared for hemodynamic studies. Both the formulations were characterized for their in-vitro characteristics and in-vivo characterization, and were

found to be comparable with the ones reported in Chapter 2 (MCP-4), and Chapter-3

(PEI-1) respectively. Mean volume diameters of the formulations were found to be in the

range of 10-11 μM for both the formulations. However, there was a significant difference

in the aerodynamic diameters of both the formulations (≈4.5μM for MCP-4; and ≈2.5μM

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 for PEI-1) making both of them suitable candidates for deep lung deposition following pulmonary administration. Decrease in aerodynamic diameter of PEI-1 can be attributed to presence of hollow pores on particles’ surface. Moreover, both the formulations showed good entrapment efficiencies (60% for MCP-4, and 85% for PEI-1); with favorable in-vitro drug release (11% for MCP-4, and 56% for PEI-1). In-vivo absorption studies of the formulations also suggested favorable drug release following intratracheal administration (Table 5.1). All these in-vitro and in-vivo characterization confirmed choice of MCP-4 and PEI-1 as optimum delivery systems for testing the efficacy of controlled release PGE1 formulations in treating PAH symptoms in MCT induced rat model of PAH.

Acute Hemodynamic Studies

Acute hemodynamic studies were performed to determine the efficacy of PLGA microparticles in providing long-term and sustained relief from PAH symptoms following a single dose treatment in MCT induced PAH rats. As can be seen in Fig. 5.1A, administration of plain PGE1 via both I.V. and pulmonary route resulted in significant

reduction in MPAP. With I.V. PGE1, there was approximately 35% reduction in MPAP, and the effects totally disappeared in 35-40 minutes, thus bringing the PA pressure back to the baseline levels. At the same time, administration of aerosolized PGE1 resulted in a sustained pulmonary vasodilation with about 32% reduction in MPAP (comparable to I.V.

PGE1). However the effects weaned off in 45-50 minutes. On the contrary, saline administration via the pulmonary route did not have any significant effect on MPAP. In case of MSAP measurement, pulmonary PGE1 showed a significant inclination toward

selective pulmonary vasodilation. As can be seen in the Fig. 5.1B, pulmonary PGE1

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Texas Tech University Health Sciences Center, Vivek Gupta, December 2010 produced ≈30% decrease in MSAP, which is significantly lower than 50% in case of I.V.

PGE1 (p<0.05).

When optimized plain PLGA microparticles of PGE1 (MCP-4) were administered to MCT induced PAH rats via the pulmonary route, a significant reduction in MPAP was observed, which was sustained up to 2 h; whereas administration of large porous PLGA microparticles (PEI-1), the vasodilatory effect in pulmonary circulation was sustained up to 3 h, with MPAP reduction of about 40% (Fig. 5.2A). Plain PLGA microparticles (MCP-

4) showed a selectivity pattern toward pulmonary vasodilation, evident form less prominent decrease in MSAP (30%) as compared to plain drug. However, to our surprise, we did not see a pulmonary selectivity pattern with large porous particles (PEI-

1), which is evident from significant reduction in MSAP after single dose treatment with

PEI-1 (Fig. 5.2B),

As can be seen in Fig. 5.3, derived from the hemodynamics data obtained from acute treatment studies, both the optimized formulations of PGE1 encapsulated PLGA microparticles certainly produce a sustained reduction in MPAP as compared to the plain drug administered via I.V./pulmonary route. As compared to plain drug administration,

MCP-4 showed a significant decrease in MPAP up to 2.5 h, whereas PEI-1 produced a sustained reduction in arterial pressure for up to 3.5 h. During this time period, the pulmonary arterial pressure was in the normal range for the same, indicating toward protective effects of PGE1 and its optimized polymeric formulations on PAH affected

pulmonary circulation.

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Chronic Treatment with Inhalable PLGA Microparticles of PGE1

As discussed in the section above, PEI-1 provided relief from PAH symptoms for the longest period of time (3-3.5 h), which is suitable for long-term PAH treatment. To determine the long-term efficacy of PEI-1 in protection against PAH symptoms, a chronic treatment study was performed in MCT induced PAH rats. In this study, plain PGE1 (I.V. or pulmonary route), or optimized microparticulate formulation (PEI-1) were administered to the animals 3 times a day for 10 days. After 10 days of treatment, animals were catheterized for MPAP and MSAP measurements, and heart-lung block was collected for further studies. As can be seen in Fig. 5.4A, treatment with I.V. PGE1 produced the

maximum decrease in MPAP (21.39±3.15 mm Hg) as compared to 39.76±3.63 mm Hg

(28 days after MCT injection) and 46.82±4.19 mm Hg (38 days after MCT injection). At

the same time, pulmonary PGE1 also produced significant reduction in MPAP as compared to MCT controls (26.92±4.75 mm Hg). More importantly, PEI-1, the optimized

PLGA microparticulate formulation of PGE1, demonstrated significant reduction in MPAP as compared to MCT controls (31.31±2.33 mm Hg). No significant difference was observed between MPAP values obtained with aerosolized plain PGE1 and PEI-1 treated

rats. However, there was a significant difference between the MPAP values obtained

with I.V. PGE1 and aerosolized formulations (plain drug or microparticles). At the same

time, there was no significant difference observed among MSAP values obtained

following I.V. or pulmonary (neither plain PGE1 nor PEI-1) (Fig. 5.4B), which indicates toward the pulmonary selective vasodilatory effects of PGE1.

Right Ventricular Hypertrophy Measurements

As discussed earlier, right ventricular hypertrophy is one of the major clinical manifestation of PAH progression and development. To determine the protective effects

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of PGE1 and its microparticulate formulations on PAH vasculopathy, extent of right ventricular hypertrophy were determined by calculating RV/LV+S ratio. Four weeks after injection of MCT, all the animals demonstrated significant right heart hypertrophy, indicated by an increase in the ratio of right ventricular weight to left ventricular plus septum weight (RV/[LV+S]) from 0.32±0.078 (control animals) to 0.649±0.069 (Fig. 5.5).

Rats receiving no treatment for 10 more days (day 28 to 38), showed further progression of right ventricular hypertrophy (RV/LV+S of 0.752±0.14). As can be seen in Fig. 5.5, inhaled formulation of PGE1 (PEI-1) not only prevented further progression of right ventricular hypertrophy, but also caused a significant regression of right ventricular hypertrophy as compared with Day 28 (MCT-Particles – 0.536±0.022).

Gelatin Zymography Measurements

As can be seen from the representative zymogram presented in Fig. 5.6, treatment with

PGE1 resulted in a significant decrease in levels of both MMP-2 and MMP-9, as compared to both MCT-28 and MCT-38 animals. Matrix metalloproteinases (MMPs) are zinc dependent endopeptidases, which are capable of degrading extracellular matrix proteins. MMPs are thought to play a major role in PAH progression by mediating several pathogenic features including cell proliferation, migration, differentiation, angiogenesis, apoptosis, and host defense. MMPs, especially, MMP-2 and -9, which are capable of degrading gelatin, are found to be 4-5 times overexpressed in PAH patients, and there are several reports efficacy of prostacyclin analogues in decreasing MMP levels, and thus knocking down the proliferation cascade. Both MMP-2 and MMP-9 are regulated by intracellular cAMP levels (Peracchia et al., 1997; McCawley et al., 2000), and are suppressed by prostacyclin analogues (Schermuly et al., 2004). As can be seen in Fig. 5.6, the protective therapy with PGE1, both as plain drug and as microparticulate

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Moreover, decrease in MMP levels by microparticulate PGE1 (PEI-1) was more

significant than plain PGE1 administered via either IV or pulmonary route.

In summary, this study is first to investigate potential applications of controlled release

drug delivery systems in delivering anti-PAH medications to the lungs via the non-

invasive pulmonary route. It can be said that inhalable PLGA microparticulate

formulations of PGE1 certainly provide a prolonged release of PGE1 due to time

dependent degradation of polymeric core of the microparticles, thus providing long-term

relief from PAH symptoms following a single dose treatment. At the same time, PLGA

microparticles also exhibit preventive effects on PAH progression in MCT induced rats

following long-term chronic treatment, which are comparable to effects of plain drug.

However, there are still more studies needed to investigate the molecular pathways

being affected in this process, and also to confirm the preventive effects of PGE1 encapsulated PLGA microparticles, which are currently underway in our laboratory.

REFERENCES

Crossno JT, Jr., Garat CV, Reusch JE, Morris KG, Dempsey EC, McMurtry IF, Stenmark KR and Klemm DJ (2007) Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling. Am J Physiol Lung Cell Mol Physiol 292:L885-897.

Gupta V, Rawat A and Ahsan F (2010) Feasibility study of aerosolized prostaglandin E1 microspheres as a noninvasive therapy for pulmonary arterial hypertension. J Pharm Sci 99:1774-1789.

Hilliker KS, Bell TG and Roth RA (1982) Pneumotoxicity and thrombocytopenia after single injection of monocrotaline. Am J Physiol 242:H573-579.

Lai YL and Law TC (2004) Chronic hypoxia- and monocrotaline-induced elevation of hypoxia-inducible factor-1 alpha levels and pulmonary hypertension. J Biomed Sci 11:315-321.

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McCawley LJ, Li S, Benavidez M, Halbleib J, Wattenberg EV and Hudson LG (2000) Elevation of intracellular cAMP inhibits growth factor-mediated matrix metalloproteinase- 9 induction and keratinocyte migration. Mol Pharmacol 58:145-151.

Peracchia F, Tamburro A, Prontera C, Mariani B and Rotilio D (1997) cAMP involvement in the expression of MMP-2 and MT-MMP1 metalloproteinases in human endothelial cells. Arterioscler Thromb Vasc Biol 17:3185-3190.

Revermann M, Barbosa-Sicard E, Dony E, Schermuly RT, Morisseau C, Geisslinger G, Fleming I, Hammock BD and Brandes RP (2009) Inhibition of the soluble epoxide hydrolase attenuates monocrotaline-induced pulmonary hypertension in rats. J Hypertens 27:322-331.

Schermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W and Grimminger F (2004) Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res 94:1101-1108.

Stinger RB, Iacopino VJ, Alter I, Fitzpatrick TM, Rose JC and Kot PA (1981) Catheterization of the pulmonary artery in the closed-chest rat. J Appl Physiol 51:1047- 1050. Todd L, Mullen M, Olley PM and Rabinovitch M (1985) Pulmonary toxicity of monocrotaline differs at critical periods of lung development. Pediatr Res 19:731-737. van Suylen RJ, Smits JF and Daemen MJ (1998) Pulmonary artery remodeling differs in hypoxia- and monocrotaline-induced pulmonary hypertension. Am J Respir Crit Care Med 157:1423-1428.

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Table 5.1: In-vitro and In-vivo Characterization of PGE1 Encapsulated Microparticulate Formulations

Formulation Particle MMAD % Drug % Cumulative Cmax t1/2 (min) Size (μM) (μM) Entrapment Release (ng/mL) MCP-4 10.66±3.71 4.3±0.8 59.44±3.61 10.49±0.16 8.48±2.45 582.39±70.3 PEI-1 10.72±0.04 2.5±0.12 83.26±3.04 55.36±0.06 20.12±3.81 362.3±50.56

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% Decrease in MPAP Following Single Dose Administration of Plain PGE1

Saline, Pulmonary Plain PGE , IV (120 μg/kg) 1 45 Plain PGE , Pulmonary (120 μg/kg) 1 40 35 30 25 20 15 10 % Decrease in MPAP % 5 0 0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

Fig. 5.1: (A) Influence of single dose administration of Plain PGE1 via either I.V. or Pulmonary Route on mean pulmonary arterial pressure. Data represent mean±SD (n = 3-5).

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% Decrease in MSAP Following Single Dose Administration of Plain PGE1

Plain PGE , IV (120 μg/kg) 60 1 Plain PGE , Pulmonary (120 μg/kg) 1 50

10 % Decrease in MSAP %

0 1020304050 Time (min)

Fig. 5.1: (B) Influence of single dose administration of Plain PGE1 via either I.V. or Pulmonary Route on mean systemic arterial pressure. Data represent mean±SD (n = 3- 5).

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% Decrease in MPAP after Administration of Aerosolized PLGA Microparticles

70 Plain PLGA Particles Large Porous PLGA Particles 60 Plain PGE1 Pulmonary

% Decrease in MPAP % 10

0 0 20 40 60 80 100 120 140 160 180 200 Time (min)

Fig. 5.2: (A) % Decrease in mean pulmonary arterial pressure (MPAP) following single administration of PLGA microparticles of PGE1. Data represent mean±SD (n = 3-5).

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% Decrease in MSAP after Administration of Aerosolized PLGA Microparticles

80 Plain PLGA Particles Large Porous PLGA Particles Plain PGE1 Pulmonary 60

0 % Decrease in MSAP %

-20 0 20 40 60 80 100 120 140 160 180 200 Time (min)

Fig. 5.2: (B) % Decrease in mean systemic arterial pressure following single administration of PLGA microparticles of PGE1. Data represent mean±SD (n = 3-5).

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Duration of Vasodilatory Effects of Plain and Microparticle Encapsulated PGE1

220 * 200 180

160 140 120 100 80

60 40 20

Duration of Vasodilatory Effects (min) Effects Vasodilatory of Duration 0 e . y s s in I.V ar le le al g n tic tic S u o ar ar Dr ulm P P in P A A la g LG LG P u P P Dr in s ain la ou Pl P or e P rg La

Fig. 5.3: Comparative profile of duration of vasodilatory effects of plain drug (I.V. and pulmonary) and microparticulate formulation (MCP-4 and PEI-1) on Decrease in Mean pulmonary arterial pressure. *p<0.05 among Plain-Pulmonary, Plain-I.V., and Microparticulate formulations. Data represent mean±SD (n = 3-5).

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Effect of Chronic Treatment on Mean Pulmonary Arterial Pressure

50 * * 40 †‡ ** 30 ** 20 MPAP (mm in Hg) in (mm MPAP 10

0 . y s am -28 -38 I.V ar le T T T- on tic Sh C C C m r M M M ul -Pa -P T T MC MC

Fig. 5.4: (A) Effect of chronic long-term treatment with PLGA microparticles on mean pulmonary arterial pressure. *p<0.05 versus sham control; **p<0.05 versus MCT-28 and MCT-38; and †p<0.05 versus MCT-28; and ‡p<0.05 versus MCT-38. Data represent mean±SD (n = 3-5).

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Effect of Chronic Treatment on Mean Systemic Arterial Pressure

120

100

MSAP (mm in Hg) in (mm MSAP

m 8 8 . ry s a -2 -3 -I.V a le h T T T on tic S C C C m ar M M M ul -P -P T T MC MC

Fig. 5.4: (B) Effect of chronic long-term treatment with PLGA microparticles on mean systemic arterial pressure. No significant difference was observed. Data represent mean±SD (n = 3-5).

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Determination of Right Ventricular Hypertrophy after 10 days Chronic Treatment

1.0 * 0.8 *

0.6 †‡ ** ** 0.4 RV/LV+S Ratio

0.2

0.0 8 8 . y s am -2 -3 I.V ar le h T T T- n tic S C C C mo ar M M M ul -P -P T T MC MC

Fig. 5.5: Influence of long-term treatment with PGE1 and PGE1 loaded PLGA microparticles on right ventricular hypertrophy in MCT induced pulmonary arterial hypertension in rats. Data represent Right to left ventricular ratio (RV/LV+S) (mean±SD; n = 3-5). *p<0.05 versus sham control; **p<0.05 versus MCT-28 and MCT-38; and †p<0.05 versus MCT-28; and ‡p<0.05 versus MCT-38.

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Determination of MMP-2 and MMP-9 by Gelatin Zymography

Fig. 5.6: Gelatin zymogram showing the efficacy of microparticulate formulation of PGE1 in decreasing the elevated levels of MMP-2 and MMP-9 in monocrotaline-induced PAH rats. Zymogram is representative of 3 separate experiments; n = 1 per treatment.

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CHAPTER 6

CONCLUSIONS

This dissertation project was designed to investigate the feasibility of a controlled release formulation of prostaglandin E1 (PGE1), a rather under-investigated therapeutic

option for PAH treatment. The proposed controlled release formulations would (i) provide

long term relief from PAH symptoms, (ii) eliminate the need for continuous infusion or

multiple inhalations per day (iii) provide pulmonary selective vasodilation and (iv) reduce

the systemic side effects and associated toxicity. Importantly, like other prostacyclin

analogues currently approved by FDA for PAH treatment, PGE1 is also a prostanoid with similar chemical and physical properties and hence the insight gained with PGE1 can

also be utilized to develop delivery systems for other prostacyclin analogs.

We first investigated the viability of a controlled release formulation of PGE1 for noninvasive pulmonary delivery. Drug loaded polymeric microparticles are one of the most investigated approaches for development of inhalable controlled release formulations.

Poly (lactide-co-glycolide) is one of the biocompatible and biodegradable polymers, which has been approved by the FDA for usage in developing novel drug delivery systems. In fact, PLGA microparticles are probably the most investigated carriers for pulmonary delivery of various large molecular weight peptides and small molecular weight therapeutic agents. We hypothesized that encapsulating PGE1 in PLGA microparticle core results in sustained time dependent release of PGE1 when

administered via the pulmonary route. To test this hypothesis, we prepared PGE1-loaded plain PLGA microparticles that can provide long term drug release following intratracheal administration in rats (Chapter 2). Initial in-vitro characterization studies suggested that although PGE1 was encapsulated in PLGA microparticulate core, the entrapment

efficiency was not very promising and several factors governed the drug entrapment in

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PLGA microparticles. These factors may include (i) type of polymer, (ii) concentration of emulsion stabilizer and (iii) osmotic properties of the internal aqueous phase. However, the in-vivo absorption profile of the optimized plain PLGA microparticles showed a very significant increase in the biological half-life of the drug following intratracheal administration−≈9.5 h as compared to ≈5 minutes following plain PGE1 administration.

Based on these results, we concluded that PLGA microparticles serve as a promising delivery system for PGE1 which however needs further optimization in terms of drug

entrapment and optimum aerodynamic properties.

The next step in this project was to develop a delivery system with enhanced drug

entrapment and improved aerodynamic properties. We hypothesized that large porous

PLGA microparticles are superior choice for delivering PGE1 via the pulmonary route.

Large porous PLGA microparticles have pores on the particles’ surface which makes them lighter and bulkier, while the aerodynamic diameter remains in the respirable range

(Chapter 3). We used polyethyleneimine (PEI)-25 kDa as a porosigen in preparing these particles. PEI, a cationic polymer, also works as an absorption enhancer. The data obtained suggest that incorporation of PEI indeed results in development of porous particles, which have large mean diameter but aerodynamic diameter in the respirable range. In addition, PGE1 also forms a charge-neutralization complex with PEI which

results in increased drug entrapment and systemic absorption. The in-vivo data

suggested that large porous microparticles of PGE1 provide prolonged release of the

drug in the circulation and provide higher Cmax.

The next study of the dissertation project has been focused on studying the effects of

incorporation of PGE1-HPβCD complex in PLGA microparticles. PGE1 is an extremely

hydrophobic molecule, which contributes to its poor absorption and low bioavailability.

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Some reports have suggested that incorporation of PGE1 into hydrophilic HPβCD toroid

like cavity results in increased aqueous solubility of PGE1 and thus better bioavailability.

We hypothesized that incorporation of water soluble PGE1-HPβCD complex into PLGA

microparticles results in development of a delivery system with increase bioavailability of

the drug (Chapter 4). Moreover, HPβCD also acts as a porosigen and a bulking agent,

creating bulky particles with low mass density and internal pores. The data suggested

that incorporation of HPβCD resulted in development of bulky particles with aerodynamic

diameter favorable for pulmonary delivery. Moreover, in-vivo data indicated that PGE1-

HPβCD inclusion complex increased the bioavailability of PGE1. When encapsulated in

PLGA microparticles, HPβCD provides a prolonged controlled release of PGE1 due to time dependent degradation of the polymeric core.

The last study of this dissertation project was directed toward determination of the efficacy of optimized PLGA microparticles of PGE1 in alleviating PAH symptoms and producing protective effects in a monocrotaline (MCT) induced rat model of PAH. MCT, when injected into animals, produce symptoms mimicking PAH symptoms in humans which include increase MPAP, right ventricular hypertrophy, increased PVR and muscularization of pulmonary arterioles. Both acute and long-term studies were performed to investigate both preventive and protective effects of PGE1 encapsulated

PLGA microparticles on pulmonary circulation upon intratracheal administration (Chapter

5). Single dose acute administration of microparticulate formulations of PGE1 (MCP-4, and PEI-1) resulted in a significant reduction in MPAP with a tendency toward pulmonary selective vasodilation. The observed effect was comparable to the plain drug administered via either I.V. or inhalable route. PLGA microparticles exhibited sustained and prolonged vasodilatory effects as compared to plain drug−2.5-3 h as compared to

35-40 minutes. The data obtained clearly suggest that PGE1 encapsulated PLGA

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microparticles provide sustained release of PGE1 which resulted in prolonged efficacy of the formulations. To determine the long-term efficacy of microparticulate formulations, we performed chronic studies for 10 days for microparticulate formulations. Chronic treatment with either the plain drug or the microparticulate formulation resulted in an improvement in pulmonary hemodynamics with little or no effect on systemic hemodynamics. The data obtained with PLGA microparticles was comparable to aerosolized plain PGE1 and no significant differences were observed. In addition, long- term treatment also exhibited protective effect on hemodynamics of PAH induced rats, which is evident from significant decrease in RV/LV+S ratio indicative of reduced right ventricular hypertrophy.

Overall, this doctoral study has provided important new information to evaluate the feasibility of controlled release anti-PAH therapeutics as a viable alternative to therapy involving continuous infusions/multiple inhalations to provide symptomatic relief. This doctoral work has also explored efficacy of PGE1 as a potential pulmonary selective anti-

PAH therapy with little or no systemic side-effects.

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Significance and Outlook:

As has been discussed in earlier chapters, there has not been any study so far which underlines the importance of sustained release drug delivery in treatment of PAH.

Despite the limitations associated with the currently available PAH therapy, there is a complete lack of studies looking in to developing new delivery systems for the currently approved anti-PAH medications. The current study tries to revitalize the efficacy of PGE1 as a potential PAH therapy by means of inhalable microparticulate drug delivery systems. In other words, it can be said that this study is the first one to highlight the feasibility of controlled release approach for sustained vasodilation in PAH patients.

This study has laid a groundwork for developing of a completely new class of controlled release inhalable anti-PAH therapies. However, this work should be considered as the first step toward development of a controlled release targeted PAH therapy with prolonged action and no unwanted systemic or local side-effects. As discussed in chapter 5, the optimized microparticulate formulation of PGE1 showed beneficial effects comparable to plain drug administration, when administered at the same dosing frequency. To further advance this project, it will be interesting to see if the controlled release PGE1 microparticles, when administered at lower dosing frequency, maintain

their beneficial effects in monocrotaline-induced PAH rats. Once this factor has been

established, the project can be moved further by performing extensive histological and

mechanistic studies so as to establish long-term safety and efficacy of inhalable PLGA

microparticles in preclinical PAH model before entering into the clinical development

phase Moreover, as PAH is a disease of small arterioles in pulmonary circulation,

strategies should be developed to target the diseased pulmonary arterioles without

affecting the entire pulmonary circulation.

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