A Thesis

entitled

Nose to Brain Delivery of Antiepileptic Using Nanoemulsions

by

Salam Shanta Taher Al-Maliki

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences,

Industrial Pharmacy

______Jerry Nesamony, Ph.D., Committee Chair

______Sai Hanuman Sagar Boddu, PhD, Committee Member

______Caren L. Steinmiller, PhD, Committee Member

______Patricia R. Komuniecki, PhD, Dean College of Graduate Studies

The University of Toledo

December 2015

Copyright 2015, Salam Al-Maliki

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Nose to Brain Delivery of Antiepileptic Drugs Using Nanoemulsions

by

Salam Al-Maliki

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in Pharmaceutical Sciences

Industrial Pharmacy

The University of Toledo

December 2015

The objective of this research work was to formulate and develop an intranasal oil-in- water nanoemulsion (NE) using spontaneous emulsification method. Based on ternary phase diagram, emulsification time, and solubility study, the composition ratio of surfactant:co-surfactant:oil was optimized to 2:1:1. The model antiepileptic drug chosen for loading was phenytoin. The NE preparation was evaluated for particle size, zeta potential, electrical conductivity, pH, % transmittance, polarized light microscopy, in vitro drug release studies using HPLC, sterility validation, differential scanning calorimetry (DSC), and in vitro nasal toxicity testing in bovine nasal mucosa. The nanoemulsion formulations were then subjected to stability studies for six months by storing formulations at 25±5°C and 5±3°C. The NE exhibited a clear and stable formulation with globule size less than 20 nm and neutral zeta potential. The absence of birefringence and high conductivity values indicated that the formulations were o/w NE.

The pH measurements confirmed compatibility of such formulation for intranasal delivery. In vitro release study showed 100% release of the drug from NE over a period iii of 48 hours. The absence of any endothermic events in the phenytoin - self nanoemulsifying mixture (SNE) when analyzed in DSC conclusively indicated that phenytoin was completely solubilized in this mixture.The histopathological studies of optimized formulation did not show signs of damage on the nasal mucosa.The results of the sterility verification test demonstrated that the sterilization of the final formulation using aseptic filtration with 0.22µm filter was sufficient for rendering the formulation aseptic. The nanoemulsion was found to be stable in all the temperature conditions when particle size distribution, drug content, pH, and conductivity were taken as stability indicating parameters during the six-month study period. Based on the obtained results, the o/w phenytoin NE was found to be stable and desirable for use in intra-nasal delivery.

iv

Dedicated to my family

Acknowledgements

I would like to express my gratitude to Dr. Jerry Nesamony for providing me an opportunity to be a part of his research group. This work would not have been possible without his valuable advice and excellent support all throughout the two years of graduate study. He provided me with all the resources and guidances that allowed me to succeed in this project.

I would like to specially thank Dr. Sai Boddu and Dr. Caren Steinmiller for serving as my committee members. I want to thank Dr. Alexander for all the knowledge gained. I thank Dr.Wissam A. AbouAlaiwi for his help with the polarized light microscopy and for agreeing to be the graduate faculty representative. I also thank thank

Dr. Andrea Kalinoski for helping me with the histopathology study. I would also like to thank my sponsorship Higher Committee for Education Development in Iraq (HCED) for supporting me financially throughout my graduate school.

I would like to thank my fellow classmate Zahraa Al-Saedi for her support, encouragement and help with my research. Special thanks to all my friends Ahmed

Fahad, Rajan, and Rinda who supported me during the research and writing.

Finally, I would like to thank my wife and my kids, my parents, my brother, cousins and my entire family, for their unlimited support and encouragement during my studies.

v

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... ix

List of Figures ...... x

1. Introduction ...... 1

1.1 Physiology and function of nose ...... 2

1.1.1. Breathing ...... 2

1.1.2. Filtration ...... 3

1.1.3. Heating and humidification...... 4

1.1.4. Olfaction and sensation ...... 4

1.1.5. Mucociliary clearance activity ...... 6

1.2 Anatomy of the nose ...... 7

1.2.1. External nose ...... 7

1.2.2. Nasal cavity ...... 7

1.2.2.1 Nasal vestibule ...... 8

1.2.2.2 Nasal valve ...... 8

1.2.2.3 The respiratory region ...... 9

vi

1.2.2.4 Olfactory region ...... 10

1.2.2.5 Nasopharynx ...... 11

1.3.Nasal ...... 12

1.3.1. Local delivery ...... 12

1.3.1.1 Nasal drugs ...... 12

1.3.1.2 Intranasal antihistamines and corticosteroid drugs ...... 13

1.3.1.3 Intranasal topical anesthetic agents ...... 15

1.3.2. Systemic delivery ...... 16

1.3.2.1. Analgesic agents (opioid and non-opioid agents) ...... 17

1.3.2.2. Cardiovascular agents ...... 20

1.3.2.3. Anti-emetic agents ...... 21

1.3.2.4. Anti-diabetic agents ...... 23

1.3.2.5. IN vaccine ...... 25

1.3.3. CNS delivery (direct Nose to brain delivery) ...... 27

1.4 Nanostructured for IN drug delivery and brain targeting ...... 30

1.4.1. Microemulsion ...... 31

1.4.2. Solid lipid nanoparticles ...... 33

1.4.3. ...... 34

1.4.4.Other colloidal carrier (polymeric micelle,dendrimer and NS) ...... 36

1.5.Antiepileptic drugs ...... 38

1.5.1. History...... 38

1.5.2 Mechanism of action ...... 39

1.5.3 Commercially available antiepileptic dosage forms ...... 39

vii

1.5.4 Alternative methods for delivery of antiepileptic drug to the brain..43

2. Significance of Research...... 46

3. Nose to Brain Delivery of Antiepileptic Drugs Using nanoemulsion system ...... 48

3.1. Abstract ...... 48

3.2. Introduction ...... 49

3.3. Materials and Methods ...... 51

4. Results and Discussion ...... 62

References ...... 85

viii

List of Tables

1.1 Commercially available dosage forms of antiepileptic drugs for treating of epilepsy ...... 41

1.2 List of the nanostructured systems used for brain delivery of antiepileptic drugs 44

3.1 Composition of optimized formulation ...... 54

4.1 Solubility of phenytoin drugs in various excipients ...... 63

4.2 Particle size and zeta potential obtained for drug loaded and blank NE ...... 66

4.3 Sterility vertification test performed on MH Agar plates indicating the presence

(+) or absence (-) of microbial growth on days 0, 7, and 14...... 77

4.4 Stability parameters of phenytoin NE ...... 80

ix

List of Figures

1-1 Anatomy of human nasal cavity ...... 2

3-1 Precision™ Reciprocating Shaker Water Baths used in vitro release study...... 61

4-1 Ternary phase diagrams of combination Cremophor RH 40 % - PEG 400 % - and

Labrafil M 2125 CS % ...... 64

4-2 A. transparent nanoemulsion B. turbid and milky ...... 65

4-3 Particle size of blank NE formulation ...... 67

4-4 Particle size of phenytoin nanoemulsion ( without filtration) ...... 67

4-5 Particle size of phenytoin nanoemulsion ( with filtration) ...... 68

4-6 Zeta potential of phenytoin nanoemulsion ...... 70

4-7 DSC thermogram of (A) Pure phenytoin drug, (B) Blank SNE mixture and

(C) phenytoin-loaded SNE mixture ...... 71

4-8 Polarized light microscope images a. optical micrograph of phenytoin NE without polarizing filter b.optical micrograph of phenytoin NE with polarizing filter at a magnification of 60x ...... 72

4-9 Polarized light microscope images a. optical micrograph of blank NE without polarizing filter b. optical micrograph of blank NE with polarizing filter at magnification of 60x ...... 72

4-10 (top) HPLC chromatogram of phenytoin drug (bottom) Calibration curve of phenytoin...... 74 x

4-11 Change in conductivity of A. phenytoin NE B. blank NE with different temperature ...... 76

4-12 Represented pictures of direct inoculation tubes a. positive control, b. negative

control, c. aseptically filtered sample d. positive sample control after 14 days of

inoculation...... 78

4-13 Represented pictures of MH Agar plates a. aseptically filtered sample b. positive control, c. negative control d. positive sample control at 14 days ...... 78

4-14 Histopathological pictures of the nasal mucosa treated with a.pbs 6.4 b. 70 %

isopropyl alcohol c. PHT NE ...... 81

4-15 In vitro drug release of phenytoin from nanoemulsion ...... 83

4-16 Kinetic model plots for (a) Zero order (b) First order (c) Higuchi and (d)

Korsemeyer-Peppas model...... 83

xi

Chapter 1

Introduction

Intranasal drug delivery is considered a viable option to deliver many types of therapeutic agents locally or systemically [1-3]. It has been used to deliver active compounds directly to the brain by passing the blood brain barrier (BBB)[4]. Intranasal drug delivery provides numerous advantages such as non-invasiveness, rapid absorption

(especially for lipophilic drugs), needle free, painless, self-administration by patients or physician, and enhanced bioavailability for drugs extensively metabolized by the liver or enzymatically degraded in the gastro intestinal tract (GIT)[5, 6]. In spite of its advantages, nasal delivery has some limitations which includes mucosal irritation due to frequent intranasal administration of drugs, the active mucociliary clearance which rapidly clears the drug from nasal cavity, and interference of nasal congestion with absorption of drug [6, 7]. Permeability of intranasally adminstered drugs are influenced by various factors such as biological, drug formulation and device related factors. The intranasal absorption and delivery rate of drugs varies with section of the nasal cavity the drug is in[8]. The nasal cavity is divided into five anatomical regions: nasal vestibule, the olfactory region, respiratory region, atrium, and nasopharynx (Fig. 1-1). Most of the drug absorption occurs in the respiratory region because of its high vasculature and large 1 surface area[4].The olfactory region offers direct access to cerebrospinal fluid so most of the intranasal drug are transported directly to CNS through this section of nasal cavity [9,

10]. It is important to understand the physiology and anatomy of the nasal cavity to address the obstacles that impact absorption and delivery of intranasal agents.

Figure 1.1. Anatomy of human nasal cavity[11].

1.1. Physiology and fuction of the nose

1.1.1 Breathing

Breathing by nose is the most important and preferred way for human newborns in the first few weeks of life. Nasal breathing in neonates offers many advantages: it prevents aspiration while swallowing during breast or bottle feeding and humidifies and warms the inspired air[12]. As the child gets older oral breathing becomes more prominent. Oral- nasal breathing becomes more active especially in intense exercise where the ventilation may increase to about 35 l/min[13]. A 42% higher loss of water was reported during oral

2 expiration when compared to nasal expiration in healthy subjects[14]. Breathing by nose can provide approximately 30 to 50% of total resistance during and this is sufficient to maintain elasticity and integrity of the lungs; while mouth breathing shows little resistance and may lead to development of lung disease such as emphysema[15]

[16]. Although life can be sustained without nasal breathing, of nasal aiflow is very dangerous as it results in the loss of air conditioning function of the nose.

1.1.2 Filtration

The nose is considered as the first filter to remove particulate matter from inspired air before reaching alveoli. Because of slow clearance activity and structural fragility of alveoli, deposition of extraneous particles in such areas may cause serious consequences in lung[17]. Several factors such as nasal disease, particle diameter, surgical alteration to the internal structure of the nose (terbinates and valves) may affect the filtration function of the nose [17, 18]. Particles with diameter of 10 um or more are trapped on the nasal mucosa and are moved toward the oropharynx to be swallowed within 30 minutes[19].

Filtering activity of the nose is decreased for small particles and gases. Particles with a diameter less than 2 μm are passed directly from the nasal cavity to the lower airways[20]. The nose can also filter and remove more than 99.9% of sulphur dioxide

(water soluble gas) that may irritate and damage air way tissues[21]. There are different mechanisms such as electrostatic interaction, mucociliary clearance, impingement, and deposition on vibrissae by which particles are filtered and cleaned from inspired air.

Deposition of a drug in the nose is affected by aerosol velocity from , and the velocity and particle diameter of the inhaled drug [20]. Nasal filtration is particularly

3 essential, especially during detrimental environmental conditions such as dusty weather as large quantity of air borne particles are needed to be filtered through the nose.

1.1.3 Heating and humidification

Another important nasal function is heating and humidification of inhaled air, both of which occurr at the same time [16]. Because of specialized vascularization in the nasal cavity, high secretary function of the epithelial gland and the goblet cells in the nose, the incoming air can be warmed and humidified efficiently and continuously[16, 20]. In the nasal mucosa, the arterial blood flows in opposite direction to the inspired air contributing to rapid and efficient heat exchange [22, 23]. It has been stated that humans can consume 300 to 400 ml of water and between 250 and 350 kcal every day during nasal breathing at mild environmental conditions[24]. These figures may be increased substantially in dry and cold climates. The normal volume of nasal secretion and exudate used to condition air was found to be one liter and this value is not constant as it changes along with changes in relative humidity and temperature of the inspired air. Seventy-five percent of this value is used to saturate inhaled air while the rest is used to enhance ciliary function of nasal mucosa such as filtering and cleaning the inspired air [16]. At room temperature, 23°C, or at very low temperatures, –18°C, the nose can effectively condition the inspired air to 30 °C and relative humidity of 98% while mouth breathing was found to be less effective in conditioning of cold air [25, 26].

1.1.4 Olfaction and sensation

Olfaction is the sense of smell which is important for humans and animals in the identification of different types of odors [23]. The mechanism of detection and

4 discrimination of smells is still unknown. The olfaction process starts when odorant binding protein binds and interacts with odorant molecules increasing their solubility several thousand times than in ambient air[23, 27]. With the aid of these proteins the hydrophobic odorants are then transported to olfactory epithelium to bind with olfactory receptor which is faster than by simple diffusion[28]. The olfactory area is located at the roof of the nasal cavity covering the superior turbinate and the nasal septum. It contains several kinds of cells such as olfactory receptor cells, supporting cells, basal cells and

Bowman’s gland [16, 17]. It is estimated that there are more than 10 million olfactory receptor cells in humans that act as peripheral sensory receptors and first-order ganglia[29]. The axons of these cells are grouped together at the glomerulus which is responsible for transmitting signals directly to the olfactory bulb. From there, extensions go to different structures such as thalamus, hippocampus, hypothalamus and to the frontal lobe[17]. The trigeminal system also plays an important role in perception of odor by enhancing or suppressing the olfactory system. It can act directly by modifying the response of olfactory receptors to chemical odorants by enhancing release of peptides from trigeminal nerves that are located in olfactory epithelium. The trigeminal nerves can also act indirectly through nasal trigeminal reflexes to decrease harmful effects of the chemicals through stimulation of secretary cells or glands[30]. Olfactory development may start in infants during the first 48 hours after birth[31]. It is thought that the sense of smell may be impaired in older people decreasing their ability to detect and discriminate some environmental hazards which can affect the quality of life such as smoke, toxins, chemicals, etc[32]. Several nasal or neurodegenerative diseases may also disturb olfactory function of the nose such that any reduction in the olfactory function

5 may not simply be an access problem as these diseases may alter olfactory processing at numerous levels[33, 34].

1.1.5 Mucociliary clearance activity

The mucociliary clearance system of the nose plays an important role in transporting mucus that contains entrapped particles from nasal epithelium to the nasopharynx with the aid of ciliated epithelial cells[35]. The nasal mucociliary activity offers a protective and defense mechanism for the lower respiratory tracts against hazards of inhaled particles and also minimize therapeutic effect of intranasal drugs by decreasing its residence time in the nasal cavity[36]. There are several methods other than mucociliary clearance through which mucus and nasal secretion are cleared from nasal cavity such as sneezing, sniffing and nose blowing, frequent nasal washing, etc. A variety of conditions and diseases may disturb mucociliary transport and lead to an increase in contact times between nasal epithelium and microbes which may increase the risk of respiratory airways infections [37]. Mucociliary clearance represent a combination of mucus and ciliary activity. The nose can secrete about 20 to 40 ml of mucus during 24 hours under normal conditions and this value may be increased in chronic inflammation of the nasal cavity where the number of goblet cells are increased [38, 39]. The ciliated cells are able to produce forceful strokes (more than 1000 per minute) causing efficient movement of the mucus toward nasopharynx and G.I.T [16]. Activity of cilia is also influenced by drugs and diseases. Drying of nasal mucus membrane or use of hypertonic saline may cause inhibition of ciliary activity and thus may impair the effectiveness of mucociliary clearance as a cleansing mechanism [40, 41]. Therefore assessing mucociliary clearance in normal and pathological conditions is very important when 6 determining the rate and extent of nasal absorption and delivery of by intranasal route.

1.2 Anatomy of the nose

1.2.1 External nose

The external nose is the visible part of the nose which is located in the midface covering both nostrils and anterior portion of the nasal cavity. Its structure is pyramidal and is composed of nasal bones which are situated superiorly and sets of cartilages inferiorly[42]. These cartilages (upper lateral cartilage, lower lateral cartilage, alar cartilage, septal cartilage and accessory cartilage) are connected to each other and to the nasal bone by fibrous tissue [43]. The nasal cartilaginous and bone frame-work are covered by groups of muscles which aid in supporting functions of the nose especially during respiration. They play important functions in decreasing total airway resistance and preventing collapse of nasal valves during exercise and thus increasing airway patency[44, 45]. The bony structure of the external nose has protective and esthetic function. It protects the internal fragile structure of the nose from accidents or injuries. It was observed that there are differences in external nose between individuals of different races. The dimensions of the nostrils and the width of the nose is found to be different in oriental, caucasian, or black adults. The average width of nose in blacks in a study was found to be 42.5 mm which is large compared to oriental or caucasian individuals[46].

1.2.2 Nasal cavity

The extension of the nasal cavity starts from the vestibule to the nasopharynx with a length of approximately 12 to 14 cm[47]. The nasal septum divides the nasal cavity into

7 two symmetrical sections with a total absorptive surface area of 150 to160 cm2 and total volume capacity of 15 to 20 ml which helps in rapid permeation and delivery of medications through nasal mucosa when a systemic effect is desired [8]. These two cavities are subdivided individually into four anatomical regions: nasal vestibule, atrium, respiratory and olfactory region as seen in Figure 1.1 and are differentiated according to their anatomical and histological functional proprieties.

1.2.2.1 Nasal vestibule

The nasal vestibule is considered the entrance part to the nasal cavity and the first region that comes in contact with the external environment. Unlike the other regions of nasal cavity, the nasal vestibule is covered with keratinized squamous epithelium which is similar to the epithelial lining of the skin[16, 23]. The nasal vestibule also contains vibrissae (thick hair) and sebaceous glands that support filtration function of the nose to filter out large particles. Within the vestibular area there are thermoreceptors that sense nasal airflow and change nasal airway resistance with inhalation of cold or warm air [48,

49]. The nasal vestibule is characterized by low vascularization, small surface area approximately 0.6 cm2, and the presence of keratinized cells. All these factors may account for low permeability and absorption of the drugs through the vestibular area[9].

1.2.2.2 Nasal valve

The nasal valve is the narrowest segment of the nasal cavity with total dimensions of 0.6 cm2[48, 49]. It is subdivided into two parts: internal and external nasal valve. The internal nasal valve is the most important portion and commonly known as the nasal valve which is responsible for more than 50 % of nasal resistance[50]. It is located posteriorly to the 8 vestibular area and is formed from overlapping of upper and lower lateral cartilages, septum, and inferior terbinates [23, 42]. The main role of nasal valve during respiration is modifying the rate and volume of the inhaled air[51]. So anything that leads to nasal valve obstruction such as trauma, surgery (rhinoplasty), or congenital alteration in the nasal structure may cause increase in total nasal resistance and even block nasal airways flow[52]. The anterior part of the nasal valve is lined with squamous epithelial cell which is converted into ciliated pseudo stratified epithelium in its posterior portion [8].

Due to small surface area and presence of such type of trans epithelia in nasal valve region, the permeability of substances and drugs is expected to be more difficult through this area.

1.2.2.3 The respiratory region

The respiratory region is one of the largest portions of the nasal cavity and it is lined with columnar pseudostratified epithelium. This type of epithelium is also known as respiratory or non-olfactory epithelium which covers 80 to 90% of nasal cavity in humans[53]. It is characterized by the presence of different types of cells and glands which includes globlet cells, basal cells, ciliated and non-ciliated cells, secretary glands and seromucus gland [54, 55]. Most of the respiratory cells are enclosed with microvilli which aid in increasing total absorptive surface area of the nasal cavity and because of that the respiratory region is considered to be the major section for systemic drug absorption[56]. Less than 20 % of the respiratory cells are covered with cilia that supports protection function of the nose through the use of their active motility in propelling the entrapped particles loaded mucus towards the pharynx and digestive system for elimination[35, 54]. Mucus is continuously secreted by goblets cells and 9 secretory glands and it is considered the major constituent of mucosal layer that covers the epithelium[57]. The respiratory region is highly vascularized and richly supplied with blood that come from network of capillaries which originate from internal and external carotid arteries[4, 58]. The rich vasculature of the respiratory zone is found to be important in warming and humidifying the inspired air in addition to enhancing permeation and absorption of topically applied drugs [8]. At the lateral wall of the nasal cavity there are special structures called turbinates or conche. The superior, middle and inferior turbinates are responsible for enlarging the nasal respiratory zone and aid in conditioning of the inspired air during respiration[59].

1.2.2.4 Olfactory region

The olfactory region/area is situated at the apex of the nasal cavity and it is covered with ciliated pseudostratified epithelium[60, 61]. The olfactory epithelium is responsible for smell perception and has a lower surface area when compared to the respiratory epithelium[62]. The percentage of olfactory epithelium area varies among animals and human beings[63]. The surface area of olfactory epithelium in humans is about 10 cm2 while in dogs it is 150 cm2 which explains the special sensing ability in dogs[4].

There are various types of cells and glands residing in the olfactory epithelium and which are not found in the nasal respiratory region such as olfactory sensory neurons, basal cells, sustentacular or supporting cells and bowman gland [60]. The olfactory sensory neurons are bipolar and consists of an axon and dendritic process[64]. Cilia arise from the dendritic process and act as receptors for chemical odorants[65]. The unmyelinated neuronal axon extends through basal lamina propria and joins with a number of axons to

10 form a group of large nerve bundles. These axons are then covered with Schwan cells which travel though cribriform plate (bone) in the brain to synapse with mitral cell or tufted cell dendrites in the glomeruli of the olfactory bulb[66]. The olfactory signals are then transmitted from the mitral or tufted cell to different regions of the brain such as the anterior olfactory nucleus, olfactory tubercle, piriform cortex, entorhinal cortex and amygdala[67]. The basal cells act as the regenerator for the olfactory epithelium every 40 days[66, 68]. Within olfactory neuro epithelium there are sustentacular cells which provide mechanical support to the other olfactory cells. In addition to the supportive function the sustentacular cells also contribute to secretion, metabolism and regulation of ionic ingredients including extracellular calcium and potassium [69-71]. The underlying lamina propria contain blood vessels, olfactory axon bundles and the secretary glands

(Bowman’s glands)[72]. The Bowman’s gland continuously secretes mucus which cover the epithelial layer. In addition, it also acts as a solvent to the sensing odorants. The histological and anatomical characteristics of the olfactory region and its vicinity to the

CNS encourages researchers to work on delivering therapeutics directly from nose to brain [73].

1.2.2.5 Nasopharynx

Nasopharynx forms the upper part of pharynx and it is situated above the soft palate and posterior to the nasal cavity. The upper part of nasopharynx is covered with ciliated epithelium while lower part is covered with squamous epithelium[20]. This structural organization acts as channel to transport the filtered, warmed air from the nasal cavity to the lungs. It has many other functions associated with vocalization and pressure

11 equalization in the middle ear and also acts as a drainage route for most of the nasal secretions[8, 74]. Increase in lipophilicity of nasally adminstered drugs leads to an increase in permeability across nasal mucosa through transcellular mechanisms.

However, it might affect the drug solubility in the nasal secretion and major proportion of the administered drugs may be drained into nasopharynx by mucociliary transport mechanism[75].

1.3 Nasal drug delivery

1.3.1 Local delivery

The aim of topical application of the drugs in the nose is to rapidly alleviate the symptoms associated with nasal diseases. The most commonly available intranasal topical agents are , antihistamines, local anesthetics and anti-inflammatory agents. To be effective, the intranasal route should be able to achieve the desired effect in low doses, thereby producing lesser side effects when compared to oral drug delivery.

1.3.1.1 Nasal decongestant drugs

The nasal decongestant drugs such as xylometazoline, oxymetazoline and naphazoline are used in the treatment and management of nasal congestion. These drugs act by constricting dilated blood vessels and reduce swelling of inferior turbinates in nasal cavity[76, 77]. Long term treatment with nasal decongestant agents might cause rebound congestion and increase the risk of rhinitis medicamentosa[78]. Due to the constrictive effect of topical xylometazoline, the temperature of nasal mucosa drops and might lead to dryness of the nose [79]. The use of topical decongestants also have pronounced effect on activity of mucociliary transport in nasal epithelium. A study conducted by Hofmann et 12 al., explains the influence of nasal agents on the ciliary activity. Research suggests that the use of xylometazoline or phenylephrine when compared to naphazoline and oxymetazoline causes reversible reduction in the activity of cilia upon nasal application.

The nasal decongestant agents are also used in management of epistaxis. It was observed that oxymetazoline alone could suppress the symptoms of 65% patients diagnosed with epistaxis[80, 81] .The therapeutic efficacy of a nasal decongestant can be further improved by incorporating a mucoadhesive substance such as hydroxyl propyl methyl cellulose (HPMC). This increases the residence time and the effect could be prolonged for more than 7 days of use[82] .

1.3.1.2 Intranasal antihistamines and corticosteroid drugs

Nasal corticosteroids and antihistamines are considered to be very effective in the management of allergic and non-allergic rhinitis[83-85]. The intranasal route delivers therapeutic agents directly to the targeted site thereby causing lesser systemic effects.

One of the most commonly available antihistamine is azelastine. It is a second-generation

H1-receptor antagonist which also has anti-inflammatory activity. The anti-inflammatory activity is due to the inhibition of synthesis of inflammatory mediators such as chemokines, kinins and free radicals[86, 87]. Another example of this class of drugs is olopatadine. It reduces the release of histamine and lysozyme triggered by nasal allergens[88]. The psychomotor retardation and other CNS effects associated with the use of oral antihistamines are overcome with topical antihistamine especially the second generation antihistamines [89]. Researchers revealed that azelastine nasal spray, 0.1%, and olopatadine nasal spray, 0.6% had faster onset of action and were therapeutically more effective in treating allergic and non allergic rhinitis. The reduction of nasal 13 congestion, rhinorrhea, sneezing and other symptoms associated was much higher when compared to orally administered anti histamines. Moreover, several studies indicated no significant clinical outcome when a combination of both oral and nasal antihistamines are administered[90]. Due to delayed action of corticosteroids, intranasal(IN) steroids are not highly effective as nasal antihistamine in management of acute symptoms of rhinitis [91,

92]. IN steroids may be used with other agents such as oral or nasal antihistamine for effective control of symptoms. Studies indicate that use of nasal steroids with oral antihistamine is more expensive with no additional therapeutic benefit in using such combination therapy. At the same time maximum clinical efficacy was observed when a combination of intranasal antihistamine and intranasal steroids are used for treating patients with seasonal allergic rhinitis (SAR)[93-95]. The most common nasal corticosteroids recommended by physicians are fluticasone propionate, mometasone furoate triamcinolone and . There are two factors that determine efficacy of topical intranasal steroids, the relative binding affinity to the binding glucocorticoid receptor and lipophilicity[96]. Compared to other corticosteroids, fluticasone propionate and mometasone furoate were found to be most preferred topical medications because they have higher affinity to bind to the glucocorticoid receptor which correlates with the potency of these steroids [97]. In addition these agents possess high degree of lipophilicity because of which only small amount of the drug is absorbed into systemic circulation[98]. Research indicates that bioavailability of fluticasone propionate and mometasone furoate was less than 2% and 0.1% respectively and no or low systemic adverse effects are seen when administered intranasally [99, 100]. Therefore low systemic adverse effects support the safety of intranasal corticosteroids (fluticasone

14 propionate, mometasone furoate and fluticasone furoate) in treatment of different nasal diseases such as allergic rhinitis, nasal polyps and rhinosinusitis [101, 102]. Their safety was established even when higher than the recommended dose was administered when compared to other older nasal corticosteroids (beclomethasone and budesonide) and oral corticosteroids[103, 104]. Increased dose of mometasone furoate about 20 times than therapeutic dose had little or no effect on adrenal function or growth rate a typical adverse effect that was observed in patients treated with oral corticosteroids[105]. The novel non aqueous aerosol formulation that contains combination of antihistamine and corticosteroids (azelastine / fluticasone propionate) which use hydrofluoroalkane as propellant could increase the treatment options for the physician in the management of patients diagnosed with allergic rhinitis[106].

1.3.1.3 Intranasal topical anesthetic agents

Nasal anesthetic agents are important especially in diagnosis or in surgical operations that involve nose or nasal cavity. Lidocaine, tetracaine, cocaine and ropivacaine are topical anesthetic agents which are commonly used by the otolaryngologist to provide adequate nasal anesthesia[107]. Many conditions require the use of a nasal anesthetic drugs such as nasa-tracheal intubation, nasal packing, management of fractured nasal bone and management of nasal abscess[108-110]. Cocaine has been utilized for a long time to provide vasoconstriction and local anesthesia in the nose[111]. In addition, cocaine showed good inhibitory and bactericidal effects against different kinds of bacteria in the nasal cavity[112]. However, cocaine is no longer preferred by the anesthesiologist due to its cost, abuse potential and decreasing availability[113]. Studies indicate that administration of a small dose of intranasal cocaine ((2 mg/kg) may increase risk of 15 microvascular myocardial infraction in healthy people [114]. The alternative local anesthetic agents such as lidocaine and tetracaine are as effective as cocaine when mixed with vasoconstrictor agents such as oxymetazoline. It was observed that effectiveness of combination of oxymetazoline and lidocaine was the same as cocaine in providing vasoconstriction and anesthesia when applied topically during intranasal surgery[115].

Due to low systemic bioavailability and longer duration of action of nasal tetracaine (> 1 hour), or tetracaine and oxymetazoline mixture was found to be most effective over lidocaine /oxymetazoline mixture for inducing local anesthesia[116]. The and delivery device of the drug also has an affect on the efficacy of these agents and patient adherence. Lidocaine 2% showed optimum anesthetic effect with less pain and comfort score when compared to atomized lidocaine and atomized cocaine. Therefore lidocaine is a drug of choice for providing anesthesia prior to insertion of a nasogastric tube[110, 117].

1.3.2 Systemic delivery

The intranasal route is considered to be an alternative way for delivery of systemic drugs into blood avoiding fist pass hepatic metabolism [118, 119]. This route has been used for a long time in management of acute or chronic diseases which require systemic exposure

[120]. The main aim of systemic intranasal delivery is to improve bioavailability with less local side effects[121]. The ideal drug candidate for intranasal delivery should have the following characteristics: 1) adequate absorption and stability profile in the nasal environment; 2)being effective at low doses ( < 25 mg per dose ); 3) limited irritation of the epithelial tissue of the nose; 4) small size ( molecular weight <1000 Da); 5) high log p value; 6) being in unionized state in the nasal environment [119]. The small volume of 16 administration of IN molecules in each nostril (150 µl) and short residence time for drug in the nasal cavity due to mucociliary clearance which happens every 15 to 20 minutes are the main challenges for intranasal delivery[122]. Thus these two factors should be considered during development of intranasal formulation to overcome these limitations.

Several drugs and hormones administered by oral or parenteral route were successfully delivered by intranasal route. Examples of the agents delivered by intranasal route include opioid analgesics, cardiovascular agents, antiviral agents, hormones and vaccines

[123-127].

1.3.2.1 Analgesic agents (opioid and non-opioid agents)

The opioid drugs are analgesic medications that are used for relieving acute and chronic pain[128, 129]. Small size and high log p value of various opioid analgesic agents could be responsible for successful IN delivery of these agents into systemic circulation[6].

Morphine, a hydrophilic compound (log p = 0.76), is generally administered by IV or oral route. After , the drug is extensively metabolized in liver and this contributes to low systemic bioavailability (20-30%) [130, 131]. Studies indicate that a formulation containing chitosan could improve bioavailability of IN morphine[132]. A recent study indicated that systemic side effects of intranasal morphine and IV morphine are similar. However, IN formulation of morphine has no significant side effects and could sustain the analgesic effect in post-operative patients[123].

Methadone is also a narcotic analgesic used to reduce symptoms associated with opioids addiction. IN formulation of methadone showed rapid onset of action, however the duration of action was slightly lower when compared to oral formulations. The local adverse effects of methadone on the nasal mucosa limits the delivery of this drug by 17 intranasal route[133]. Tramadol has been used over the past three decades for controlling acute and chronic pain[134]. It is administered by different routes oral, parenteral, rectal, etc [135, 136]. A study was conducted in healthy mice for evaluating efficacy of 20mg/kg of tramadol solution administered through oral and nasal routes by comparing pharmacokinetic parameters. The results showed that Tmax of the dug delivered by intranasal route (0.17 hr) is lower than Tmax for the same drug when administered orally

(1 hr) confirming the rapid onset of action of IN tramadol[137]. There are many advantages of using IN opioids over IV opioids in emergency department (ED). Firstly

IN administration is easy and no special training is necessary. IN administration eliminates the need of IV equipment and reduces risk of needle injuries and infection[138]. The analgesic efficacy of IN sufentanil has been studied in adults and children[139, 140]. IN sufentanil offers immediate pain relief in post-operative patient and also can be used in adults with extreme injuries indicating the possibility of use of opioid drugs via this in emergency department[141]. Another analgesic compound that is generally used in emergency department is . Several studies indicate the effectiveness of IN ketamine in providing well tolerated analgesia in short time with fewer adverse effects[138, 142, 143]. In addition to efficacy of IN route for the delivery of substances into systemic circulation, IN administration is also a well suited route for children. IN ketamine or IN sufentanil have been used for preinduction of anesthesia while combination of IN ketamine and sufentanil are successfully used for management of pain in 74% of children undergoing painful procedures[144, 145].

Fentanyl is a selective opioid agonist and it is 100 times more potent as an analgesic than morphine[146]. It relieves pain in a few minutes so it is highly preferred among pediatric

18 patients. The safety profile of IN fentanyl, rapid analgesic effect, and its analgesic equivalency to IV opioids makes it more preferable for controlling pain in children in a variety of clinical settings[147]. The commercial formulations of IN fentanyl are aqueous solution (Instanyl®) and mucoadhesive formulations (PecFent®). Several clinical trials and studies has been done to evaluate the importance of IN opioids on both animals and humans. The most commonly used IN narcotic analgesic are [148], [149], hydromorphone[150], and remifentanil[151]. Numerous articles suggest

IN application of is as effective as the IV route in reversing CNS symptoms due to opioid overdose[152, 153]. Nonsteroidal anti-inflammatory drugs ( NSAIDs ) are group of medications that possess analgesic, anti-inflammatory, and antipyretic effects

[154, 155]. NSAIDs are generally administered by oral, IV, rectal, or topical routes. The most commonly used IN NSAID approved by FDA is ketorolac tromethamine spray

(SPRIX®)[156]. SPRIX® is used in management of moderate to severe pain. However, because of its adverse effects, the duration of treatment should not be more than five days. Inhalation of SPRIX® in upright position could increase proportion of the drug deposition in the nasal cavity without any deposition in the lungs[157]. A novel thermosensitive gel containing a mixture of carrageenan, poloxamer and 407 hydrogel was used for increasing bioavailability and to prolong residence time of IN ketorolac tromethamine [158]. Research indicates the administration of 30 mg of IN ketorolac could significantly decrease need of morphine consumption in controlling pain especially after major surgery[159]. The other examples of analgesic NSAIDs investigated for IN delivery are indomethacin[160] and meloxicam which require further studies to prove their clinical advantage in humans[161].

19

1.3.2.2 Cardiovascular agents

IN cardiovascular agents have been used for a long time in treatment of clinical conditions that require rapid onset of drug effect, as an alternative way to parenteral administration[162]. Carvedilol is non selective β –blocker used in management of patients diagnosed with hypertension and angina pectoris[163, 164]. It is a small lipophilic molecule and it is well absorbed from GIT when administered orally. The systemic bioavailability of carvedilol is low (25%) because of first pass metabolism

[165]. The small size and lipophilic characteristics of carvedilol makes it a good candidate for IN delivery. Research indicates that alginate microspheres have an important role in enhancing absorption of the carvedilol across nasal mucosa and in improving bioavailability[166]. In a recent study, researchers used chitosan polymer instead of alginate in the IN formulation. Results indicate that after IN administration of chitosan microsphere to rabbits, the absolute bioavailability of carvedilol is increased to

72.29% indicating increased drug absorption through nasal mucosa[165]. The mucoadhesive properties of chitosan and sodium alginate polymers increased the residence time of the drug in the nasal cavity and that may lead to prolonged therapeutic effect of carvedilol[165, 166]. Valsartan another cardiovascular agent which is an angiotensin II receptor antagonist used in the treatment of hypertension[167]. The systemic bioavailability of oral valsartan is 23%[168]. The IN administration of Valsartan microsphere provides increased bioavailability and maximum therapeutic index with less dosing frequency when compared to conventional dosage form[169]. Another example of an antihypertensive drug with potential for IN delivery is verapamil HCL. Verapamil

HCL is a calcium channel blocker frequently used for the treatment of high blood

20 pressure and cardiac rhythmic disorder such as supraventricular tachycardia[170].

Despite the fact that more than 90% of the drug is absorbed from GIT the bioavailability is low and this is due to hepatic metabolism and its high affinity to bind with plasma proteins[171, 172]. In a recent study, intranasal administration of an aqueous solution of verapamil HCL was shown to increase its bioavailability to 47.8% when compared to

13% when administered orally[173]. Thus IN application of various cardiovascular drugs may offer alternative and a promising approach to overcome restrictions associated with other delivery routes.

1.3.2.3 Anti-emetic agents

Anti-emetic drugs are classes of medications that are used to treat nausea and vomiting.

Majority of patients undergoing chemotherapy generally experience nausea and vomiting[174]. Continuous and persistent vomiting may lead to nutritional, metabolic and mental disorders. Therefore, the main reasons of using these medications are for controlling vomiting and improving the quality of the life[175]. Most of the commercial antiemetic agents are available as oral or parenteral formulations. Variable absorption and bioavailability was observed with oral antiemetic agents in patients suffering from vomiting leading to inadequate control of emesis. Erratic absorption, invasiveness of parenteral , and patient hospitalization have been found to reduce patient adherence to drug treatment either by parenteral or oral administration route [176]. The nasal route provides rapid absorption of antiemetic agents with constant dosing.

Therefore IN administration of these agents could enhance bioavailability and increase patient adherence. Metoclopramide is a potent antiemetic agent used to control nausea and vomiting due to different pathological conditions. IN metoclopramide offers rapid 21 relief from symptoms especially in crisis conditions like severe nausea and vomiting.

Alteration in pH, use of absorption enhancers, or mucoadhesive polymers increases the amount and rate of drug absorption through nasal mucosa. Research indicates that metoclopramide nasal spray is more effective than commercial oral tablets in controlling symptoms of diabetic gastroparesis[177]. In another study, the efficacy of metoclopramide nasal spray was tested in both genders. It was found that the effect of pharmacological treatment with metoclopramide nasal spray is significantly different in females when compared to males. This is due to hormonal differences and the variety of gastric emptying times between men and women[178]. Ondansetron hydrochloride is a 5-

HT3 receptor antagonist that is used for management of nausea and vomiting associated with cancer chemotherapy or symptoms developed after surgical operations[179, 180].

Delivery of ondansetron HCL by intranasal route was expected to attain rapid therapeutic antiemetic effect even when a small dose of the drug was administered[181]. Due to the short elimination half-life of ondasetron HCL (3 hrs), several trials have been done to prolong therapeutic effect of the drug in systemic circulation[182, 183]. Pectin based microsphere was found to be a promising formulation in delivery of ondasetron hydrochloride through IN route because of its good stability against enzymatic degradation and increase in contact time between the formulation and the nasal mucosa at the absorption site [183]. Granisetron HCL is another antiemetic drug of 5-HT3 receptor antagonist family. It was formulated as IN droppable gel. The in vivo studies confirmed that the use of a droppable gel could double bioavailability and prolong release of granisetron HCL in comparison to the oral dosage forms[184]. Promethazine is a neuroleptic drug of the phenothiazine family. It was considered the drug of choice for the

22 management of symptoms of space motion sickness in astronauts [185, 186]. Cognitive disturbance, short term memory loss and sedation are the main side effects associated with promethazine therapy[187, 188]. IN administration of scopolamine was found to be a good alternative to promethazine in reducing symptoms of space motion sickness disease with minimal possibility of undesirable adverse effects such as sedation or cognitive function impairment[188].

1.3.2.4 Anti-diabetic agents

Anti-diabetic agents are class of medications used to control blood glucose levels [189,

190]. They include insulin and oral antidiabetic agents. Insulin has been used frequently in patients with type 1 diabetes mellitus(DM) [191]. It is also used in type 2 DM patients where oral antidiabetic drugs failed to reduce high blood glucose levels [192].

Subcutaneous injection is the common route for insulin administration [193]. Daily insulin injection is associated with many undesired effects such as tissue invasion and infection reducing patient adherence [194]. In addition, the normal pattern of postprandial insulin secretion is difficult to be achieved with different insulin SC preparations [195,

196]. Therefore a tremendous amount of work has been done to develop alternative routes for insulin administration. The common alternative routes are: nasal, oral, pulmonary and [197] [198]. The hydrophilic nature and large molecular weight are major challenges that affect delivery of the insulin by intranasal route. To increase the permeability of the insulin across nasal mucosa and improve bioavailability, various mucoadhesive and absorption enhancer agents were used in formulations such as chitosan polymers[199], bile salts[200], chelators[201], and cyclodextrin molecules[202].

Nanotechnology based delivery systems which include microemulsion, nanoemulsion, 23 and nanoparticles, were found to be the most promising approach in enhancing delivery of insulin molecules by IN route [203, 204]. Research indicates that IN PEG-grafted chitosan nanoparticles increases nasal absorption of insulin to a greater extent than IN insulin solution [205] . A study indicated bioavailability of insulin was increased to 21% after nasal administration of a w/o microemulsion indicating efficiency of microemulsion

[206]. Several studies proved the efficacy of intranasal insulin not only in normalizing hyperglycemia but also in treating cognitive impairment due to Alzheimers disease or other diseases that impair brain insulin signaling [207, 208]. Glucagon-like peptide-1

(GLP-1) is an injectable anti hyperglycemic agent used in DM type 2. Its likelihood to produce hypoglycemia is low in comparison to older oral antidiabetic agents [209]. The gastrointestinal adverse effects of GLP-1 and the need of frequent injections restrict their clinical use [210]. IN GLP-1 showed less adverse effects with rapid glycemic control but further studies are needed to evaluate their safety and efficacy with long term treatment of type 2 DM [211]. Repaglinide is an oral antidiabetic agent and it belongs to the class of meglitinides. Its oral bioavailability is low, 50% of the drug is extensively metabolized in the liver [212]. Polysaccharide microsphere and degradable starch microspheres were used in separate studies to increase bioavailability and duration of action of the IN repaglinide [212, 213]. Both of the formulations showed an increase in relative bioavailability in comparison to nasal solution. Polysaccharides microspheres showed full dissolution of the repaglinide with 100% release after 2 hrs. The duration of hypoglycemic was extended to reach up to 6 hrs[212].

24

1.3.2.5 IN vaccine

IN delivery of vaccine has received great attention as most of the infectious agents such as viruses, bacteria and parasites access the body through mucosal surface[214]. IN mucosal vaccination has potential to provide immunity in both the local(mucosal) and systemic compartments[215]. The non–invasiveness, nasal permeability and vascularization are major advantages that help in successful IN delivery of different agents including vaccines[216]. In addition, the nasal cavity is rich with nasopharynx - associated lymphoid tissue(NALT) which contain number of immune cells such as dendritic cell, macrophage, T-cell, B-cell and M-cells and considered the primary inductive part for mucosal immunity in the upper part of the respiratory tract [217, 218].

The antigen is taken by M cell and transported to the other immunocompetent cells for presentation thus inducing mucosa immunity[219]. In spite of these advantages, there are some challenges with IN vaccination. The presence of degradation enzymes in the nasal cavity which degrade vaccines based on biological materials such as DNA vaccines and rapid clearance of the IN vaccine can be due to nasal clearance mechanism [35, 220]. In many cases, purified antigens alone are not capable to evoke immune response after their delivery by IN route[221]. Adjuvants are incorporated in the IN formulations to improve the immunogenicity of some antigens such as peptide and proteins[222, 223]. Some common examples of adjuvants are saponin[224], liposomes[225] and bacterial toxin or cytokines[226]. The main aspects of any effective mucosal vaccination is to offer a potent defense against various diseases and provide long term immunity after single vaccination[221]. Various approaches have been applied for the development of effective nasal vaccine such as using a mucoadhesive polymer with an absorption enhancer. This is

25 considered a logical approach to increase residence time of the antigen in the nasal mucosa thereby increasing interaction with immune cells[9, 227]. Studies indicate that administration of IN application of alginate microsphere of serum albumin (PSA) could effectively enhance mucosal and systemic immunity in cattle[228]. Chitosan based formulations have been utilized to improve delivery of IN vaccine and also serves as adjuvant to improve immunogenicity of different vaccines[227, 229]. The safety profile of chitosan derivatives was established when administered intranasally as adjuvant with ovalbumin (OVA) antigen to the cynomolgus monkeys and mice[230]. In a recent study, a novel thermal-sensitive hydrogel consisting of propyl chitosan chloride and glycerophosphate was formulated with adenovirus antigen for IN vaccination. Results indicate that application of hydrogel may increase residence time of the antigen in nasal mucosa increased level of immunoglobulins in serum and lung mucosa[231]. Various particulate delivery systems were also utilized for IN administration of vaccines[232].

The benefits of using such systems are increased stability of antigens through their protection from nasal enzymes, enhancing their uptake by M cells and promoting sustained release of the vaccine at NALT tissue to increase duration of action[221, 233].

Examples of these type of systems are polymeric particles, liposomes, virosomes and immune-stimulating complexes (ISCOMs)[234]. ISCOMs are colloidal structures with particle size of 40 nm and consist of saponin and cholesterol [235]. The immunstimulatory effect and small particle size (within size range of viruses) could make the ISCOMs a viable option for IN delivery of vaccines and provide good protection against different viruses such as hepatitis B, influenza, and respiratory syncytial virus[225, 236-238]. The other particulate vesicles such as microparticles/nanoparticles

26 liposomes, virosomes were also used for IN administration of vaccines. They are effective in terms of antigen presentation and inducing humoral and cellular responses[233]. DNA-based vaccines has been studied frequently in animal models to show their potency in stimulating immune response. Obtaining good stability and entrapment efficiency are issues with developing IN DNA vaccinations due to the negatively charged of DNA molecule[239]. To overcome these disadvantages, a cationic polymer such as chitosan could be used with DNA for effective delivery of IN vaccine[240, 241]. Results indicate that using polyethylenimine (PEI) with DNA- influenza A H5N1 (H1N1) antigen for IN vaccination could increase the titer of IgA antibodies to a great extent while the mucosal immune response was not observed after parenteral or IN vaccination with naked DNA[242]. The only IN vaccine approved by

FDA is FluMist®. FluMist® is a type of live attenuated vaccine which is able to provide immunization against influenza virus A and B without inducing flu-like symptoms[243].

Even when data shows promising results in animal models the efficacy and safety of IN vaccine should be confirmed rigorously in humans.

1.3.3 CNS delivery (direct nose to brain delivery)

Delivery of IN drugs directly to the brain to treat different CNS diseases has a long history of research. Studies indicate that both small and macromolecules could be effectively transported by IN route to the brain for treating various CNS disorders[244-

246]. There are three suggested pathways by which drugs can reach the brain[247, 248].

The first pathway is by the systemic route where IN drug could be transported from blood circulation to the brain tissue and CNS by crossing BBB[249]. The major challenges that could decrease CNS presentation of the therapeutic agents by this 27 pathway are tight junctions of BBB and presence of efflux protein transporters such as P- glycoprotein (P-gp)[250, 251]. To cross the BBB, the drug should not be a good substrate for the P-gp efflux proteins and must be driven by active/passive transport. Most of the lipophilic drugs with high log P value prove to be good candidates for targeted brain delivery by this route[252]. The second pathway is by olfactory route where the drug is transported to the CNS either by intracellular or extracellular mechanism[253, 254]. The intracellular mechanism involves axonal transport of the drug where it is actively taken up by neuronal cells by endocytosis or pinocytosis[255]. Despite successful delivery of macromolecules like peptides and proteins[256] and small particles like viruses[257] and metals[258], transport to the brain tissues is very slow through this route [248, 253]. The extracellular or extra neuronal mechanism allows the direct delivery of the drug to the

CSF or the brain parenchymal tissue within minutes following IN administration[247,

259]. This mechanism is also called the olfactory epithelial pathway where the therapeutic agents are absorbed from the olfactory epithelium and diffuses into the perineural channels or lymphatic channels to the CSF surrounding the brain[260]. The solutes are rapidly eliminated from CSF due to the CSF replacement which occurs up to four times a day[261]. The rapid CSF replacement affects diffusion of large molecules to the brain tissue while small, highly diffusible substances have a small/low impact [248].

Research indicates that horseradish peroxidase (HRP), a protein tracer, could be rapidly delivered to the nasal bulb of monkeys and mice following IN administration [262].

Several metals tend to accumulate in the olfactory bulb after their instillation in nasal cavity indicating the importance of olfactory pathway in delivery of metals via intracellular or extracellular transport[263]. siRNA is a nucleic acid material used to

28 suppress expression of genes responsible for CNS diseases such as brain tumors ,

Parkinson's disease and Alzheimer's disease [264]. It was observed that IN labeled siRNA successfully targeted the olfactory mucosa and olfactory bulb via olfactory nerve pathway in 30 minutes after administration in mice[265]. So the olfactory pathway is responsible for delivery of many therapeutic agents such as neurotrophins (NGF)[266], and insulin growth factor[IGF]-1[247], anticonvulsants such as carbamazepine and lamotrigine [244, 267], inotropic agents (dopamine)[255], analgesic agents such as morphine[268], and anti-diabetic drugs such as insulin to different sites in the brain tissue[247]. The third pathway is the trigeminal pathway, which may be involved in transport of solutes and drugs to the spinal cord and caudal brain regions following IN administration[247]. The trigeminal nerves innervate both respiratory and olfactory regions such that IN drugs could reach the trigeminal nerve after their absorption from these regions[269]. The trigeminal nerve travels and enter the CNS sites in the pons which offer entrance to other brain regions[270]. It was reported that the trigeminal nerve pathway played important role in direct nose to brain transport of different therapeutics such as small interfering RNAs, IGF-1, and interferon-β1b[247, 271, 272]. In addition to direct brain targeting, the trigeminal nerve pathway could effectively transport IN drugs to the orofacial structures innervated by trigeminal nerve and this could be utilized for the treatment of different conditions such as trigeminal neuralgia and postoperative dental pain[269]. Numerous studies show effectiveness of IN route for the delivery of molecules directly to the brain. Most of these studies were done on animals especially in mice. The anatomy and physiology of nose and brain tissue vary greatly between various species of animals and humans, and this results in a lack of clinically relevant correlation. Olfactory

29 mucosa covers about half of total nasal epithelium in rodents while the percentage of olfactory mucosa that covers human nasal epithelium is 5%. Because of this difference, the transport of drugs and solutes from nose to brain by olfactory pathway could be more notable in rodents than human [273, 274]. In comparison to humans, the total CSF volume in rats is 0.25 ml and is renewed every 1 hr. So these differences should be considered when interpreting the animal data and translating them into human applications[275].

1.4 Nanostructured system for intranasal drug delivery and brain targeting

Successful treatment of CNS diseases depends on the ability of therapeutic agents to cross BBB in sufficient quantity and without causing permanent tissue damage. The BBB and blood–cerebrospinal fluid barrier (BCSFB) are the major barriers that restrict entry of different molecules into the CNS especially hydrophilic and macromolecules[276].

Many approaches have been utilized for direct delivery of molecules and drugs into various CNS sites such as intrathecal, intracerebral, transcranial, and intraparenchymal

(intra CSF) delivery. The major disadvantages with these approaches are invasiveness of the procedure and unsuitability for treatment of chronic CNS conditions that require continuous administration of the drugs[277]. Intranasal delivery is considered an alternative and non-invasive route for brain targeting. Despite the IN route being considered as effective in the delivery of many therapeutic agents via different pathways to the brain, some studies have shown that the total quantity of a particular drug reaching the brain and CSF is very low [278]. This was found to be especially true during IN administration of large hydrophilic molecules[279]. The low CNS uptake of these agents

30 could be overcome by loading the drugs in nanostructured systems[277]. Encapsulation of the drugs in colloidal carriers could increase bioavailability in various brain regions by allowing their access across BBB and protecting them from enzymatic inactivation[280,

281]. Nanostructured systems like microemulsion, solid lipid nanoparticles, liposomes and polymeric micelles were tested in vitro and in animals models to prove their efficacy in brain delivery of therapeutic and diagnostic agents when administered intranasally[282].

1.4.1 Microemulsion

Microemulsions (ME) are isotropic formulations consisting of oil, surfactants and/or co- surfactants. The droplet size of the dispersed phase in microemulsions is about 20 to 200 nm[283]. MEs provide numerous advantages including high entrapment efficiency, targeted drug delivery and are also well suited for both hydrophilic and lipophilic drugs[284]. The ME mixtures provide uniformity of dose and can also be developed into a spray formulation.[285]. Therefore MEs have been used for oral[286], topical[287], parenteral [288], pulmonary and IN drug delivery[289]. IN ME’s provide an economical and effective route of administration by allowing direct delivery of the entrapped/dissolved drug to CNS. The solubility of poorly water soluble intranasal agents can be improved by formulating them into an o/w microemulsion. These microemulsions have demonstrated an increased permeation rate across cell membranes thereby providing effective drug delivery to the CNS[206]. Nimodipine is a calcium channel blocker used mainly for clinical management of cerebrovascular spasms. The effectiveness of an oral formulation of nimodipine was limited because of first pass metabolism related low

31 systemic bioavailability [290]. An o/w microemulsion formulation successfully enhanced the bioavailability of nimodipine after nasal administration and showed an increased drug uptake by olfactory neuron to three folds in comparison to IV injection[291]. IN microemulsion could be used as alternative to IV administration in enhancing the effect of therapeutics and management of acute CNS diseases such as status epilepticus.

Diazepam showed a faster onset of effect in 2 or 3 minutes and good bioavailability when incorporated in ethyl laureate-based microemulsion[285]. Delivery of drugs from nose to brain could also be improved by inhibition of P-glycoprotein (P-gp) efflux transporters

[292]. Most anti-HIV drugs including saquinavir are substrates to P-glycoprotein. High concentrations of saquinavir was found in the rat brain after IN administration of drug loaded nanoemulsion(NE). The NE effectively decreased the viral load at CNS sites and could be used in the treatment of neuronal AIDS [293]. ME constituents such as long chain triglycerides or polyunsaturated fatty acid(PUFA) also play important role in the transport of drugs and molecules across BBB[282]. Addition of PUFA like docosahexaenoic acid to the oil phase of the MEs was found to improve the IN delivery of albendazole sulfoxide and curcumin to the brain and could maintain sustained release for up to one day [294]. Studies indicate the incorporation of mucoadhesive polymers like chitosan prolong the contact time between nasal mucosa and ME thereby increasing drug absorption and bioavailability[295]. Several drugs including buspironehydrochloride[295], risperidone[296], clonazepam[203], olanzapine[297], mirtazapine[298], carbamazepine[299], paliperidone[300] and sumatriptan[301] showed good bioavailability and better cerebral targeting efficiency when delivered as mucoadhesive ME by IN route. A number of histopathological studies were done on

32 nasal epithelium tissue to confirm the safety profile of the MEs during IN drug delivery.

It was observed that most of these MEs were safe and non-toxic especially for nanostructured systems formulated with non-ionic surfactants[302, 303]. The most common side effect associated with IN ME is nasal irritation. It could be reduced by increasing water content to more than 50%, decreasing the surfactant concentration and by avoiding alcohols as a component in ME formulations [303, 304].

1.4.2 Solid lipid nanoparticles (SLN)

SLN are colloidal drug carriers consisting of solid lipids stabilized by different types of surfactants and co-surfactants [305]. SLN have many advantages over other nanostructured systems (liposomes, , and polymeric particles) because they can incorporate hydrophilic and lipophilic substances with a high drug loading capacity

[306]. They have been found to provide sustained release of the drug with little or no cytotoxicity[307]. In addition, scaling up the process and large scale manufacturing of

SLNs is easy and economical [308]. Due to their lipophilic nature and small diameter,they are used widely in delivering molecules and drugs from nose to brain[309].

SLN loaded with risperidone were developed with Compritol 888 ATO and Pluronic F-

127 using a solvent diffusion–solvent evaporation method. It was observed that the brain/blood ratio (BBR) of the IN SLN risperidone was 5-10 fold higher when compared to IV risperidone indicating the effectiveness of IN SLN for brain targeting [306].

Rosmarinic acid (RA) is a compound having antioxidant and neuroprotective properties.

It was successfully incorporated and optimized in SLN formulations using glyceryl monosterate (GMS) and tween 80 [310]. Tween 80 is a nonionic surfactant widely used

33 as an emulsifier and stabilizer in pharmaceuticals. Use of tween 80 in IN SLN formulation is advantageous as it improves drug delivery to the brain by inhibition of P- gp present in the intranasal membrane and BBB and by interacting with endothelial cell membrane and increasing membrane fluidity[311, 312]. In vivo studies from IN administration of RA-loaded solid lipid nanoparticle on Wistar rats, showed that the drug appeared in optimum concentration in the brain and significantly controlled behavioral abnormalities and attenuated induced oxidative stress resulted [310]. These results prove the ability of SLN in brain targeting and treating CNS disorders like Huntington Disease.

In a recent study, SLN were used for the direct CNS delivery of antitubercular agents for treating tubercular meningitis. Streptomycin is a first line antitubercular drug administered by IM injection. Bacterial resistance, invasiveness, nephrotoxicity and ototoxicity related with high dosing, are the main disadvantages associated with parenteral therapy using streptomycin sulfate [313]. Permeability of streptomycin across the mycobacterium cell wall was enhanced when incorporated in SLN formulation reducing the risk of developing resistant strain of bacteria. Moreover, biodistribution studies confirmed that levels of streptomycin were higher upon IN administration of streptomycin SLN when compared to free drug injection[204]. IN SLN preparations used in a study evaluating its safety did not exhibit any signs of cell toxicity or cell necrosis indicating safety of these formulations [314].

1.4.3 Liposomes

Liposomes are colloidal vesicles consisting of outer phospholipid bilayers which are similar to cell membrane phospholipids and an inner aqueous compartment[315]. These

34 special structural characteristics could facilitate loading both of hydrophilic and lipophilic drugs. Targeted delivery to brain using liposomal formulations has been reported in a number of studies[316, 317]. There are several disadvantages associated with liposomes including short half-life, rapid systemic elimination, inability to release the drugs in sustained manner and fast enzymatic degradation of the phospholipid components[318].

These limitations could be overcome by using cationic or PEGylated liposomes[319,

320]. Encapsulation of molecules like proteins in cationic liposomes increases the residence time in nasal epithelium, and improves the drug uptake to brain following IN administration. A study conducted by Migliore et al., suggested ovalbumin loaded cationic liposomes could efficiently deliver the protein to the brain up to 6 hrs after IN administration in rats. In a recent study, a novel peptide (H102) was incorporated in

PEGylated liposomes to increase the drug uptake to the brain for treating Alzheimer’s

Disease. After IN administration of these liposomes the AUC of H102 peptide in the brain of rats used in this study was found to be three fold higher than that IV and IN drug solution group[321]. Several trials has been done for effective delivery of different acetyl cholinesterase inhibitors for treating Alzheimer’s Disease using liposomes. Flexible liposomes were used instead of conventional liposomes for IN delivery of drugs and chemicals. Fluidity and flexibility characteristics of the lipid bilayers of this type of could enhance delivery of drugs across various cellular membranes[322].

Studies indicate that IN galanthamine hydrobromide was successfully delivered to the brain of the rat by employing flexible liposomes[323]. Rivastigmine is another acetyl choline esterase inhibitor used for management of patients diagnosed with Alzheimer’s

Disease. IN administration of rivastigmine liposomes was found to provide higher AUC

35 and ten fold higher Cmax and higher levels of drug concentration in brain compared to oral and IN free drug administration[324]. Delivery of drugs using liposomes can also be improved by the addition of a permeability enhancer to the formulations[325]. Giuseppe

Corace et al. studied the effect of α-tocopherol on IN delivery of tacrine HCL loaded liposomes to CNS sites. The study indicates that permeability of tacrine HCL was markedly enhanced due to the fusion of phospholipid vesicles with cell membrane. In addition, a-tocopherol has antioxidant and neuroprotective properties which could further increase efficiency of liposomes for treating Alzheimer’s disease.

1.4.4 Other colloidal carriers ( polymeric micelle, dendrimer and nanosuspension)

Polymeric micelles are colloidal structures which consists of amphiphilic copolymers.

They have a hydrophobic core which is responsible for incorporating water insoluble drugs and hydrophilic shell which provides good stability to the drugs from the physiological environment[326]. The diameter of polymeric micelles is less than 100 nm[327]. Due to their nanometer size range, ability to solubilize and transport drug in optimum amounts across BBB was observed in in vivo and in vitro studies. High stability, and sustained release of incorporated drug are the major advantages of polymeric micelles over surfactant based micelles [328]. It was reported that polymeric micelles containing copolymers such as poly(ethylene oxide) and poly(propylene oxide) were able to increase bioavailability of efavirenz( anti HIV drug) in CNS to four fold in comparison to the IV drug administration [329]. Olanzapine was successfully loaded in mixture of Pluronic®L121 as a hydrophobic copolymer and Pluronic®P123 as a hydrophilic block to form polymeric micelle. It was found that at a drug:Pluronic®

36

L121:Pluronic® P123 ratio of 1:8:32, the naonmicelle showed good brain targeting efficiency without stability issues or tissue toxicity following IN administration[330].

Modification of the nano micelles surface with cell penetrating peptides such as HIV-1

Tat peptide could add additional benefit for direct transport of the drug from nose to brain[331]. T. Kanazawa et al. developed a nanomicelle formulation consisting of polyethylene glycol polycaprolactone co-polymers and conjugated with Tat peptide. In vivo studies showed a considerable amount of the material was observed in brain tissue following IN administration suggesting efficiency of the formulation in direct brain delivery of genetic materials like siRNA.

Dendrimers are branched macromolecules in the nanometer size of less than 10 nm[332].

Unique characteristics of dendrimeres are small and uniform particle size, high degree of water solubility, high encapsulation efficiency, and well defined homogeneous structure.

Polyamidoamine (PAMAM) was one of the first introduced dendrimers in drug delivery[333]. They are characterized by high drug solubilizing effect and excellent tissue targeting. It was found that IN administration of PAMAM dendrimer could effectively deliver haloperidol directly to the brain without systemic absorption[334]. Despite of a large number of studies using dendrimers as a novel drug delivery system, their clinical use in human has been limited because of toxicity problems [335].

Nanosuspensions are colloidal dispersions of very fine particles of drug in a liquid vehicle. These dispersed particles are within sub-micron size and could be stabilized by adding surfactants or polymers[336]. Nanosuspensions have been utilized for delivering hydrophobic drugs for optimizing drug delivery[337]. The large surface area of the fine particles could contribute to enhanced bioavailability of the drugs in the brain and

37 systemic circulation. Bhavna et al. developed a mucoadhesive nanosuspension for the brain delivery of donepezil for management of Alzheimer’s Disease. In vivo studies conducted on Sprague–Dawley rats showed the concentration of donepezil in the brain after IN administration of a nanosuspension formulation was three times higher than drug suspension and this confirms the importance of such formulations in direct CNS delivery of neuro therapeutic agents.

1.5 Antiepileptic drugs

1.5.1 History

Epilepsy is a serious CNS disease which affects more than 50 million people around the world. A large number of drugs have been evaluated and used for a long time to control epilepsy and reduce seizure frequency. In the early twentieth century, the first drug introduced for management of epilepsy was phenobarbital. The reduced cost and high therapeutic efficacy of this drug are the main reasons of its wide clinical use even now[338]. Other antiepileptic drugs such as phenytoin, ethosuximide, sodium valproate, carbamazepine, and some benzodiazepines were utilized as antiepileptic agents since

1970. Most of these medications like valproic acid are preferred in clinical use over barbiturate drugs due to their utility in treating various types of epilepsy[339]. New antiepileptic drugs introduced over the last decade include felbamate, topiramate, vigabatrin, gabapentin, levetiracetam, lamotrigine, tiagabine, oxcarbazepine and zonisamide. These medications offer advantages over older drugs due to pharmacokinetic and pharmacodynamics effects associated with these drugs[340]. There are more than twenty anti-epileptic drugs currently available today. Selection of 38 appropriate drug and dosage form depends on many factors such seizure type and frequency, patient age, drug adverse effect, drug-drug interactions etc.

1.5.2 Mechanism of action

The mechanism of action of most antiepileptic drugs are not completely understood but in general the antiepileptic action is attributed to their effect on the balance between neuronal excitation and neuronal inhibition[341]. Therefore drugs could exert their antiepileptic effect through blockade of ion channels (phenytoin, carbamazepine, lamotrigine, ethosuximide) or through potentiation of gamma-aminobutryic acid (GABA) neurotransmission (barbiturates, benzodiazepines, vigabatrin) [342] .In addition, there are many drugs that use multiple mechanisms to suppress seizure activity (valproic acid and gabapentin) [342].

1.5.3 Commercially available antiepileptic dosage forms

Antiepileptic drugs are generally administered orally for treating patients with chronic epilepsy. The oral formulations of antiepileptic medications are available in the market in the form of , , solution, suspension, and delayed release tablet or capsule dosage forms as shown below in table 1.1. In general tablets can be formulated as a coated or uncoated tablet, delayed release or disintegrating tablet. Clonazepam and lamotrigine are the only antiepileptic drugs formulated as oral disintegrating tablets[343].

Oral disintegrating tablets are designed to be solubilized in saliva before swallowing therefore it is a good alternative for patients having difficulty swallowing. Sprinkle 39 capsules are a new generation capsule which could be administered orally by sprinkling their contents into food. Topiramate and sodium valproate sprinkle capsules are also given to epileptic patients diagnosed with dysphagia[344, 345]. It was reported that systemic bioavailability of various antiepileptic oral formulations are more than 80%, except stiripentol capsules which have a bioavailability of approximately 30 to 70%[346].

Parenteral formulations are used as an alternative to the oral route in conditions requiring rapid onset of action. IV administration of diazepam or lorazepam are considered the most preferred choice for initial management of status epilepticus patients[347]. Other antiepileptic drugs administered by IV or IM route are phenytoin sodium, , and valproate. of the drug in the form of or gel could also provide an alternative route especially in cases of severe nausea and vomiting, infant patients, or instances that prevent oral administration of drugs. The only approved rectal antiepileptic drug is Diastat®( Diazepam). Diastat® is a rectal gel formulation widely used for treatment of patients with repetitive seizure[348]. Many trials have been done for evaluating delivery of antiepileptic drugs through the buccal or sublingual route.

Uncontrolled and excessive salivation which may occur during seizure episodes could be detrimental to the successful delivery of the drug in sufficient quantity to the targeting site.

40

Table 1.1 commercially available dosage forms of antiepileptic drugs for treating of epilepsy Brand names Scientific Available forms name Acetazolamide® Acetazolamide Oral tablets, 125, 250mg. Diamox Sequels® Diamox SR: capsules (prolonged-release) Diamox® 500mg. IV injection: 500mg/ml Ativan® lorazepam Tablets : 0.5, 1, and 2 mg IV: 2 mg/ml, 4mg/ml Oral solution Concentrated 2 mg/mL Apotium® Eslicarbazepin Oral tablets :200,400,600,800 mg e acetate Depakene®, Valproate Delayed Depakote® releasetablet:125,250,500Extended release tablets:250,500mgIV: 100 mg/ml : 250 mg/5ml (Dilantin®) Phenytoin Infatabs (chewable): 50 mg Suspension: 125 mg/5ml Dilantin® Phenytoin IV injection:50 mg/mLExtended-release Phenytek® sodium capsule:30,100,200,300 Felbatol® Felbamate Tablets: 400 and 600 mg Suspension: 600 mg/5ml Inovelon® Rufinamide Oral tablets 100mg, 200mg, 400mg, oral Banzel® suspension 40mg/ml Klonopin® Clonazepam Tablets: 0.5, 1 and 2 mgorally Rivotril® disintegrating tablets:0.25,0.5,0.1,2 Keppra® Levetiracetam Oral tablets 250mg, 500mg, 750mg, 1000mg, oral solution 100mg/ml. injection:100mg/ml Lamictal® Lamotrigine Oral tablets 25mg, 50mg, 100mg, 200mg, chewable dispersible tablets 2mg, 5mg, 25mg. orally disintegrating tablets: 25,50,100,200mg Luminal® Phenobarbital Oral tablets: 15mg, 30mg, 60mg, phenobarbital® 15mg/5ml Lyrica® Pregabalin Oral Capsules 25mg, 50mg, 75mg, 100mg,150mg, 200mg, 225mg, 300mg, oral solution 20mg/ml midazolam® Midazolam IV: 1mg/ml and 5mg/ml HCL Oral solution: 2 mg/ml Mysoline® Primidone Oral tablets 50mg, 250mg

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Table 1.1( continued)

ONFI® Clobazam tablets 5,10,20mg oral liquid suspension 2.5mg/ml POTIGA® Ezogabine Oral tablet:50,200,300,400mg Tegretol® Carbamazepine Chewabletabs:100mg Carbitrol® Tablets:200mg XR tabs: 100, 200, and 400 mg Extended-release caps: 100, 200 and300mg Suspension: 100 mg/5ml Topamax® Topiramate Oral tablets:25mg, 50mg, 100mg, 200mg Topamax® Sprinkle Oral capsule: 15mg, 25mg, 50mg. Qudexy® XR Extended release capsule:25,50,100,150,200 Trileptal® Oxcarbazepine Oral film tablets 150mg, 300mg, 600mg Oxtellar® XR XR tablets 150,300,600 oral suspension 60mg/ml Valium®, Diazepam Tablets: 2, 5, and 10 mg Diastat® Oral Solution: 5 mg/5ml l IV: 5 mg/ml rectal gel: 2.5, 5, 10, and 15 mg fixed unit- doses Vimpat® Lacosamide Oral tablets 50mg, 100mg, 150mg, 200mg, syrup 10mg/ml. Zarontin® Ethosuximide Oral capsule:250 Oral solution:250mg/ml Zonergan® zonisamide Oral capsule:25,50,100

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1.5.4 Alternative method for delivery of antiepileptic drugs to the brain

Approximately one third of epileptic patients are resistant to medical therapy with commercial antiepileptic dosage forms[349]. The drug resistance could be due to the drug not reaching the target area or due to low drug concentration in the brain. Extensive hepatic metabolism, drug-dug interactions, high plasma protein binding, and presence of multi drug resistance efflux transporters in GIT and BBB could be the major factors that restrict transport of antiepileptic drugs effectively to the CNS and for the low therapeutic concentration [350, 351]. In addition, systemic toxicity of most of the antiepileptic drugs when administered via current dosage forms is a limitation during treatment of seizures

[352]. Finding an alternative route of administration such as IN, buccal and rectal delivery could overcome drug resistance by circumventing liver metabolism and increase penetration of the drugs through BBB[353, 354]. Direct delivery of the medications into brain by intracerebral or intracerebro ventricular (ICV) are considered important approaches to the management of epilepsy especially in drug resistant patients[355].

Several studies have used nanostructured systems for the targeted delivery of antiepileptic drugs to the receptor site [356-358]. It was found that incorporation of antiepileptic drugs in these systems could effectively release the drug in the epileptogenic area with minimal systemic toxicity. Table 1.2 summarize a list of the colloidal systems being evaluated in the management of epilepsy.

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Table 1.2 list of the nanostructured systems used for brain delivery of antiepileptic drugs

Drug Carrier type Study model Relevant results Phenytoin SEDDS CD®IGS rats The AUC after administration of ( oral route) PHT SEDDS was increased 2.3 times more than dilantin suspension[359] O/W Sprague- Gamma Scintigraphy showed good microemulsion dawley rats level of phenytoin drug in brain (IN route) tissue [360] Carbamazepine Nanoemulsion beagle dogs Showed good pharmacokinetics ( NE) (I.V data in terms of AUC , Cp0 and route) t(1/2) [361] Microemulsion Wistar rats Brain to blood ratio of CBZ after (IN route) IN administration of CBZ ME was 2 times higher than after iv administration of CBZ ME [299] Mucoadhesive Swiss Albino CBZ loaded MNEG exhibited a Nanoemulgel mice excellent bio adhesion to the (MNEG) bovine nasal mucosa with high (IN route) protection to the mice against chemical and electrical induced convulsion [362] Solid lipid Albino rats The chitosan SLN formulation nanoparticle showed faster onset of recovery (SLN) (oral) when compared to control group[363] Ultrafine The solubility was increased up to nanoparticles In vitro ten times than pure CBZ and (oral) model: 100% of the drug was from the dissolution formulation in less than 1 hr[364] apparatus phenobarbital Microemulsion Wistar rats The phenobarbital ME was (Transdermal) successfully able to prevent seizure episodes in up to 70% of the tested rats[365] Clonazepam Nanoparticle Swiss The seizure crisis was prevented in ( oral route) Webster mice even after oral mice administration of low dose of Wistar rats clonazepam SLN [366]

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Table 1.2( continued)

Gabapentin Albumin Wistar rats Increased the concentration of nanoparticles gabapentin drug in the brain (I.P) three fold in comparison to the drug alone[358] Microemulsion Rat skin The w/o ME enhanced higher (transdermal) flux and better permeability for the drug through rat skin than w/o ME.[367] lamotrigine Polymeric Sprague Increase brain uptake of micelles (I.V) Dawley rats lamotrigine loaded polymeric micelle two times than free drug administration[357] Nanosuspension In vitro In vitro release study provided model ( controlled release of the drug dialysis bag) with up to 98% in period of 24 hours.[368] Oxcarbazepine Polymeric in vitro No significant differences was nanoparticles models of the observed between permeability 1- BBB values of the free drug and nano (hCMEC/D3 carrier loaded drugs across cells) 2- hCMEC/D3 cells and BeWo human cells[369] placental trophoblast (BeWo cells)

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Chapter 2

Significance of Research

Systemic delivery of antiepileptic drugs by oral or parenteral route is considered the most common approach for treating epilepsy disease .The BBB has series of anatomical and physiological features that prevent transport of various drug from circulation to the brain to exert their therapeutic actions .Due to impervious nature of BBB, high concentration of the drug should be achieved in the blood to even small amount of medications to reach brain regions[370].Increased parenteral or oral dosing of antiepileptic drugs to maintain desired therapeutic levels in epileptogenic areas could be associated with increased drug concentrations in blood and therefore potential adverse effects are probable .Systemic adverse effects, like skin rash, weight loss, and bone marrow suppression, may lead to decreased adherence of epileptic patients with medical treatment and therefore failure of full management of epilepsy[371].The numerous disadvantages of systemic drug delivery have encouraged researchers to search for alternative approaches for efficient delivery for antiepileptic drug to seizure region without prominent tissue damage.IN delivery has gained much attention from researcher as it provide direct and efficient brain targeting and reduce dose of the drug required to achieve therapeutic effects[372].Encapsulation of the drugs by colloidal systems could protect the drugs from the physiological

46

environment. The significance of our study to develop nanostructured formulations loaded with antiepileptic drug phenytoin with characteristics make it suitable for brain targeting by IN route with potential minimal local and systemic side effects .To achieve this, the NE formulations was prepared using polyethoxylated 40 hydrogenated castor oil(Cremophor RH 40), as surfactant, labrafil M 2125 Cs as oil and polyethylene glycol,

as co-surfactant .It was found that cremophor RH 40 is able to improve bioavailability of various hydrophobic drugs either by increasing fluidity of cell membrane or by

inhibition of p-glycoprotein efflux mechanism especially for drugs that are substrate of this protein transporter. Phenytoin is chosen as model drug and incorporated in the optimized mixture of labrafil M 2125 Cs, cremophor RH 40 and polyethylene glycol.

.

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Chapter 3

Nose to Brain Delivery of Antiepileptic Drugs Using Nanoemulsions

3.1. Abstract

The objective of this research work was to formulate and develop an intranasal oil-in- water nanoemulsion (NE) using the spontaneous emulsification method. Based on ternary phase diagram, emulsification time, and drug solubility studies the composition ratio of surfactant:co-surfactant:oil was optimized to 2:1:1. The model antiepileptic drug chosen for loading was phenytoin. The NE preparation was evaluated for particle size, zeta potential, electrical conductivity, pH, % transmittance, polarized light microscopy, in vitro drug release studies using HPLC, sterility validation, differential scanning calorimetry (DSC), and in vitro nasal toxicity testing in bovine nasal mucosa. The nanoemulsion formulations were then subjected to stability studies for six months by storing formulations at 25±5°C and 5±3°C. The NE exhibited a clear and stable formulation with globule size less than 20 nm and neutral zeta potential. The absence of birefringence and high conductivity values indicated that the formulations were o/w NE.

The pH measurements confirmed compatibility of such formulation for intranasal delivery. The in vitro release study showed 100% release of the drug from NE over a

48

period of 48 hours. The absence of any endothermic events in the phenytoin - self microemulsifying mixture (SME) when analyzed using DSC conclusively indicated that phenytoin was completely solubilized in this mixture. The histopathological studies of the optimized formulation did not show signs of damage to the nasal mucosa. The results of the sterility verification test demonstrated that the sterilization of the final formulation using aseptic filtration with a 0.22µm filter was sufficient for rendering the formulation aseptic. The nanoemulsion was found to be stable at all temperature conditions evaluated when particle size distribution, drug content, pH, and conductivity were taken as stability indicating parameters during the six-month study period. Based on the obtained results, the o/w phenytoin NE was found to be stable and desirable for use via intra-nasal delivery.

3.2. Introduction

Intranasal (IN) drug administration is considered as one of the most important routes by which drugs can be either absorbed into systemic circulation or transported directly to the brain. IN drugs traverse directly from the nasal cavity to various brain regions through either olfactory or trigeminal nerves [247]. IN route has been used for treatment of acute conditions that require rapid therapeutic effect as an alternative to intravenous administration[285, 373]. IN delivery has numerous advantages over oral or parenteral route including large absorptive surface area, bypassing first pass metabolism, increased systemic and brain bioavailability and increased patient adherence and it is needle free and painless [4, 374, 375]. However, the rapid clearance of the drugs or formulations from the nasal cavity and poor nasal permeability are considered major factors that limit 49

successful therapy by IN route. Therfore alternative technologies have been used to overcome these limits using approaches such as mucoadhesive or colloidal systems.

Nanoemulsion (NE) is a nanostructured drug delivery system with small droplet size

(<100 nm)[376]. O/W NEs are fine dispersions of oil-in–water, stabilized by surfactant and/or co-surfactant molecules. Compared to emulsions, NEs are kinetically stable without showing any sign of flocculation during their storage, even for long periods of time[377]. They offer several advantages making them suitable for drug delivery including ease of preparation and optimization, high drug loading capacity, enhanced solubility of poorly water-soluble drugs, and improved drug permeability across mucosal membranes which leads to increased bioavailability [378]. In addition, the colloidal system provides good protection for drugs against biological degradation. Many reports have indicated use of nano-emulsion formulations for IN delivery of various neuro- therapeutic drugs for treatment of CNS disorders like epilepsy[296, 379].

Phenytoin is a lipophilic (log P, 2.47) compound that belong to BCS class II drugs[380].

It is an antiepileptic drug used for treatment of all types of epilepsy except absence seizure. It also has antiarrhythmic and muscle relaxant properties. Phenytoin is administered orally in the form of tablets, capsules, or suspension. Due to its low solubility in water (about 100 μg/mL at 25°C), the gastrointestinal absorption is slow and erratic after oral administration [381]. In addition, phenytoin follows non-linear and saturable pharmacokinetics which may cause bioavailability problems and large variations in blood concentration of the drug among patients [382]. For IM administration, phenytoin should be formulated with high alkaline solvents such as 50

propylene glycol. Slow and incomplete absorption was observed with IM dosing as most of the drug crystalizes at the site of injection [383]. Therefore incorporation of phenytoin in NE formulations is considered as an interesting way to overcome problems associated with phenytoin solubility and facilitate its brain targeting following IN administration.

In the present study we formulated an oil-in-water nanoemulsion using spontaneous emulsification method with potential for intranasal delivery. Phenytoin was loaded successfully in the self-nanoemulsifying mixture using labrafil M 2125 Cs as oil, cremophor RH 40 as surfactant and polyethylene glycol (PEG) 400 as co-surfactant.

These ingredients are considered safe and non-irritating in animals and humans. The efficiency of the nanemulsion formulation as a potential delivery system for phenytoin was evaluated and characterized by studying various properties such as particle size, pH, zeta potential, sterility study, thermal and stability characteristics, in vitro nasal mucusa toxicity studies, and in vitro release.

3.3. Materials and Methods

3.3.1 Materials

Labrafil M 2125® CS, Labrafac Lipophile WL 1349, Labrafac™ PG and, Capryol™ 90, were provided by Gattefosse, St. Priest, France. Tween 80(Lot 106188), ethyl oleate (Lot

XT0253) and phenytoin were purchased from Spectrum Chemicals. Polyethylene Glycol

400 (PEG 400) was obtained from Hampton Research Company, USA. Cremophor® RH

40 was supplied by BASF Chemicals, USA. Tryptic soy broth (Soyabean Casein Digest medium-BactoTM, Lot 2030828) and Mueller-Hinton broth (Lot 3240477) were 51

purchased from Fisher Scientific (Pittsburgh, PA). Methanol (HPLC grade) was supplied by Fisher Scientific (Pittsburgh, PA). Deionized water was obtained from our laboratory.

3.3.2 Methods

3.3.2.1 Drug solubility determination

The solubility of phenytoin in various oils and surfactants was determined by adding 300 mg of phenytoin (PHT) to 3ml of each vehicle in microcentrifuge tubes and mixing using a vortex mixer for 5 minutes. Drug-excipient mixtures were equilibrated in a water bath and heated to 40°C to facilitate drug solubilization followed by continuous shaking on an isothermal shaker for 48 hours at ambient room temperature (25°± 0.5 ◦C). The samples were then centrifuged at 3000 rpm for 20 min to remove undissolved phenytoin. Aliquots of supernatants were diluted with methanol and filtered through a 0.22 μm filter and analyzed by HPLC for phenytoin concentration.

3.3.2.2 HPLC analysis of phenytoin

For the solubility, the drug content and in vitro release studies, reversed phase-high- performance liquid chromatography (RP-HPLC) was used for the determination of phenytoin concentration. The HPLC system (Waters Alliance e2695 separation module,

Milford, MA) was equipped with a reverse phase C18 column (Symmetry C18 column -

3.5μm, 4.6×75 mm) and photodiode array detector. A mixture of methanol and water

(45:55) was used as the mobile phase at a flow rate of 0.5 ml/min and the column temperature was set to 27˚C. Samples were properly diluted with methanol and injected directly into the HPLC system using a run time of 7 minutes. The retention time of 52

phenytoin was 5.4 min with maximum absorption wavelength (λmax) of 210 nm. A series of standard phenytoin solutions were prepared in methanol by serial dilution at concentrations between 50µg/ml to 0.195313 µg/ml. The calibration curve was constructed by plotting the average peak area against concentration used for quantification.

3.3.2.3 Preparation of blank nanoemulsion

PHT exhibited maximum solubility in Labrafil M 2125 CS, PEG 400, and Cremophor 40 among various excipients tested [384]. After careful evaluation of phase diagrams, a mixture of Cremophor RH 40 (50%) – PEG 400 (25%) – Labrafil M 2125 CS (25%) was considered as the optimum formulation for drug delivery because it resulted in a large nanoemulsion area, short emulsification time, and transparent appearance when mixed with aqueous medium. The NE formulation was prepared by a spontaneous emulsification technique that involved mixing of Cremophor RH 40 (surfactant) and PEG

400 (co surfactant) together using a vortex mixer for 45 minutes to produce a homogenous mixture then the oil phase (Labrafil M 2125 CS) was added and vortexed for 5 minutes to form the final SNE mixture. Deionized water was added gradually to the mixture with gentle stirring to form a clear NE formulation.

3.3.2.4 Preparation of phenytoin NEs

The phenytoin incorporated NE was prepared by adding 20 mg of phenytoin to 4 g of self nanoemulsifying mixture and vortexed until a homogenous and isotropic mixture was obtained. 0.6 ml of SNE mixture was added per 2 ml of deionized water with continuous 53

gentle stirring which resulted in the formation of transparent and clear phenytoin nanoemulsion. The NE prepared was placed in sealed glass tubes away from light and stored at room temperature. No heat was required during preparation of NEs. Table 3.1 shows the composition of the optimized formulation

Table 3.1 Composition of the optimized formulation Excipient Quantity (mg) Phenytoin 20 Cremophor RH 40 2000 PEG 400 1000 Labrafil M 2125 1000

3.3.2.5 Polarized Light Microscopy

Polarized light microscope (PLM) is an analytical technique commonly used in pharmaceutics to differentiate between crystalline and amorphous active pharmaceutical ingredients. It is also used to characterize various phenomena such as crystal twining, solubility of crystals in different solvents, sublimation, and particle size distribution. The major advantage of PLM over other screening techniques is its degree of sensitivity. The aim of application of PLM in our research is to differentiate between isotropic nanoemulsions and potential anisotropic liquid crystalline systems. In order to obtain accurate analyses, the formulation should be clean, free from bubbles, and form a thin film as much as possible. Samples of blank NE and drug loaded NE were investigated under PLM. A drop of NE sample was taken and placed between cover slip and glass slide and then observed under normal optics with and without polarizing filter. The images were procured by a digital camera (a Nikon model TiU coupled with photometric 54

Cools nap EZ 20 MHz monochrome camera) at a magnification of 60x. MetaMorph software was used for image analysis.

3.3.2.6 Droplet size measurements of nanoemulsion

Dynamic light scattering (DLS) is an analytical technique used widely for characterizing the size and size distribution of nanoparticles in liquid medium. The particle size measurement by DLS utilizes small amounts of sample (approximately 1 ml). The samples prepared for dynamic light scattering should be clear to slightly hazy solution.

Some liquid samples require further dilution to reduce or suppress interdroplet interactions. 0.6 ml of unloaded SNE mixture was taken and dispersed with different volumes of water (400ml, 200ml, 100 ml, 50 ml, 25 ml, 10 ml, 5 ml, and 2ml). The droplet size of resultant NEs was determined using the DLS instrument (Nicomp

380ZLS) equipped with a 100mW He-Ne laser of wavelength 658 nm at a scattering angle of 90°C and a temperature of 23°C. NE samples containing 400, 200, 100, 50, and

25 ml of water were taken directly for measurement without further dilution while samples containing 25 ml, 10 ml, 5 ml, and 2ml were diluted with deionized water before measurements. The particle size of optimized drug loaded NEs was measured before and after filtration. All samples were transferred to disposable borosilicate glass culture tubes (Kimble Chase, Vineland, NJ). Three cycles were run at 5 minutes per cycle. Mean volume weighted diameter was then determined from the average of three runs for each sample.

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3.3.2.7 Zeta potential measurements

Electrophoretic light scattering (ELS) allow measurement of electrophoretic mobility and zeta potential of colloids like micelle, ME, NE, and proteins. The sample prepared should be clean and dust free as dust may cause Doppler shifted peaks and change the shape and width of the observed spectrum of the measured samples [385]. Nicomp 380 ZLS instrument was also used for of zeta potential measurements but in the ELS mode. A number of drug loaded and unloaded NEs samples were prepared in the same manner as in DLS measurements. Three ml of diluted sample was transferred to the plastic cuvette and filled to ¾th its volume. To ensure uniform distribution in the temperature of the sample, the cuvette was kept in sample holder for 5 to 10 minutes prior to measurements.

The measurements were taken in triplicates at a scattering angle of 14.06° and temperature of 23°C.

3.3.2.8 Conductivity measurement

The electrical conductivity measurements of NEs were performed using a Mettler Toledo

Seven Multi™ Meter equipped with a conductivity probe. Conductivity studies play an important role in determining the phase systems of NE formulation (oil-in-water or water-in-oil). Samples of blank and phenytoin NEs were analyzed using a conductivity meter. The instrument was calibrated with conductivity standard solutions before each use. The conductivity probe was dipped in 5 ml of each standard until equilibrium was reached and a stable reading was obtained. The conductivity probe was cleaned properly after each sample reading. The measurements were done in triplicate at 25 ± 2 °C.

Typically temperature has a pronounced effect on the phase behavior of naonemulsions 56

formulated with a non-ionic surfactant [386]. As temperature increases, the electrical conductivity of the nanoemulsion is also increased to the extent where o/w NE is converted to a w/o emulsion. The temperature of conversion is called the phase inversion temperature (PIT). The conductivity meter was also used to determine the phase inversion temperature (PIT) of the NE formulations. The conductivity electrode was immersed in 25 ml of the formulations and the beaker was heated gradually using a

Fisher Scientific™ Isotemp™(Stirring Hotplate: Ceramic Top). The conductivity values were recorded after each 1 °C increase in temperature of the formulation [387].

3.3.2.9 pH measurement ,% transmittance

The pH values for NE were determined by placing the pH electrode into 10 ml of undiluted blank and phenytoin nanoemulsion using a digital pH meter. Buffer solutions of pH 4.0, 7.0, and 10 were used to calibrate the pH meter before each use. All measurements were done in triplicate for each sample at 25 ± 2 °C and the data were expressed as mean ± SD. The transparency of NE samples was measured using a UV- visible spectrophotometer (Agilent 8453, UV-Vis Spectroscopy System) at 633 and 700 nm [388, 389]. The clarity was also observed after diluting the SNE mixture 10X and

100X with deionized water [390]. Deionized water was used as blank. The transparency was expressed as % transmittance and triplicate measurements were taken for each sample.

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3.3.2.10 Thermodynamic properties

Differential scanning calorimetry (DSC) is one of the most applied and preferred techniques for thermal analysis. Any thermal transition of the substances and materials will be associated with absorption or release of heat[391]. Before DSC measurements, the sample was placed in a suitable pan. Hermetically sealed pans are recommended for volatile while non-hermetic and/or open pans are used for non-volatile samples.

For accurate quantitative DSC measurements, the sample and reference pan should be similar in thermal mass and type of metal used. The thermal properties of phenytoin-

SNE mixture was examined in comparison to pure PHT drug and blank SNE mixture using a Mettler Toledo DSC822e Star-e system. Approximately 5 to 10 mg of a particular sample was weighed and placed in standard 100 μl aluminum pans. After weighing the pan was quickly sealed using a mechanical crimper. An empty pan was used as a reference. The samples were scanned at a temperature range starting from 25 °C to 350

°C using a heating rate of 10°C /min under a stream of nitrogen gas. Star-e software was used for acquisition and analysis the data.

3.3.2.11 Sterility testing

Sterility testing was carried out to assess whether the final IN formulation was free from viable microorganisms. It was done via the aseptic filtration method. One ml of the PHT

NE sample was passed through a 0.22 μm sterile nylon membrane filter (Millex ® syringe filter) to obtain a sterile formulation. The sterility testing was validated using tube/direct and plate inoculation methods. Tryptic soy broth (TSB) was prepared and used for the direct inoculation method. In the direct inoculation method, there was a 58

negative control, positive control, positive sample control, and aseptically filtered PHT

NE. The positive controls were prepared using staphylococcus aureus (ATCC BAA

1692). This bacteria was grown and incubated in the nutrient medium (TSB) at a temperature of 37°C for 24 hours. Serial dilutions were made to get the final concentration of bacteria to 102 CFU/ml. The negative control tube contained 1 ml of sterile water and 9 ml of the uninoculated medium. The aseptically filtered sample tube contained 1ml of the filtered NE and 9 ml of the uninoculated medium. The positive control tube contained 1 ml of sterile water with a bacteria count of 102 CFU/ml and 9 ml of uninoculated medium while positive sample control tube contained 1 ml of NE sample containing a bacterial count of 102 CFU/ml and 9 ml of uninoculated medium. All tubes were prepared in duplicate and incubated at 35°C to promote growth of bacteria. For the plate method, Mueller Hinton (MH) agar plates were prepared and stored in the refrigerator. They were equilibrated at room temperature for 30 minutes prior to use.

Samples of 100 μl were withdrawn from each of the tubes prepared by the direct inoculation method on days 0, 7 and 14 and uniformly spread onto MH agar. The plates were prepared in duplicate and incubated at 35°C for 24 h to detect any bacterial growth.

All tests were performed under aseptic conditions in a laminar air flow hood. All glassware was packaged and autoclaved before being used in this study.

3.3.2.12 Stability study

In order to assess the physical and chemical stability of the optimized formulation, NE samples were subjected to long term storage conditions ( 25± 2 °C , 5± 2 °C ) for a period of 6 months. The stored samples were centrifuged after each sample withdrawal and 59

characterized every month for clarity, phase separation, particle size, drug content, and pH values. The data obtained were expressed as mean ± SD. One-way ANOVA test was used to detect significant differences between characteristics of NEs at day 1 and 180th day of storage.

3.3.2.13 Nasal histopathology study

Histopathological studies were carried out on bovine nasal mucosa to confirm safety of the phenytoin nanoemulsion when administered intranasally. Fresh cow nasal mucosa was collected from a local slaughter house (Kastel Slaughter House, Riga, MI) and transported in phosphate buffer solution (pH 6.5). It was cleaned with phosphate buffer and cut into nine symmetrical pieces with uniform thickness. The first 3 pieces were treated with phenytoin NE and the next 3 pieces were treated with the positive control

70% isopropyl alcohol, while the last three pieces were treated with negative control

(PBS 6.4). After 1 hour of treatment, all samples were washed thoroughly with phosphate buffer solution and stored directly in 10% formalin solution for 24 hours. It was important to ensure that the volume of formalin used was ten times more than the volume of the fixed sample. After 24 hours, the formalin solution was replaced with 70 % ethanol and the samples stored at 4°C for dehydration. The dehydrated sections were then embedded in agar and paraffin block and cut to 5 μm thickness by using a microtome.

Finally, the samples were stained with a combination of hematoxylin and eosin (H&E) dye and observed under optical microscope to detect any damage.

60

3.3.2.14 In vitro drug release study

In vitro release profile of phenytoin from nanoemulsion was evaluated using a dialysis bag technique. A mechanical shaker (Thermo Scientific™ Precision Reciprocating

Shaker Bath, USA) was used in this study at a rotational speed of 60 rpm/min. 0.6 ml of phenytoin SME mixture was dispersed in 2ml of phosphate buffer solution (USP phosphate buffer, release medium, 0.2 M, pH 6.4). The final concentration of phenytoin in the final NE was 1.1 mg/ml. A dialysis membrane (Spectra/Por® Dialysis Membrane) having a pore size of 2.4 nm and molecular weight cutoff between 12,000–14,000 Da was used. The dialysis membrane was hydrated in phosphate buffer solution for 12 hours before use. 3 ml of the NE sample was placed in the dialysis bag and then both sides of the dialysis bag were sealed. The dialysis bag was then placed in a beaker containing 150 ml of phosphate buffer solution and the temperature was maintained at 37°C ± 0.5.

Aliquots of the release medium (0.5 ml) were taken at fixed time intervals and replaced with fresh buffer solution to maintain constant volume. The tests were done in triplicates, amount, and cumulative percentage release of drug was quantified using HPLC method.

Figure 3.1 Precision™ Reciprocating Shaker Water Baths used in vitro release study

61

Chapter 4

Results and Discussion

4.1 Drug solubility determination

Phenytoin is a lipophilic molecule that is poorly soluble in water and thus it was expected to show good solubility when incorporated into lipid based formulation. It belongs to the class II drugs of BCS [392]. Oil-in-water (o/w) NE have been used to improve delivery of water insoluble drugs across nasal mucosa by increasing their solubility in the oil phase

[393]. Characteristics of (o/w) NE such as low globule size, fluidity, high solubilization capacity, and lipophilic nature could make them appropriate for intranasal drug delivery[295]. Solubility of drug in oil phase greatly affects its loading capacity in the final NE system. It is important for the drug to have greater solubility in different NE excipients to be able to eventually provide the required dose. The aim of the solubility study was to determine appropriate SNE components that have good solubility for the selected drug. The solubility of phenytoin in various oils, surfactants ,and co-surfactants are shown in table 4.1

62

Table 4.1: Solubility of phenytoin in various excipients Excipients Function Solubility (mg/ml ) ± SD Cremophor RH 40 Surfactant 51.37 ±3.75 Tween 80 Surfactant 11.46 ± 1.29 PEG 400 Co –surfactant 58.44± 6.5 Caprypol 90 Co-surfactant 7.2± 0.09 Labrafil 2125 Cs Oil 4.43±0.6 LL WL 1349 Oil 0.77±0.09

Ethyl oleate Oil 0.85±0.02 Labrafac pg oil 0.89±0.1 Pbs 6.4 Solvent 0.076±0.013 Water Solvent 1.17±0.1 methanol Solvent 17.2±1.9

Labrafil M 2125 CS was chosen as oil phase among other vehicles because maximum amount of the phenytoin was solubilized in this oil. The drug showed highest solubility in cremophor RH 40 and PEG 400 among others surfactants and co-surfactants tested.

Selection of these excipients are not only based on their drug solubilizing capacity but also on their ability to form transparent and stable NE with oil phase. The excipients used should be non-toxic and categorized as generally regarded as safe (GRAS) materials

[394]. Nonionic surfactants like cremophor RH 40 and PEG 400 are usually used in preparation of o/w NEs as they are safe, biocompatible and less influenced by changes in pH. Additionally, the selected surfactants and co-surfactant materials should provide high hydrophilic-lipophilic balance (HLB) values (greater than 10) which is important to form appropriate o/w NEs[395]. The efficiency of NE formulations is linked with size of NE area. The components cremophor RH 40, PEG 400 demonstrated shortest emulsification time and largest NE regions when plotted on a ternary phase diagram shown in figure 4.1 63

[384] . Therefore based on these results, the ratio of surfactant to co-surfactant to oil was taken as 2:1:1 and selected as an optimum SNE mixture for loading phenytoin.

Figure 4.1 Ternary phase diagrams of combination ofCremophor RH 40 % - PEG 400 % and- Labrafil M 2125 CS % [384]

4.2 Drug loading

Self-nanoemulsifying mixtures (SNE) are isotropic mixture of oil, surfactant and co- surfactant which are able to form o/w NEs upon dilution with water. The order of mixing of the NE components is very important when preparing NEs as it forms only when the surfactant and co-surfactant are mixed together with the oil followed by dilution with the aqueous phase [396]. The maximum drug loading capacity was determined by preparation of SNE mixtures with various concentration of the drug. The final concentration was chosen depending on the ability of the SNE mixture in maintaining drug in solubilized form without showing any sign of drug precipitation or crystallization.

64

20 mg of phenytoin was considered to be the maximum amount of phenytoin that can be dissolved in 4 gm of SNE mixture. The drug loaded SNE mixture was able to form stable

NEs without phase separation over a storage period of 48 hours. It was observed that there was no difference in the physiochemical properties of blank and phenytoin NEs.

A B

Figure 4.2 A. Transparent nanoemulsion B. turbid and milky emulsion

4.3 Characterization of Nanoemulsion

4.3.1 Droplet size analysis.

Droplet size distribution of NE is an important parameter than can be used to assess both stability and biopharmaceutical aspects. The small particle size of NEs provide large interfacial surface area for absorption or permeation of the drug across biological membrane and thus the relative bioavailability of the incorporated drug is improved. The higher stability and optical clarity of NE system compared to classic emulsion preparation can be attributed to their low particle size[377]. Droplet size of NEs is affected by the type and concentration of surfactants used. An increase in concentration of cremophor RH 40 results in decrease in particle size of CBZ NE to 30.3 nm[397]. An 65

increase in concentration of surfactant may cause breakdown of oil globules to smaller sizes molecules leading to decreased droplet size [398]. The drug loaded NE was filtered through a 0.2 μm Nalgene® syringe filter and the particle size of the filtered PHT NE was also determined to check for the effect of filtration on the size of the droplets. The results from the droplet size determination of blank NE are presented in table 4.2 and

.

Table 4.2 Particle sizes and zeta potential obtained for drug loaded and blank NE

Formulation Volume of Particle size Zeta potential water (ml) (nm ± SD, n=3 ) (mV ± SD, n=3 )

Cremophor RH 40 400 20.2 ± 0.23 0.15 ± 0.1

PEG 400 200 21.6 ± 0.78 0.067 ± 0.05

Labrafil 2125 cs 100 19.9 ± 0.95 0.35 ± 0.16

75 21.3 ± 1 0.18 ±0.02 50 21.6 ± 0.5 0.13 ± 0.07 25 20 ± 1.29 0.16 ± 0.05

10 18.5 ± 0.51 0.12 ± 0.07

5 18.4 ± 0.9 0.18 ± 0.076

2 18 ± 1.1 0.3 ±0.26

PHT NE ( without 2 20.2 ± 0.9 0.09±0.02 filteration) Filtered PHT NE 2 19.6 ± 0.3 0.2±0.05

66

Figure 4.3 Particle size of blank NE formulation

Figure 4.4 Particle size of phenytoin nanoemulsion ( without filtration)

67

Figure 4.5 Particle size of phenytoin nanoemulsion ( with filtration)

Base on the results shown in table 4.2 it can be concluded that there was no difference in the particle size of NE prepared with different amounts of water indicating that an increase in volume of water has little or no effect on the droplet size of NE [399]. The mean diameter of the blank and drug loaded NE was 18 ± 1.1nm and 20.2 ± 0.9 respectively and both samples displayed a Gaussian distribution of particle sizes( figure

4.3,figure 4.4). Slight increase in particle size of drug loaded NE could be due to incorporation of phenytoin (Mol. Wt.: 252.26 g/mol) within the core of micelles [400].

No significant difference in the droplet size and size distribution was observed between the filtered and unfiltered drug loaded formulations confirming the ability of NE to pass through syringe filter without changing its size.

68

4.3.2 Zeta potential measurements

Surface charge (zeta potential) of NE can be measured using Nicomp 380 ZLS instrument in electrophoretic light scattering mode. Due to nonionic nature of the excipients used to preparation of NE system, the zeta potential of blank and drug loaded

NE was found to be neutral. In comparison to negative or neutral charged particles, the positive charged particles are rapidly attracted to mucosal membrane[401]. Although this type of electrostatic interaction could improve bioavailability of the drug it may produced irritation and/or other toxicity in the membranes [402]. The particle charge also affects pharmacokinetics of the drug in the body. Research indicates that neutral nanoparticles were able to stay longer in blood circulation than charged particles as it has less affinity to bind to circulating proteins [403]. A stable dispersion of the particles could be obtained when values of zeta potential are above ±30 mV. This is due to presence of repulsion forces acting between particles[404]. Since none of the excipients used have any charge, a low or neutral zeta potential was expected for the final nanoemulsion. Lower zeta potential values may lead to interparticulate interaction and unstable formulations could be formed. However, many studies have reported that a stable NE can be formed even when using non-ionic excipients [405, 406].

69

Figure 4.6 Zeta potential of phenytoin nanoemulsion

4.3.3 DSC analysis

DSC was performed to investigate the solid-state nature of the drug in the SNE mixture.

The pure drug showed a sharp endothermic peak at 296°C ( fiqure 4.7) which corresponds to its melting transition. The DSC thermogram of blank and drug loaded

SNE mixture did not exhibit any peaks at this temperature indicating that the phenytoin drug was either converted to an amorphous form or molecularly dispersed in the formulation.

70

Figure 4.7 DSC thermogram of (A) Pure phenytoin drug, (B) Blank SNE mixture and (C) phenytoin-loaded SNE mixture

4.3.4 Polarized light microscopy

The isotropic behavior of NE system can be easily identified and characterized with help of PLM. The main difference between polarized and ordinary light is that the polarized light vibrates in only one direction. The polarized light was allowed to interact with various samples. If the materials contain anisotropic structure, it will rotate some of polarized light and thus be bright and visible when viewed under a light polarizing filter.

Isotropic material will not rotate polarized light resulting in a dark background when viewed under a light polarizing filter. In this study, none of the formulation samples showed birefringence suggesting that they are true NE[407]. The presence of liquid crystals can be identified by presence of birefringence. The identical results obtained for

71

both blank and drug loaded formulations indicated that phenytoin did not change the optical properties of NE system (figures 4.8,4.9) .

a b .

Figure 4.8 Polarized light microscope images a. optical micrograph of phenytoin NE without polarizing filter b. optical micrograph of phenytoin NE with polarizing filter at a magnification of 60x

a b

Figure 4.9 Polarized light microscope images a. optical micrograph of blank NE without polarizing filter b. optical micrograph of blank NE with polarizing filter at magnification of 60x

72

4.3.5 HPLC analysis of phenytoin

A simple and accurate HPLC method was successfully developed for quantification of phenytoin. This method was validated by determining linearity, accuracy, limit of detection(LOD), limit of quantification (LOQ), and precision. The chromatogram of phenytoin showed a sharp peak with a retention time of 5.4 min( figure 4.10A). By plotting the area under the curve against each standard drug concentration, a calibration curve that was found to be linear in the range of 50µg/ml to 0.195313 µg/ml with R2 value of 0.9999 was obtained. According to the ICH Q2(R1) recommendations [408],

LOQ and LOD were calculated and found to be 1.45645 ng and 0.480 ng, respectively.

The percentage recovery of phenytoin ranged from 97.5% to 104.5% indicating the high accuracy of the developed HPLC method. The method also showed a good intra-day precision with RSD not exceeding 2%. In conclusion, the developing HPLC method was found to be suitable for the analysis of phenytoin in bulk form and/or nanoemulsion dosage form.

73

3500000 y = 65171x - 11596 R² = 0.9999 3000000

2500000

2000000

1500000 Peakarea

1000000

500000

0 0 10 20 30 40 50 60 Concentration (ug/ml)

Figure 4.10: (top) HPLC chromatogram of phenytoin drug (bottom) Calibration curve of phenytoin

74

4.3.6 pH measurement ,% Transparency

IN formulations should be within physiological pH range in order to avoid nasal irritation. The pH of blank NE was found to be 5.71 ± 0.27. Loading of phenytoin may cause a slight decrease in the pH of the final formulation. The final pH of optimized NE formulation was within range of intranasal secretion (4.5 to 6.5) which confirms that the

NE prepared was potentially compatibile for intranasal delivery. The NEs remained clear and transparent even after diluting 10 to 100 times with water. The percentage transmittance (%T) was taken as indicator for NEs clarity. The %T of all formulated NEs was found to be more than 90%. This indicates the ability of SNE mixture to form a transparent formulation when diluted with water. The observed clarity of NE system is due to their extremely small droplet size (less than 100 nm) which is not exceeding 1/4th of the wavelength of visible light [409].

4.3.7 Conductivity measurements

High conductivity values were obtained for both blank and phenytoin NE indicating that the formulations were o/w type. The NE formulated with polymeric surfactants tend to form a w/o milky emulsion at high temperature[386]. Polyethoxylated castor oil

(cremophor RH 40) is a hydrophilic nonionic surfactant used in preparation of o/w nanoemulsion. Its affinity to oils and aqueous phase is highly influenced by changes in temperature of the system. With heating, these polyethoxylated derivatives become more lipophilic due to removal of water molecules from their structure. Therfore the phase behavior of NE formulated with these surfactants could also change with increasing temperature. Phase inversion temperature is defined as the temperature at which the 75

phase behavior of o/w NE is changed to w/o type. As shown in figure 4.11, the conductivity of the system increases with increasing temperature until it reached a point at which conductivity values were sharply decreased and this was refered to as the PIT.

The aim of measurement of PIT is to ensure that the temperature of storage or preparation of NE is not close to the PIT of the o/w NE. If the PIT of NE is within the range of 25 to

50 °C, this could be detrimental for the use of NE as vehicle for IN delivery of drugs.

The results of PIT obtained for drug loaded and blank NE were 83°C and 88 °C respectively. The PIT of PHT NE was slightly less than blank NE as presence of electrolyte or high log P drugs could lower the PIT of the system [410].

500 Formulation 83°C

/cm 400 A

uS 300 200 100 0 Conductivity in Conductivity 0 20 40 60 80 100 120 Temperature in Celsius 600 Blank 88 °C

500 B /cm

uS 400 300 200 100 0 conductivity in conductivity 0 20 40 60 80 100 120 Temperature in Celsius

Figure 4.11 change in conductivity of A. phenytoin NE B. blank NE with different temperature 76

4.4 Validation of sterility

The sterility of PHT NE samples were validated using direct inoculation as well as plate inoculation methods. In the direct inoculation technique the samples were prepared and stored as per the method described in USP. The negative, aseptically filtered sample control did not show any sign of bacterial growth while positive control and positive sample control showed turbidity throughout the 14 days study period (fiqure 4.12).

Samples were taken from direct inoculation test tubes on days 0, 7, and 14 for plating. All the plates were incubated at 35°C to facilitate growth of bacteria. After 1day of incubation, the plates were observed for the presence or absence of microbes. Except for the positive sample control and the positive control, other plated samples did not show any microbial growth (Table 4.3, figure 4.13). The results of the sterility verification test on days 0, 7, and 14 indicated that sterilization technique using aseptic filtration with a

0.22µm filter was sufficient for sterilizing the PHT NE formulation.

Table 4.3: Sterility vertification test performed on MH Agar plates indicating the presence (+) or absence (-) of microbial growth on days 0, 7 and 14

Days Negative Positive control Positive sample Aseptically control control filtered sample 0 - + + -

7 - + + - 14 - + + -

77

a b c d . . . .

Figure 4.12 Represented pictures of direct inoculation tubes a. positive control, b. negative control, c. aseptically filtered sample d. positive sample control after 14 days of inoculation

a a b . . .

c d

Figure 4.13 Represented pictures of MH Agar plates a. aseptically filtered sample b. positive control, c. negative control d. positive sample control after 14 days

78

4.5 Stability study

Stability study provide us information about how the quality and quantity of the drugs remain constant for a period of time under influence of temperature, light and humidity.

Nanoemulsion systems are characterized by their high stability which is due to their steric stabilization especially when formulated with polymeric surfactants like cremophor RH

40. In addition, the small particle diameter of such systems could also increase stability through decreasing gravity forces, preventing coalescence and flocculation to enhance free dispersion of the system without phase separation[377]. In this study the optimized

NE showed no drug precipitation or flocculation or phase separation when it was subjected to centrifugation for 30 min. The physiochemical stability of PHT NE was monitored by measuring particle size, pH, % drug content, and % transmission of the NE stored at various temperatures. Results of the stability study are shown in Table 4.4. The physiochemical parameters were evaluated for statistical significance by one-way

ANOVA analysis using SPSS software. No significant change (p> 0.05) was observed in the samples stored at 5°C and 25°C over the 180 days of storage. There was a slight increase in the pH of the PHT NE during storage at room temperature. In general, the degradation of the drug is slow at these temperature which indicate its stability in NE formulations. Based on results obtained from this study it is concluded that the physical and chemical stability of the drug and the formulation was maintained when loaded into nanoemulsion system.

79

Table 4.4 : Stability parameters of phenytoin nanoemulsion

(T) Parameters 0 month 1 month 2 month 4 month 6 month Drug 97.27±2.1 102± 1.9 98.05±1.5 97.39±0.8 94.84±0.75 content % RT Particle 18.3±1.014 21.1±1.3 19.3±0.5 17. 3±0.4 19.7±0.11 size pH 5.5±0.5 5.2±0.07 5.6±0.01 6.4±0.12 5.97±0.4 % T 90.38±0.1 93.05±0.49 91.9±1.5 94.3±0.62 91.4±0.5 5 °C Drug 97.5±1.3 96±4.2 100.6±3.8 99.5±2 96.3±2.4 content % Particle 19.75±1.1 19.2±0.72 14.53±1.3 20.5±0.05 18.4±0.2 size pH 5.21±0.01 5.35±0.25 5.16±0.19 5.02±0.1 5.028±0.2 % T 94.2±2.3 92.1±0.14 95.52±1.7 94.28±3 95.6±0.9

4.6 Nasal histopathology study

Mucociliary clearance is considered one of the most important physiological function of the nose. It is a function of the mucus and cilia activity so any damage to the cilia could affect clearance mechanism of the nose. Nasal histopathological study was done to ensure that the optimized NE formulations has no significant damage on the nasal mucosa or cilia activity. As shown in figure 4.14, the samples treated with the positive control (70% isopropyl alcohol) showed cilia loss and damage in the internal structure of the tissue compared to the normal conditions observed in the nasal mucosa treated with phosphate buffer solution (pH 6.5). However, no significant change in cilia and intact nasal mucosa were observed in the nasal mucosal tissues treated with PHT nanoemulsion thus confirming safety of PHT NE for intranasal administration.

80

No epithelium damage, intact cilia a

Loss of cilia, internal tissue damage Intact cilia, no significant tissue damage

b c

Figure 4.14 Histopathological pictures of the nasal mucosa treated with a.pbs 6.4 b. 70 % isopropyl alcohol c. PHT NE

4.7 In vitro drug release studies

The results of in vitro drug release from the NE formulation is shown in figure 4.15. A dialysis bag technique was used to ensure accurate release of the drug from the NE. The release profile of the drug can be obtained from plotting percentage of cumulative release versus time. Approximately 50% of the drug was released in 8 hours and a plateau was reached within 48 hours from starting the experiment. The sustained release of the drug 81

could be due to its affinity to the oil phase which is linked with its high partition co- efficient. The small membrane surface area is also possibly affecting the amount of the drug diffusion form the continuous phase to the release medium. Additionally, use of polymeric surfactants like cremophor RH 40 in high concentrations could also hinder release of the drug due to its ability to form strong interfacial films that surround oil globules. Therefore the rate limiting step for drug release was its ability to diffuse across the interfacial film. It was reported that nanoemulsion formulations containing cremophor

RH 40, Span80 and canola oil could sustain the release of vitamin E acetate up to 10 days [411]. The high partition co-efficient of phenytoin (log P is approximately 2.47), dialysis membrane characteristics, and presence of cremophor RH40 in the formulation could explain the sustained release of phenytoin from the NE formulation. The drug release kinetics were fitted into four different models (zero order, first order, Higuchi and

Korsmeyer-Peppas model). The highest R2 value was obtained for the Korsmeyer-Peppas model. The n exponents in the Korsmeyer-Peppas equation is related to the mechanism of drug release. Fickian diffusion is expected when the “n” exponent is equal to 0.45 and non fickian model when “n” is equal to 0.89. In our study the n exponent was 0.7158 as shown in figure 4.16 C which indicates that the drug was released by non fickian diffusion (anomalous transport).

82

120

100

80

60

40

% cumulative % cumulative release 20

0 -20 0 20 40 60 80 100 120 Time(hrs)

Figure 4.15 In vitro drug release of phenytoin from nanoemulsion

a b b

c d

Figure 4.16 Kinetic model plots for: (a) Zero order (b) First order (c) Higuchi and (d) Korsemeyer-Peppas model.

83

4.8.Conclusion

In this study, an SNE mixture was successfully prepared using cremophor RH 40, PEG

400 and labrafil M 2125 Cs in a ratio of 2:1:1 respectively and phenytoin was successfully incorporated in it. The particle size of blank and drug loaded NE was found to be less than 20 nm. The DSC results showed complete conversion of phenytoin drug from crystalline form to amorphous or monomolecular dispersion form. The pH of the final formulation was found to be optimum for intranasal delivery. The isotopic nature of

NE system was confirmed using polarized light microscopy. Sterilization of the final formulation with aseptic filtration was found to be sufficient for sterilization of formulation. The system showed sustained release of phenytoin from oil in water nanoemulsion. The physiochemical characteristics at normal and cold conditions remained stable for six months. Based on these characteristics, the NEs prepared could be a promising new approach for intranasal delivery of the antiepileptic drug for treatment of epilepsy and other types of seizures.

84

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