Physiology Of Blood Brain Barrier And Cerebrospinal Fluid Biology Essay

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Of all cancer in children, brain tumor is the most frequently occurring Johnson, Wright and Gilberston, 2009 and is the main cause of cancer related mortality in children (Levy and Thorburn, 2012; Khatua et al., 2012). Anticancer agents are somehow ineffective in treating malignant brain tumours (Sarin, 2009) due to low penetration through the Central Nervous System (CNS) barriers, particularly blood-brain barrier (BBB) and blood cerebrospinal fluid barrier (BCSFB) (Nigaraj, 2006; Yan and Liu, 2012). Therefore, efforts in developing effective anticancer delivery to brain tumour is highly important.

Olfactory region is a novel alternative route for brain tumour therapy (Shingaki et al., 2010) with possibility to bypass the BBB (Chen et al., 2008). In situ gelling system provides a novel strategy to enhance contact with mucosa and release profile (Cai et al., 2011; Han et al., 2008) with a transition from solution to gel phase at around 32 to 35°C (Nazar et al., 2011). In addition to thermoresponsive property, mucoadhesiveness of hydrogels is beneficial to enhance mucosal contact (Han et al., 2008).

Objectives

In this project, thermoresponsive gel for nose-to-brain delivery of anticancer 5-Fluorouracil will be developed.

Proposed Methodology

The project involves development of formulation, characterisation and evaluation. In the developmental stage, a series of formulations containing thermoresponsive polymers such as Pluronic F127 and Pluronic F68, in combination with a mucoadhesive polymer such as sodium alginate, gellan gum and HPMC will be generated using cold method and screened for the gelation temperature. The selected formulations will then be characterised by FT-IR spectroscopy, thermogravimetric analysis (TGA), and Differential Scanning Calorimetry (DSC); and evaluated for viscosity, clarity, texture, and drug release. For viscosity study, rotational viscometer and controlled shear rheometer will be used while dialysis tubing method will be used for drug release study. Successful formulation will satisfy the characterisation and evaluation.

Anticipated Results

In situ gel formulation consists of Pluronic F127, Pluronic F68, and sodium alginate (22:3:1 %) w/w is expected to have the desired gelation temperature and mucoadhesiveness and be the best of all the formulas.

CHAPTER i

Background

Introduction

Of all cancer in children, brain tumor is the most frequently occurring, with 20-25% incidence (Johnson, Wright and Gilberston, 2009) and being the main cause of cancer related mortality in children (Levy and Thorburn, 2012; Khatua et al., 2012). In treating malignant brain tumours, anticancer agents are used along with surgery and radiotherapy (Sarin, 2009). The anticancer agents can interrupt the cell cycle, such as alkylating the DNA, or interact with the surface receptors and affect certain pathways, such as inhibiting tyrosin kinase (Sarin, 2009). Anticancer agents are somehow not able to provide favourable efficacy in treating malignant brain tumors (Sarin, 2009). For an instance, temozolomide showed 3-years event-free survival (EFS) rate of less than 13% in children with high grade glioma (Khatua et al., 2012). Moreover, irinotecan, combined with bevacizumab, in treating children malignant gliomas yielded a very low progression free survival of 9.7% at six months (Khatua et al., 2012). This is in part due to low penetration through the Central Nervous System (CNS) barriers, particularly blood-brain barrier (BBB), blood cerebrospinal fluid barrier (BCSFB) (Nigaraj, 2006; Yan and Liu, 2012).

Recently developed chemotherapy drugs which work efficiently outside the brain have been unsuccessful in clinical trials against brain tumours (Nigaraj, 2006). Currently, more than 98% of CNS drug are dislodged by the BBB (Nigaraj, 2006). This problem can be explained by the change in the way a CNS drugs are discovered. Unlike the former approach of CNS drug discovery, which involved both structure-activity and structure-transport relationships through trial and error, nowadays it has been replaced by high throughput screening, where the selection relies on structure-activity relationships at the target receptor (Pardridge, 1997). Drugs might show desired activity on CNS but fail when administered systematically, because drug permeability on CNS barrier is not considered (Pardridge, 1997).

There is lack of effective CNS drug delivery strategy which can be seen from the fact that less than 1% of the effort in CNS drug development globally is given towards delivery strategy (Pardridge, 1997). Therefore, efforts in developing effective anticancer delivery to brain tumour is highly important.

1.2. Physiology of Blood Brain Barrier and Cerebrospinal Fluid

The brain is protected and dynamically regulated in a way that an isolated compartment of CNS is reserved (Yan and Liu, 2012). This area constituted by the brain interstitial fluid (ISF) and cerebrospinal fluid (CSF), and surrounded by the brain ventricles and subarachnoid spaces (Begley, 2004). Chemical substances can enter the brain from blood circulation and CSF circulation, regulated by the BBB and the BCSFB (Yan and Liu, 2012).

The BCSFB, by interfacing the brain and CSF, combined with the high turnover rate of CSF contribute in pushing the chemical substances back into systemic circulation (Yan and Liu, 2012). Meanwhile, the BBB prevents the penetration of chemical substances to the CNS by either hindering the passage or expelling them from brain through the efflux transporters (Begley, 2004).

The BBB is regarded as the most substantial barrier in drug penetration into the brain (Yan and Liu, 2012). The BBB composed of endothelium, extracellular base membrane, pericytes, astrocytes, and microglia (Yan and Liu, 2012). This in combination with the peripheral neurons, constitute a neurovascular unit, as shown on figure 1 (Yan and Liu, 2012).

Figure 1.The BBB and peripheral neurons forming a neurovascular unit (Yan and Liu, 2012)

Features of Blood Brain Barrier

Yan and Liu (2012) reviewed the BBB characteristic, which results in a very low and selective permeability to chemicals, as listed below:

The endothelium is not fenestrated and has very small number of pinocytotic vesicles;

The tight junctions which present in between the endothelial cells are very tight because they are occupied by complex transmembrane proteins interacting with cytoplasmic accessory proteins;

There are a number of transporters such as GLUT1 and LAT1, which specifically enables glucose and amino acid penetration into the brain, and efflux transporters such as p-glycoprotein (P-gp) and multidrug resistance-related proteins MRPs which prevents the drug entry to the brain;

Other components such as astrocytes, astrocytic perivascular end-feet, pericytes, perivascular macrophages and neurons also regulate the BBB

Features of Cerebrospinal Fluid

The CSF is secreted continuously by the choroid plexi, and it circulates within the surfaces and convexities of the brain, followed by drainage through the arachnoid vili into the peripheral bloodstream (Illum, 2000). The CSF flow characteristic and drug diffusion rate into the brain are two important factors in drug penetration into the brain, causing a functional barrier for the drugs that reach the CSF to go further to the brain compartment (Illum, 2000).The interaction between CSF and brain is illustrated on figure 2 (Illum, 2000).

Figure 2. The interaction between CSF, brain compartment, and systemic circulation (Illum, 2000)

1.3. Drug Transport to Central Nervous System

Generally, soluble materials can enter the brain across the BBB through several pathways (Yan and Liu, 2012). Substances can diffuse into the brain either by paracellular and transcellular route. Paracellular pathway (figure 3a) transports small water-soluble molecules, but not to a great extent while transcellular pathway (figure 3b) commonly facilitates small lipophilic compounds such as alcohol and steroid hormones to enter the brain (Yan and Liu, 2012).

Compounds that are not able to diffuse across BBB need active carriers or receptor-mediated or vesicular mechanisms (adsorptive transcytosis) to move across the BBB (Yan and Liu, 2012). Active carriers selectively transport molecules such as glucose (figure 3c), with supplied energy from the ATP (Yan and Liu, 2012). However, there are efflux transporters such as ATP binding cassette (ABC) transporter P-gp and multidrug resistant protein (MRP) (figure 3d) which expel compounds, regardless the lipophilicity, from the BBB (Begley, 2004a, 2004b, Yan and Liu, 2012). This is a major obstacle for delivering drugs to the brain (Yan and Liu, 2012).

Macromolecules such as growth factors and enzymes can pass the BBB through receptor-mediated transcytosis (RMT) (figure 3e) where the molecules are engulfed by the cells within a vesicle and then passed to the other side of membrane (Yan and Liu, 2012). Ionised compounds can interact electrostatically with negatively charged plasma membrane surface (for instance, heparin sulphate proteoglycans) and thus move across the BBB via adsorptive-mediated transcytosis (AMT), or pinocytosis route (figure 3f) (Yan and Liu, 2012). In cell-mediated transcytosis (figure 3g) the immune cells such as monocytes or macrophages are utilised to pass the BBB. This route can be used for particulate carrier systems (Yan and Liu, 2012).

Figure 3.Various pathways for substances to penetrate the brain (Yan and Liu, 2012)

1.4. Strategies for Drug Delivery to Central Nervous System

In this part, available strategies for CNS drug delivery are outlined. It includes pharmacochemistry approach, alternative routes and the use of carriers.

Physicochemical Properties Optimisation

Generally, most of CNS drugs have molecular weight ranging from 150 to 500 Da and, and log P of 0.5 to 6.0 (Begley, 2004). In contrast, when a compound has a great polarity, strong Lewis bond, rotatable bonds, and potentially forms a hydrogen bond, the compound is unlikely to penetrate the brain (Begley, 2004).

Therefore, careful drug design based on the mentioned criteria might be reasonable (Begley, 2004). However, there are some limitations in increasing drug lipophilicity, including poor solubility and bioavailability, substantial plasma protein binding and liver metabolism (Begley, 2004). Computer-based study to predict the BBB permeability has been developed but it is inapplicable for predicting drug-transporters interaction (Begley, 2004).

Prodrugs and chemical delivery systems

A drug can be designed in a way that it is inactive and lipophilic when administered, so as to penetrate the brain, but following metabolism is converted to the polar and active form, allowing it to give pharmacological effect and be locked in the brain (Begley, 2004). For an instance, diacetyl morphine, a prodrug-form of morphine is developed to improve brain penetration (Begley, 2004). Following brain penetration,diacetyl morphine is transformed back to morphine, which is polar and can actively interact with its target, opioid receptor (Begley, 2004). The polarity of morphine prevents it to be discharged from the brain (Begley, 2004).

Intracerebral injection/infusion

Direct injection or infusion into the brain or through CSF is useful for implant settlement or delivery of stem cells into the brain (Begley, 2004). However, the brain tissues are in high risk of damage and infection when intracerebral administration is used (Begley, 2004).

Blood-brain barrier modulation

Paracelullar pathway for drugs can be improved by widening the tight junctions (Begley, 2004).One of the techniques is termed osmotic opening, the regulation of tight junction permeability by using osmotic agent like hypertonic mannitol administered into a carotid artery(Begley, 2004). Brain penetration of methotrexate (MTX) was reported to be 10- to 100-times better with osmotic opening method (Begley, 2004). The drawback of this method is the possibility of foreign matter entry such as microorganisms during the opening of the tight junctions (Begley, 2004).

Delivery via endogenous transporters

As mentioned earlier, BBB has various transporters taking up molecules selectively into the brain (Begley, 2004). Therefore drugs can be designed to mimic the substrates of certain transporters, especially those for large neutral amino acid, or L-system, in order to have a high brain penetration (Begley, 2004). As an example, L-DOPA can be transported by the L-system due to its affinity to the L-system transporter, despite the potential competition with the other endogenous amino acids (Begley, 2004).

Inhibition of efflux mechanisms

In contrast to the aforementioned transporter, efflux transporters reject drugs form the brain (Begley, 2004). To escape these transporters, drugs can be designed to be not similar to the substrates of efflux transporters or can be delivered together with specific efflux transporter inhibitor (Begley, 2004). Cyclosporin A, probenecid, and fumitremorgin C have been used as inhibitors for Pgp, MRP, and BCRP with satisfactory results (Begley, 2004). Efflux transporter inhibitor is not recommended for long-term therapy since the down-regulated transporter might not able to prevent harmful substances to penetrate the CNS (Begley, 2004).

Cell-penetrating peptide vectors

Currently, cell-penetrating peptides have been attempted to be used as drug carrier due to its ability to pass through the cells by virtue of their structure or possibly via endocytosis (Begley, 2004). In situ brain perfusion study on rat has shown 3- to 8-fold increase of brain penetration of doxorubicin when contained in cell-penetrating peptide, penetratin and SynB1 compared to doxorubicin alone (Begley, 2004).

Liposomes and nanoparticles

Liposomes and nanoparticles can deliver drugs preferably to the brain, and by surface modification of the particulate system, such as attaching targeting moieties, the drug-containing particles can enter the CNS by a specific BBB pathway (Begley, 2004). Liposomal digoxin with OX 26 mAb on the surface have been constructed to improve brain penetration overcoming the problem that digoxin is a substrate of Pgp efflux transporter (Begley, 2004).

The olfactory route

Olfactory region is a new alternative route for CNS delivery which is fascinating due to injection-free and possibility to avoid the BBB (Chen et al., 2008). There is a growing interest of the absorption route through the nasal cavity, CSF, and finally into the CNS (Illum, 2000). A number of drugs have been administrated intranasally to target the brain, such as sulfonamides, cephalexin, progesterone, zidovudine, insulin and hyaluronidase (Illum, 2000). The various pathways for drugs to penetrate the CSF and brain through nasal route are shown on figure 6 (Illum, 2000).

Figure 6. The biodistribution of drug administered nasally to the brain tissue, CSF and systemic circulation (Illum, 2000).

Lipid soluble drugs with low molecular weight (< 500 Da) can be quickly absorbed by nasal epithelial cells with maximum concentration achieved in 1 to 20 minutes after administration (Chen et al., 2008). Some of the drug will undergo clearance by the mucociliary clearance system, some will be absorbed into the systemic circulation and cleared out of the body (Hussain et al., 1980;Illum, 2000), and some can either reach the brain via BBB or CSF, or can be expelled by the BBB and CSF back into the blood circulation (Illum, 2000).

The drug absorption by nasal route can take place transcellularly, paracelularly or through olfactory nerve (Illum, 2000). The latter allows the drug uptake by the neuron cells and transfer by intracellular axonal towards the olfactory bulb (Illum, 2000). The extent of absorption by this route is influenced by the physicochemical properties of the drug, mainly lipophilicity and molecular weight, and the formulation aspect (Illum, 2000). According to previous study, molecular weight affects nasal absorption more obviously than lipophilicity (Chen et al., 2008), being restricted to a range of 20 to 40 kDa (Sakane et al., 1995).

1.5. Intranasal Delivery for Brain Tumour

CNS delivery via nasal route can be used for brain tumor therapy (Shingaki et al., 2010). Shingaki et al. (2010) has evaluated this strategy in delivering methotrexate (MTX) on rats with brain tumour and the result showed that MTX successfully reached the CSF and CNS to a great extent and significantly inhibited in vitro growth of glioma cells. Another study examined nasal route for brain tumor chemotherapy was performed by Taki et al. (2012). Anticancer agent camptothecin in a polymeric system based on methoxy poly(ethylene glycol) /poly(ε-caprolactone) block copolymer and Tat analog-modified, a cell penetrating peptide was delivered intranasally to rats with intracranial glioma tumors (Taki et al., 2012). Sakane et al. (1999) has developed intranasal delivery to the brain for 5-fluorouracil.

1.6. Formulation Strategy for Intranasal Preparation

Intranasal preparation for pediatric is acceptable since it is pain-free and easy to use (Wolfe and Braude, 2010). For pleasant use, drug concentration in the preparation must be adjusted to allow administration of minimum volume, preferably 0.2 to 0.3 mL for each nostril (Warrington and Kuhn, 2011).

Generally, conventional nasal preparations have low viscosity so that easily running off, resulting in short mucosal residence time and poor bioavailability (Cai et al., 2011). In situ gelling system, which is liquid upon administration but then congealed into a gel in the nasal cavity, provides a novel strategy to enhance contact with mucosa and release profile (Cai et al., 2011; Han et al. , 2008). Such preparations evolve through a sol-gel transition which is modulated by temperature, usually at 32-35°C (Nazar et al., 2011).

Of all the viscoelastic polymers used in pharmaceutical preparation, chitosan has a great potential since they assist drug penetration via paracelullar pathway by modulating tight junctions (Nazar et al., 2011). Besides chitosan, thermoresponsive gel of chitosan/glycerophosphatehas been designed by Chenite et al. (Nazar et al., 2011).

In addition to thermoresponsive property, mucoadhesiveness of hydrogels is beneficial to enhance mucosal contact (Bertram and Bodmeier, 2006). Bioadhesive material such as carrageenan, hydroxypropyl methylcellulose (HPMC) and sodium alginate has been used to decrease nasal mucociliary clearance of incorporated drugs (Bertram and Bodmeier, 2006).

CHAPTER ii

Intended design and methods of investigation

In this project, thermoresponsive gel for nose-to-brain delivery of anticancer 5-Fluorouracil will be developed. Briefly, the project consists of two steps, which are development of formulation and evaluation. In the developmental stage, a series of formulations containing thermoresponsive polymers such as Pluronic F127 and Pluronic F68, in combination with a mucoadhesive polymer such as sodium alginate, gellan gum and HPMC will be generated and screened for the gelation temperature. The selected formulations will then be characterised by FT-IR spectroscopy, thermogravimetric analysis (TGA), and Differential Scanning Calorimetry (DSC); and evaluated for viscosity, clarity, texture, and drug release. Successful formulation will satisfy the characterisation and evaluation.

The preparation of gel uses a technique referred as cold method. The ingredients are dissolved one by one in an aqueous solvent, kept in a low temperature overnight, and heated in a water bath to form a gel. The formation of gel is ideally close to, and not higher than body temperature. This is achieved by optimising the ratio of thermoresponsive and mucoadhesive polymers used in formulation.

Subsequent to formulation development, characterisation is essential to investigate the interaction of polymers with drug. FT-IR, TGA, and DSC analysis of gel preparation will provide specific spectra to be studied and compared with pure compounds.

Some of the evaluation of gel employs various methods. For viscosity study, rotational viscometer and controlled shear rheometer will be used, where variables affecting the viscosity such as shear force, shear frequency, and temperature will be applied. Dialysis tubing method, carried out in USP Apparatus 2 will be used for drug release study.

The detail of experimental design is as described below, starting from the rationale of formulation, preparation, charactersation, to evaluation.

Materials

The materials will be used in this research are Pluronic F127 (P127), Pluronic F68 (P68), Sodium alginate (AL), Gellan gum (GG), Hydroxypropyl methylcellulose (HMPC), benzalkonium chloride, Sorensens phosphate buffer, sodium chloride, glycerin, 5-Fluorouracil (5-FU). The function of these materials can be seen in table 1.

Table 1. Function of materials

Ingredients

Function

Pluronic F127

Thermoresponsive agent

Pluronic F68

Thermoresponsive agent

Sodium alginate

Mucoadhesive material

Gellan gum

Mucoadhesive material

HMPC

Mucoadhesive material

Benzalkonium chloride

Preservative

Sorensens phosphate buffer pH 5.8

Maintains a stable pH

sodium chloride

Isotonicity agent

Glycerin

Emollient

5-FU

Active compound

Thermoresponsive polymer is used to produce a dosage form with high viscosity, with the aim of preventing mucosal ciliary clearance. Mucoadhesive polymer is used in addition to thermoresponsive polymer to prolong the contact of dosage form with nasal mucosal surface. To achieve convenient use, isotonicity and pH of formulations must be taken into consideration and therefore the use of buffer and tonicity agent will be applied. Emollient is necessary to minimise the possibility of irritation upon intranasal administration, while preservative is used to prevent microbial contamination of the product which can lead to infection.

Preparation of PluronicGels (the Cold Method)

The preparation of Pluronic gels uses cold method. Into a beaker containing 35 mL of Sorensens phosphate buffer pH 5.8, benzalkonium chloride, sodium chloride and glycerin are added individually, followed by continuous mixing by using magnetic stirrer. Next, P127, P68, and a mucoadhesive polymer (AL, GG or HPMC) are added slowly, while keep stirring the mixture. These steps apply for all the formulas, and the intended final concentrations of the ingredients are shown on table 2. The mixtures are then kept overnight at 4 °C and added with Sorensens phosphate buffer pH 5.8 to a weight of 50 gram. The preparations are then stored at cold temperature for 48 hours prior to evaluation.

(method adapted from Pisal et al., 2004)

Table 2. Formulations of Pluronic Gels

Formula

P127 (%)

P68 (%)

AL (%)

GG (%)

HPMC (%)

Benzalkonium Chloride (%)

Glycerin (%)

NaCl

(% equivalence)

F1

23

3

0.6

-

-

0.02

0.5

0.9

F2

23

3

1

-

-

0.02

0.5

0.9

F3

23

3

-

0.6

-

0.02

0.5

0.9

F4

23

3

-

1

-

0.02

0.5

0.9

F5

23

3

-

-

0.6

0.02

0.5

0.9

F6

23

3

-

-

1

0.02

0.5

0.9

F7

18

15

0.6

-

-

0.02

0.5

0.9

F8

18

15

1

-

-

0.02

0.5

0.9

F9

18

15

-

0.6

-

0.02

0.5

0.9

F10

18

15

-

1

-

0.02

0.5

0.9

F11

18

15

-

-

0.6

0.02

0.5

0.9

F12

18

15

-

-

1

0.02

0.5

0.9

F13

6

27

0.6

-

-

0.02

0.5

0.9

F14

6

27

1

-

-

0.02

0.5

0.9

F15

6

27

-

0.6

-

0.02

0.5

0.9

F16

6

27

-

1

-

0.02

0.5

0.9

F17

6

27

-

-

0.6

0.02

0.5

0.9

F18

6

27

-

-

1

0.02

0.5

0.9

Gelation temperature

The gelation temperature is determined visually by tube inversion method. An aliquot of each formula (1 mL) is placed in a glass vial within a water bath. The temperature is set at 4 °C for 30 minutes, and is then raised to 20 °C. The sol-gel transition is examined within a range of temperature from 20 to 50 °C, with 5 minutes interval after a rise of 1 °C. The vials are taken out every minute and inverted to observe the phase of the polymer mixtures. The gelation point is reached when the mixtures do not flow following tube inversion, at least for 30 seconds.

(method adapted from Cha et al., 2011; Wu et al., 2007)

Clarity and texture analysis

The clarity of polymer mixture solutions is examined visually against a black background and white background. The texture analysis including firmness, consistency and cohesiveness is carried out assessed using texture analyzer.

(method adapted from Kant et al., 2011)

Characterisation

FT-IR Spectroscopy

To identify the presence of interaction of polymers during gelation process (Kant et al., 2011), the polymer mixtures are analysed using FT-IR spectrophotometer (wave number 650 - 4000 cm-1) with KBr disk method. A dried measured sample is mixed with KBr (contains 1% w/w of polymers) and compressed into a disk. The FT-IR spectrum is then obtained.

(method adapted from Abruzzo et al., 2013; Rejinold, 2011)

Thermogravimetric Analysis

Thermogravimetric analysis (TGA) of the polymer powders and polymer mixtures is performed to confirm the interactions between polymers and drug and to determine water content in hydrogel (Kant et al., 2011). During analysis, temperature is set within a range from 5 to 1200 °C, with heating rates from 0.01 to 40 K min-1, in a controlled atmospheres.

(method adapted from Abruzzo et al., 2013; Kant et al., 2011)

Differential Scanning Calorimetry

Differential Scanning calorimetry (DSC) analysis investigates any difference in heat flow of materials in comparison with pure compound (Kant et al., 2011).The sample in solution phase (50 mg) is placed in the pan and sealed. Sample is then analysed with scanning rate of 0.5 K min-1. The sample is heated from 25 to 40°C and then cooled back to the initial temperature and is reheated to 40°C. Distilled water is used for temperature and enthalpy calibrations and as a reference. The change in temperature curves in heating cycle is compared to the baseline and is examined to determine the starting of gel-sol transition temperature (Tsol-gel). Once the endothermic peak is back to the baseline, it is considered as the end ofTsol-gel.

(method adapted from Iijima et al., 2012)

Viscosity and Rheology

The viscosity of polymer mixtures is evaluated with a Brookfield's rotational viscometer. A 10 mL aliquot of sample is placed into a small sample container. With a circulating water bath, the temperature of sample is then increased to 40 °C and measurements of viscosity are carried out using suitable spindle at various temperature.

(method adapted from Pisal et al., 2004)

The rheological property of polymer solution is examined by dynamic mechanical analyzer. The samples are kept between two parallel plates with 25 mm diameter and set 0.5 mm apart. The thickness of sample is approximately 1 mm. Measurements are performed under a controlled stress of 4.0 dyne cm-1 and a frequency of 1.0 rad s-1. A circulating water bath is used to adjust the temperature with a heating rate of 0.5 °C min-1.

(method adapted from Cha et al., 2011; Xuan et al., 2010)

The rheological property of polymer solution is examined by controlled stress rheometer which has cone/plate geometry (diameter 40 mm, angle 2° and gap 54 µm) for applying a sinusoidal shear. The elastic modulus (G') and viscous modulus (G'') are obtained from the measurement. The point where G' undergoes a drastic change can be referred as the gelation temperature.

(method adapted from Grah et al., 2010)

The gel texture beyond the gel point is characterised by plotting the G' values against shear frequency. This is performed under a specified stress where G' and G'' values remain constant and the sample retains a stable structure.

(method adapted from Grah et al., 2010)

Drug Release

The best nasal gel formula is prepared and loaded with 5-FU 0.3%. The method of preparation is the same like the aforementioned except the addition of 5-FU into the mixture is done before dissolving the polymers. A drug release study is then performed using dialysis tubing method.

A measured amount of the solution (1 g) is transferred into a 10 mL glass vial and incubated in a water bath at its gelation temperature for 10 minutes to allow the gel formation. An aliquot of gel preparation equivalent to 300 µg of 5-FU is then placed into a hydrated dialysis cell (width 25 mm, cellulose membrane). The cell is incubated in USP Apparatus 2 vessel with 500 mL medium, under a constant rotation of 50 rpm. Samples are taken from the vessel at specified time point (30, 60, 120, 240, 360, and 480 minutes) and replaced with an equal volume of buffer/media. The concentration of drug is determined with UV/VIS spectrophotometer at 266 nm.

(method adapted from Kant et al., 2011; Chaibva and Walker, 2007)

Annotated bibliography

A. Abruzzo,F. Biguccia, T. Cerchiara, B. Saladini, M.C. Gallucci, F. Cruciani, B. Vitali, B. Luppi (2013). Chitosan/alginate complexes for vaginal delivery of chlorhexidine digluconate. Carbohydrate Polymers. 91, 651- 658.

The study examines the characteristic of gels containing low molecular weight sodium alginate intended for mucosal delivery, and applies the use of FT-IR spectroscopy and Thermogravimetric Analysis for gel characterisation.

A. Aka-Any-Grah, K. Bouchemal, A. Koffi, F. Agnely, M. Zhang, M. Djabourov, G. Ponchel (2010). Formulation of mucoadhesive vaginal hydrogels insensitive to dilution with vaginal fluids. European Journal of Pharmaceutics and Biopharmaceutics. 76, 296-303.

The research suggests the optimal molar ratios of Poloxamer F127, Poloxamer F68, and HPMC to produce an ideal thermoresponsive gel. It also describes the use of controlled stress rheometer and micro-DSC for rheology study and characterisation of gel hydrogels.

C.R. Behl, H.K. Pimplaskar, A.P. Sileno, J. deMeireles, V.D. Romeo (1998). Effects of physicochemical properties and other factors on systemic nasal drug delivery. Advanced Drug Delivery Reviews. 29, 89-116.

The paper discusses the excipients commonly used in nasal formulation, physical characteristic of nasal preparation, nasal products which are currently available and those in clinical trials.

D.J. Begley (2004). Delivery of therapeutic agents to the central nervous system: the problems and the possibilities. Pharmacology & Therapeutics. 104, 29- 45.

The paper describes the characteristic of blood-brain barrier and possible alternatives for brain delivery.

G. Dumortier, J.L. Grossiord, F.Agnely, and J.C. Chaumeil (2006). A Review of Poloxamer 407 Pharmaceutical and Pharmacological Characteristics. Pharmaceutical Research. 23 (12), 2709-2725.

This paper reports the effect of sodium alginate on the sol-gel transition temperature of poloxamer gel preparation, an optimisation of sol-gel transition temperature using combination of Poloxamer 407 and Poloxamer 188.

H. Nazar, D.G. Fatouros, S.M. van der Merwe, N. Bouropoulos, G. Avgouropoulos, J. Tsibouklis, M. Roldo (2011). Thermosensitive hydrogels for nasal drug delivery: The formulation and characterisation of systems based on N-trimethyl chitosan chloride. European Journal of Pharmaceutics and Biopharmaceutics. 77, 225-232.

The paper mentions the possibility of tight junction opening on by chitosan.

H. Sarin (2009). Recent progress towards development of effective systemic chemotherapy for the treatment of malignant brain tumors. Journal of Translational Medicine. 7, 77.

The paper mentions about the inefficiency of currently available systemic chemotherapy.

H. Taki, T. Kanazawa, F.I Akiyama, Y. Takashima and H. Okada (2012). Intranasal Delivery of Camptothecin-Loaded Tat-Modified Nanomicells for Treatment of Intracranial Brain Tumors. Pharmaceuticals. 5, 1092-1102.

The research shows that brain chemotherapy through nasal route is possible.

J. Chen, X. Wang, J. Wang, G. Liu, X. Tang (2008). Evaluation of brain-targeting for the nasal delivery of ergoloid mesylate by the microdialysis method in rats. European Journal of Pharmaceutics and Biopharmaceutics. 68, 694-700.

The paper mentioned that molecular weight of drug has more effect compared to lipophilicity in order to be used in nasal preparation.

J.M.M. Levy, A. Thorburn (2012). Modulation of pediatric brain tumor autophagy and chemosensitivity. J Neurooncol. 106, 281-290.

The paper mentions about the death risk associated with pediatric brain tumour.

J. Ryu, S. Chung, M. Lee, C. Kim, C. Shim (1999). Increased bioavailability of propranolol in rats by retaining thermally gelling liquid suppositories in the rectum. Journal of Controlled Release. 59, 163-172.

The research suggests the optimal molar ratios of Poloxamer F127, Poloxamer F68 and various mucoadhesive polymer to produce an ideal thermoresponsive gel. It also studies the effect of sodium alginate on the gel strength.

J. Xuan, P. Balakrishnan, D.H. Oh, W.H. Yeo, S.M. Park, C.S. Yong, H.G. Choi (2010). Rheological characterization and in vivo evaluation of thermosensitive poloxamer-based hydrogel for intramuscular injection of piroxicam. International Journal of Pharmaceutics. 395, 317-323.

The paper examines the effect of sodium chloride on the viscosity and gelation time of Pluronic gels, suggest the optimal molar ratios of Pluronic F127 and Pluronic F68 to produce an ideal thermoresponsive gel. It also describes the use of rheometer to evaluate rheological property and semipermeable membrane tube for dissolution study.

L. Illum (2000). Transport of drugs from the nasal cavity to the central nervous system. European Journal of Pharmaceutical Sciences. 11, 1-18.

The paper describes the olfactory route for drug delivery to the brain.

M. Iijima, M. Takahashi, T. Hatakeyama, H. Hatakeyama. (2012). Detailed investigation of gel-sol transition temperature of j-carrageenan studied by DSC, TMA and FBM. J Therm Anal Calorim. 10, 1-7.

The paper describes the method of Differential Scanning Calorimetry used in determining gelation temperature.

N.S. Ningaraj (2006). Drug delivery to brain tumours: challenges and progress. Expert Opin. Drug Deliv. 3 (4), 499-509.

The paper mentions the blood-brain barrier as the main obstacle in CNS drug development.

R. Johnson, K.D. Wright, and R.J. Gilbertson (2009). Molecular Profiling of Pediatric Brain Tumors: Insight Into Biology and Treatment. Current Oncology Reports. 11, 68-72.

The paper mentions the incidence rate of brain tumour among children.

S. Cao, X. Ren, Q. Zhang, E. Chen, F. Xu, J. Chen, L. Liu, X. Jiang (2009). In situ gel based on gellan gum as new carrier for nasal administration of mometasone furoate. International Journal of Pharmaceutics. 365, 109-115.

The study develops thermoresponsive gel formulation using gellan gum and describe the use of rotational viscometer to evaluate viscosity of in situ gel preparation in both solution and gel phases.

S.E. Warrington, R.J. Kuhn (2011). Use of Intranasal Medications in Pediatric Patients. Orthopedics. 34 (6), 456-459.

The paper suggests the ideal volume of solution to be administered via nostril.

S.Khatua, Z.S. Sadighi, M.L. Pearlman, S. Bochare, T.S. Vats (2012). Brain Tumors in Children- Current Therapies and Newer Directions. Indian J Pediatr. 79 (7), 922-927.

The paper mentions about the inefficiency of currently available systemic chemotherapy, in terms of event-free survival rate.

S. Pisal (2004). Pluronic gels for nasal delivery of Vitamin B12. Part I: Preformulation study. International Journal of Pharmaceutics. 270, 37-45.

The research studies the effect of benzalkonium chloride on the sol-gel transition temperature of Poloxamer gel preparation, describes the cold method for gel preparations, inversion method for gelation temperature determination, and the use of rotational viscometer for viscosity study.

T. Chung, S. Lin, D. Liu, Y. Tyan, J. Yang (2009). Sustained release of 5-FU from Poloxamer gels interpenetrated by crosslinking chitosan network. International Journal of Pharmaceutics. 382, 39-44.

The research suggest the concentration of 5-FU incorporated into hydrogels, describes the use of cone/plate viscometer for viscosity study and osmosis membrane method for dissolution study.

T. Chung, D. Liu, J. Yang (2010). Effects of interpenetration of thermo-sensitive gels by crosslinking of chitosan on nasal delivery of insulin: In vitro characterization and in vivo study. Carbohydrate Polymers. 82, 316-322.

The research suggests the optimal molar ratios of Poloxamer F127 and Poloxamer F68 to produce an ideal thermoresponsive gel, describe the use of cone/plate viscometer for viscosity and rheology study and the use of dialysis method for drug release study.

T.R. Wolfe and D.A. Braude (2010). Intranasal Medication Delivery for Children: A Brief Review and Update. Pediatrics. 126, 532.

The paper mentions the acceptability of nasal preparations among pediatric patients.

T. Shingaki, D. Inoue, T. Furubayashi, T. Sakane, H. Katsumi, A. Yamamoto, and S. Yamashita (2010). Transnasal Delivery of Methotrexate to Brain Tumors in Rats: A New Strategy for Brain Tumor Chemotherapy. Molecular Pharmaceutics. 7 (5), 1561-1568.

The research evaluates the feasibility of brain chemotherapy of methotrexate through nasal route.

U. Bertram and R. Bodmeier (2006). In situ gelling, bioadhesive nasal inserts for extended drug delivery: In vitro characterization of a new nasal dosage form. European Journal of Pharmaceutical Sciences. 27, 62-71.

The study compares the bioadhesive strength of sodium alginate, HMPC and PVP90 in the nasal preparations.

W.M. Pardridge (1997). Drug Delivery to the Brain. Journal of Cerebral Blood Flow and Metabolism. 17, 713-731.

The paper discusses the current progress in CNS drugs and reasons for the poor CNS drug development.

Y. Chen, L. Liu (2012). Modern methods for delivery of drugs across the blood-brain barrier. Advanced Drug Delivery Reviews. 64, 640-665.

The paper describes the main function of blood-brain barrier and brain and cerebrospinal fluid barrier.

Z. Cai, X. Song, F. Sun, Z. Yang, S. Hou and Z. Liu (2011). Formulation and Evaluation of In Situ Gelling Systems for Intranasal Administration of Gastrodin.  AAPS PharmSciTech. 12 (14), 1102-1109.

The paper mentions the rationale of in situ gel preparation for nasal preparation.

Project management plan

Tasks

May

June

July

August

September

6th - 10th

13th - 17th

20th - 24th

27th - 31st

3rd - 7th

10th - 14th

17th - 21st

24th - 28th

1st - 5th

8th - 12th

15th - 19th

22nd - 26th

29th - 31st

1st - 31st

2nd

3rd

10th

Discussion with supervisor

A

 

B

 

 

 

C

 

 

 

D

 

 

 

 

E

 

Writing up draft - Introduction

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Progress report to supervisor

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Preparation of materials

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Formulation development

Formulation: F1 - F18

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Additional formulation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Evaluation of selected formulas:

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Tsol-gel

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Clarity

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Texture

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rheology - rotational viscometer

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Rheology - cone/plate viscometer

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Characterisation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FT/IR

TGA

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DSC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Preparation of drug loaded gels

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Optimisation of drug release study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Drug Release Study

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Submission of Report and Abstract

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Poster presentation

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Discussion matter: A- objectives; B - materials and methods; C introduction; D - results and discussion; E - poster

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