الفهرس 
General IntroductionOnly 14 pages are availabe for public view
Abstract
Hypertension (HTN) is a major public health issue in both developing and wealthy countries. Worldwide, HTN affects around one billion individuals. It is a serious public health problem in Egypt, with a frequency of 26.3% among the adult population. The majority of the existing oral antihypertensive medications for HTN control have undesirable side effects. As a result, finding alternate routes to overcome the limitations of the oral route became necessary, one of the most important limitations are the destruction of the drugs by digestive enzymes and/or stomach acid, and the first pass metabolism which affect the bioavailability of most of these drugs.
The intranasal route is now widely used as an alternative to oral and parenteral use, as it offers several advantages; being non-invasive, painless, and simple to use, self-administered and overcome the undesirable side effects obtained from either oral or parenteral use.
Olmesartan medoxomil (OLM) ((5-methyl-2-oxo-1, 3-dioxolen-4-yl)- methoxy-4-(1-hydroxy-1-methylethyl)-2-propyl-1-{4-[2-(tetrazol-5-yl)-phenyl] phenyl methylimidazol-5carboxylate) is a novel active and selective non-peptide angiotensin II receptor blocker. OLM acts by inhibiting angiotensin II type 1 receptor sites in the smooth muscles of the vascular system, preventing the vasoconstrictor effects of angiotensin II. However, it has a very low oral bioavailability; this in turns increases its daily dose with subsequent increase in its side effects. Therefore, several trials have been conducted in order to improve OLM therapeutic potential by formulating it in adequate delivery systems, hence overcome the constraints involved in its delivery.
Chitosan is the most widely utilized natural polymer because of its non-toxicity, biodegradability and mucoadhesive properties that make it the best polymer for intranasal administration to overcome the mucociliary clearance. Chitosan nanoparticles (CS NPs) were effectively employed as carriers to deliver the therapeutic payload to the desired target location at the optimal dosage, to achieve drug sustained release due to its bio-adhesive properties, to improve cellular and tissue absorption and to lower systemic toxicity.
The aim of this thesis was to investigate the effectiveness of OLM in the treatment of hypertension via the intranasal route while being encapsulated in a natural polymeric nanoparticulate system as CS NPs. This was aimed to overcome the side effects encountered with its oral delivery and boost its systemic bioavailability and hence its therapeutic efficacy via pharmacokinetic and pharmacodynamic investigations.
The work in this thesis is divided into two chapters:
Chapter I: Preparation and characterization of Olmesartan medoxomil loaded chitosan nanoparticles.
Chapter II: In vivo Evaluation of Olmesartan medoxomil loaded chitosan nanoparticles: Pharmacokinetic and Pharmacodynamic studies
Chapter I: Preparation and characterization of Olmesartan medoxomil loaded chitosan nanoparticles.
This chapter deals with the formulation, and characterization of OLM loaded CS NPs. CS NPs were prepared using ionic gelation method using TPP as a cross linker, and then characterized for their morphology by transmission electron microscopy (TEM), particle size (PS), polydispersity index (PDI), zeta potential (ZP), and entrapment efficiency (%EE). A preliminary study was conducted to investigate the effect of different variables; CS pH, TPP pH. CS: TPP mass ratios, and TPP concentration, applying a systematic one-factor-at-time approach in order to optimize the formulation conditions. Following the preliminary study, D-optimal design was applied, and three dependent variables were considered: CS concentration, TPP pH, and the preparation method (magnetic stirrer and homogenization techniques). An ex-vivo permeation study of OLM from the optimized CS NPs formulations was carried out using nasal sheep mucosa in simulated nasal fluid. Moreover, the physical stability study was performed on the selected formula over a storage period of 3 months at refrigeration temperature (5 ± 3°C) and at room temperature (25 ± 3 °C). The effect of storage on the PS, PDI, ZP, and %EE of the CS NPs was investigated.
The results of this chapter revealed the following:
1. A sensitive, precise, and accurate method for the quantitation of OLM in distilled water and phosphate buffer saline PBS (pH 6.8) containing 10% (v/v) ethanol was developed and validated using UPLC.
2. Chitosan (CS) concentrations had a positive impact on PS and PDI, as increased CS concentration from 0.1 to 0.4% w/v lead to an increment in PS and PDI accordingly. However, the lowest CS concentration (0.1%) showed a percent transmittance values >50 which indicates the inability to form NPs.
3. The optimum CS concentration succeeded in producing uniform and homogenous NPs without aggregation was 0.3% at pH = 5.
4. As TPP concentration was increased from 0.1 to 0.2 % w/v, a significant size enlargement with heterogeneity (p<0.05) was obtained. However, further increase in TPP concentration above 0.1% w/v resulted in aggregation formation.
5. For surface charge, ZP was a non-significant parameter with p>0.05 as all prepared CS NPs showed highly positive ZP values attributed to the CS amino groups.
6. Decreasing CS:TPP mass ratio from 9:1 to 2:1 lessens the percent transmittance to <50%, which would indicate the successful formation of the CS nano-dispersions, therefore the optimum CS:TPP mass ratio was 3:1 at higher TPP concentration range (0.1 – 0.2%).
7. Homogenization technique produced more homogenous PS with an acceptable PDI values than magnetic stirrer method.
8. The optimized OLM loaded CS NPs highly showed EE% values (up to 75.20 ± 7.07%).
9. The ex-vivo permeation study of the selected OLM loaded CS NPs through sheep nasal mucosa revealed that a significant higher permeability of F1 than the other formulae (p<0.05) translated to its higher Q24 value (189.02 ± 7.08 µg/cm2).
10. The selected OLM loaded CS NPs formulation (F1) was composed of 0.3% CS at pH 5, 0.1% TPP at pH 9 and CS:TPP mass ratio was 3:1.
11. The TEM photomicrographs for the selected formulae (F1) showed a predominant spherical shape with a smooth surface.
12. Physical stability study proved that the selected CS NPs was able to maintain the same physicochemical properties (PS, PDI, ZP and EE%) upon storage for three months under refrigeration (5 ± 3°C) and at room temperature (25 ± 3°C).
Chapter II: In vivo Evaluation of Olmesartan medoxomil loaded chitosan nanoparticles: Pharmacokinetic and Pharmacodynamic studies
In this chapter, a pharmacokinetic study was performed to investigate the biological availability of OLM in plasma from the chosen CS NPs when administered through the nasal route against OLM suspension administered by oral and nasal routes. Blood samples were collected from the orbital vein and kept for further analyzing using a validated HPLC assay to obtain the pharmacokinetic parameters including (Cmax), (Tmax), (AUC0-24), (AUC0-∞), (MRT), (Kel) and (t1/2). The safety of CS NPs on the nasal tissue was examined through a histopathological study by staining the nasal tissue samples with hematoxylin and eosin (H&E) and Trichrome Stain (Masson). In addition, a pharmacodynamic study was employed to study the effect of the intranasal administration of OLM loaded CS NPs in reducing the BP compared with OLM susp administered by oral and nasal routes, negative and positive controls as well. All rats were injected intraperitoneally (IP) with 75 mg/kg/day nitro-L-arginine-methyl ester (L-NAME) solution for 10 days before treatment and continued till the end of the experiment to ensure the occurrence of HTN. Assessment of hypertension was performed by measuring systolic blood pressure (SBP), diastolic blood pressure (DBP), electrocardiogram (ECG), and heart rate (HR) for all groups.
The results of this chapter revealed the following:
1. CS NPs had the ability to increase the bioavailability of OLM nearby 13 folds when administered intranasally compared to OLM suspension administered by intranasal or oral routes.
2. CS NPs had mucoadhesive properties which played an important role for decreasing the clearance of OLM and by sequences increasing the MRT and the t1/2 in comparison with nasal and oral OLM suspension.
3. OLM suspension showed a significant irritation of nasal mucosa with mild edema when administered intranasally as revealed from histopathological structures stained with (H&E) stain while numerous congested blood vessels were noticed from images stained with (MTC), however when OLM was given in the form of CS NPs, an improvement in the histological structures of the nasal tissue was noticed with an apparently normal nasal mucosa and mild congestion.
4. Hypertension was produced by IV of L-NAME with a daily dose of 75mg/kg/day for 10 days.
5. The ability of intranasal OLM loaded CS NPs to reduce the blood pressure back to the normal range was achieved, as there was no significant difference in the measured parameters (SBP, DBP, ECG and HR) between group I (normal group) and group V (IN OLM NPs).
from the previously stated results, it can be concluded that the encapsulation of OLM in CS NPs offered a safe, biocompatible nanocarrier system. Intranasal route proved to be an alternative route for oral route for OLM CS NPs as it succeeded to protect the drug from GIT degradation, increase its distribution & bioavailability and as a result can decrease the dose frequency and decrease the drug possible side effects.