Doctor of Philosophy

dissertationWith the steadily increasing prevalence of endometrial hyperplasia (EH) and endometrial cancer (EC) among premenopausal women, the pursuit of improved conservative therapeutics is vitally important. Work in this dissertation attempts to optimize treatments for EH and EC through three different approaches: 1) formulating an enhanced progesterone (P4) vaginal formulation, 2) providing a new in vivo tool (animal model) for EH, and 3) testing the potential of imiquimod (IQ) as a treatment for primary EC. In the first approach, glycol chitin, a polymer with thermosensitive properties, was used as the delivery vehicle for the new progesterone (P4) formulation (GC-P4). Thermogelling, mechanical, and dynamic studies using rheology showed that GC-P4 gelled and maintained suitable physical characteristics in the vaginal environment. The formulation was capable of releasing the drug in a controlled manner with no effect on the drug's bioactivity. GC-P4 was safe to the vaginal environment as the in vitro studies depicted. Local safety was confirmed in mice, as well is the ability of GC-P4 to inhibit EH disease progression. The induction of EH after implantation of subcutaneous estrogen (E2) pellets in mice was the second approach explored. The main motive for this innovative approach was the lack of an in vivo model that effectively represents EH as seen in human. Analysis of endometrial tissues using H&E and several immunohistochemistry stains reflected different stages of disease development, hormonal receptors status, and common genetic abrasions as seen in the human disease. In order to widen the treatments options for patients with low grade EC who do not respond well to conventional treatments yet desire a conservative therapy, the potential role of IQ in the management of EC was evaluated. In vitro studies using Ishikawa and HEC-1A cells showed that IQ is capable of inducing apoptosis in cells, possibly through inhibiting expression of the BCL-2 family of proteins. Moreover, IQ was able to inhibit EC progression in a mouse xenograft model. The endeavor described in this dissertation aims to address some of unmet needs in researching and treating abnormal endometrial proliferations, i.e. EH and EC. The studies and tools developed here lay the foundation for future studies aiming at understanding and advancing therapies for EH and EC

Evaluating the bioequivalence of levetiracetam brand and generic oral tablets available in the Saudi market in vivo

Background: Epilepsy is a common global neurological disorder. About 30% of epileptic patients are managed with anti-epileptic Drugs (AEDs). Since 2000, Levetiracetam (LEV) has been marketed around the world as an AED under the brand name Keppra, and recently more generics are found in the Saudi market as cheaper alternatives. The objective of this study is to evaluate the bioequivalence of LEV brand and generics available in the Saudi market in mice. Methods: Pharmacokinetics (PK), liver function test, and behavioral studies were conducted for LEV brand and generic in different groups of Blab/c mice. Results: PK results show a significance difference in PK parameters mostly evidenced with generic 3, then generic 2. The only significant different between Keppra and generic 1 was in T1/2. In addition, Keppra did not significantly increase liver enzymes in comparison to other generics. On the other hand, other generics showed less favorable results in increasing liver enzymes. Keppra reduced the number and intensity of epileptic attacks, had no mortality rate due to epilepsy, and was associated with less sever seizures attacks. Conclusion: Keppra, the brand form of LEV, has better safety and efficacy profiles in mice compared to 3 generics found in the Saudi market. Therefore, we recommend evaluating the same parameters tested in this study in patients utilizing similar generics and brand to establish the existence of bioequivalence between LEV brand and generics

Imiquimod-Loaded Chitosan-Decorated Di-Block and Tri-Block Polymeric Nanoparticles Loaded In Situ Gel for the Management of Cervical Cancer

Background: Cervical intraepithelial neoplasia, the predisposing factor for cervical cancer (CC), is caused by human papillomavirus (HPV) infection and can be treated with imiquimod (IMQ). However, poor water solubility and side effects such as local inflammation can render IMQ ineffective. The aim of this study is to design a prolonged release nano system in combination with mucoadhesive–thermosensitive properties for an effective vaginal drug delivery. Methods: Polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL), poly lactide-co-caprolactone (PLA-PCL), and poly L-lactide-co-caprolactone-co-glycolide (PLGA-PCL) were used to create IMQ nanoparticles. Chitosan (CS) was then added to the surfaces of the IMQ NPs for its mucoadhesive properties. The NPs were then incorporated into poloxamer hydrogels. The NPs’ size and morphology, encapsulation efficiency (EE), in vitro drug release, gel characterization, ex vivo drug permeation, and in vitro safety and efficacy were characterized. Results: Two batches of NPs were prepared, IMQ NPs and CS-coated NPs (CS-IMQ NPs). In general, both types of NPs were uniformly spherical in shape with average particle sizes of 237.3 ± 4.7 and 278.2 ± 5.4 nm and EE% of 61.48 ± 5.19% and 37.73 ± 2.88 for IMQ NPs and CS-IMQ NPs, respectively. Both systems showed prolonged drug release of about 80 and 70% for IMQ NPs and CS-IMQ NPs, respectively, within 48 h. The gelation temperatures for the IMQ NPs and CS-IMQ NPs were 30 and 32 °C, respectively; thus, suitable for vaginal application. Although ex vivo permeability showed that CS-IMQ NPs showed superior penetration compared to IMQ NPs, both systems enhanced drug penetration (283 and 462 µg/cm2 for IMQ NPs and CS-IMQ NPs, respectively) relative to the control (60 µg/cm2). Both systems reduced the viability of cervical cancer cells, with a minimal effect of the normal vaginal epithelium. However, IMQ NPs exhibited a more pronounced cytotoxic effect. Both systems were able to reduce the production of inflammatory cytokines by at least 25% in comparison to free IMQ. Conclusion: IMQ and CS-IMQ NP in situ gels enhanced stability and drug release, and improved IMQ penetration through the vaginal tissues. Additionally, the new systems were able to increase the cytotoxic effect of IMQ against CC cells with a reduction in inflammatory responses. Thus, we believe that these systems could be a good alternative to commercial IMQ systems for the management of CC

Flavoured water consumption alters pharmacokinetic parameters and increases exposure of erlotinib and gefitinib in a preclinical study using Wistar rats

Background Erlotinib (ERL) and Gefitinib (GEF) are considered first line therapy for the management of non-small cell lung carcinoma (NSCLC). Like other tyrosine kinase inhibitors (TKIs), ERL and GEF are mainly metabolized by the cytochrome P450 (CYP450) CYP3A4 isoform and are substrates for transporter proteins with marked inter-/intra-individual pharmacokinetic (PK) variability. Therefore, ERL and GEF are candidates for drug-drug and food-drug interactions with a consequent effect on drug exposure and/or drug-related toxicities. In recent years, the consumption of flavoured water (FW) has gained in popularity. Among multiple ingredients, fruit extracts, which might constitute bioactive flavonoids, can possess an inhibitory effect on the CYP450 enzymes or transporter proteins. Therefore, in this study we investigated the effects of different types of FW on the PK parameters of ERL and GEF in Wistar rats. Methods ERL and GEF PK parameters in different groups of rats after four weeks consumption of different flavours of FW, namely berry, peach, lime, and pineapple, were determined from plasma drug concentrations using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). Results Data indicated that tested FWs altered the PK parameters of both ERL and GEF differently. Lime water had the highest impact on most of ERL and GEF PK parameters, with a significant increase in Cmax (95% for ERL, 58% for GEF), AUC0–48 (111% for ERL, 203% for GEF), and AUC0–∞ (200% for ERL, 203% for GEF), along with a significant decrease in the apparent oral clearance of both drugs (65% for ERL, 67% for GEF). The order by which FW affected the PK parameters for ERL and GEF was as follows: lime > pineapple > berry > peach. Conclusion The present study indicates that drinking FW could be of significance in rats receiving ERL or GEF. Our results indicate that the alteration in PKs was mostly recorded with lime, resulting in an enhanced bioavailability, and reduced apparent oral clearance of the drugs. Peach FW had a minimum effect on the PK parameters of ERL and no significant effect on GEF PKs. Accordingly, it might be of clinical importance to evaluate the PK parameters of ERL and GEF in human subjects who consume FW while receiving therapy

A Facile Synthesis and Molecular Characterization of Certain New Anti-Proliferative Indole-Based Chemical Entities

Cancer cells frequently develop drug resistance, which leads to chemotherapeutic treatment failure. Additionally, chemotherapies are hindered by their high toxicity. Therefore, the development of new chemotherapeutic drugs with improved clinical outcomes and low toxicity is a major priority. Several indole derivatives exhibit distinctive anti-cancer mechanisms which have been associated with various molecular targets. In this study, target compounds 4a–q were obtained through the reaction of substituted benzyl chloride with hydrazine hydrate, which produces benzyl hydrazine. Subsequently, the appropriate substituted benzyl hydrazine was allowed to react with 1H-indole-2-carboxylic acid or 5-methoxy-1H-indole-2-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a coupling agent. All compounds exhibited cytotoxicity in three cell lines, namely, MCF-7, A549, and HCT. Compound 4e exhibited the highest cytotoxicity, with an average IC50 of 2 µM. Moreover, a flow cytometry study revealed a significantly increased prevalence of Annexin-V and 7-AAD positive cell populations. Several derivatives of 4a–q showed moderate to high cytotoxicity against the tested cell lines, with compound 4e having the highest cytotoxicity, indicating that it may possess potential apoptosis-inducing capabilities

The Design of Anionic Surfactant-Based Amino-Functionalized Mesoporous Silica Nanoparticles and their Application in Transdermal Drug Delivery

Melanoma remains the most lethal form of skin cancer and most challenging to treat despite advances in the oncology field. Our work describes the utilization of nanotechnology to target melanoma locally in an attempt to provide an advanced and efficient quality of therapy. Amino-functionalized mesoporous silica nanoparticles (MSN-NH2) were developed in situ through the utilization of anionic surfactant and different volumes of 3-aminopropyltriethoxysilane (APTES) as a co-structure directing agent (CSDA). Prepared particles were characterized for their morphology, particles size, 5-flurouracol (5-FU) and dexamethasone (DEX) loading capacity and release, skin penetration, and cytotoxicity in vitro in HT-144 melanoma cells. Results of transmission electron microscopy (TEM) and nitrogen adsorption–desorption isotherm showed that using different volumes of APTES during the functionalization process had an impact on the internal and external morphology of the particles, as well as particle size. However, changing the volume of APTES did not affect the diameter of formed mesochannels, which was about 4 nm. MSN-NH2 showed a relatively high loading capacity of 5-FU (12.6 ± 5.5) and DEX (44.72 ± 4.21) when using drug: MSN-NH2 ratios of 5:1 for both drugs. The release profile showed that around 83% of 5-FU and 21% of DEX were released over 48 h in pH 7.4. The skin permeability study revealed that enhancement ratio of 5-Fu and DEX using MSN-NH2 were 4.67 and 5.68, respectively, relative to their free drugs counterparts. In addition, the accumulation of drugs in skin layers where melanoma cells usually reside were enhanced approximately 10 times with 5-FU and 5 times with DEX when delivering drugs using MSN-NH2 compared to control. MSN-NH2 alone was nontoxic to melanoma cells when incubated for 48 h in the range of 0 to 468 µg/mL. The combination of 5-FU MSN-NH2 and DEX MSN-NH2 showed significant increase in toxicity compared to their free dug counterparts and exhibited a synergetic effect as well as the ability to circumvent DEX induced 5-FU resistance in melanoma cells

Novel Metoprolol-Loaded Chitosan-Coated Deformable Liposomes in Thermosensitive In Situ Gels for the Management of Glaucoma: A Repurposing Approach

Glaucoma is a long-term eye disease associated with high intraocular pressure (IOP), which seriously damages the eyes, causing blindness. For successful therapy, potent drugs and delivery systems are required. Metoprolol (MT) is believed to help reduce elevated IOP. The paradigm of ocular therapeutics may be changed by the integration of chitosan-coated liposomes (CLPs) with thermosensitive in situ gel (ISG). Therefore, MT-CLPs were developed and characterized and compared to uncoated ones (MT-LPs). Furthermore, MT-LP- and MT-CLP-loaded ISGs were prepared and characterized in in vitro, ex vivo, and in vivo studies. MT-LPs and MT-CLPs displayed spherical shapes with nanosize range, reasonable EE%, and significant bioadhesion. The zeta potential changed from negative to positive after CS coating. The extended in vitro drug release of MT-CLPs showed significant mucin mucoadhesion. The formed ISGs were homogeneous with a pH range of 7.34 to 7.08 and a rapid sol–gel transition at physiological temperature. MT-ISG1 (MT-LP) and MT-ISG2 (MT-CLPs-0.5) could increase ocular permeability by 2-fold and 4.4-fold compared to MT-ISG (pure MT). MT-ISG2 demonstrated significantly reduced IOP in rabbits without causing any irritation. In conclusion, MT-ISG2 markedly enhanced corneal permeability and reduced IOP. They would be promising carriers for MT for glaucoma management

Aspartames Alter Pharmacokinetics Parameters of Erlotinib and Gefitinib and Elevate Liver Enzymes in Wistar Rats

Background: Erlotinib (ERL) and gefitinib (GEF) are extensively metabolized by CYP450 enzymes. Aspartame (ASP), an artificial sweetener, induces CYP2E1 and CYP3A2 enzymes in the brain and could increase liver enzymes. In this work, the influence of ASP on the pharmacokinetics (PK) of ERL and GEF in Wistar rats was evaluated. Methods: The PKs of ERL and GEF were evaluated after receiving 175 mg/kg or 1000 mg/kg of ASP for four weeks using UPLC-MS/MS. Levels of liver enzymes after four weeks of ASP consumption were also evaluated. Results: ASP 175 mg/kg was able to significantly alter levels of Cmax (36% increase for ERL, 38% decrease for GEF), AUC0–72 (205% increase for ERL, 41% increase for GEF), and AUC0–∞ (112% increase for ERL, 14% increase for GEF). Moreover, ASP 175 mg/kg decreased the apparent oral clearance ERL and GEF by 58% and 13%, respectively. ASP 1000 mg/kg increased Cmax of ERL by 159% and decreased GEF’s Cmax by and 73%. Both AUC0–72 and AUC0–∞ were increased by ASP 1000 for ERL and decreased for GEF. CL/F decreased by 64% for ERL and increased by 38.8% for GEF. Moreover, data indicated that ASP significantly increased levels of liver enzymes within two weeks of administration. Conclusions: Although ASP 175 and 1000 mg/kg alter ERL and GEF PKs parameters, ASP 1000 mg/kg has the highest impact on most parameters. ASP 1000 mg/kg also can significantly increase activities of liver enzymes indicating the possibility of inducing liver injury. Therefore, it might be of clinical importance to avoid the administration of aspartame containing products while on ERL or GEF therapy

Mesoporous Silica Nanoparticles Coated with Carboxymethyl Chitosan for 5-Fluorouracil Ocular Delivery: Characterization, In Vitro and In Vivo Studies

This study investigates the development of topically applied non-invasive amino-functionalized silica nanoparticles (AMSN) and O-Carboxymethyl chitosan-coated AMSN (AMSN-CMC) for ocular delivery of 5-Fluorouracil (5-FU). Particle characterization was performed by the DLS technique (Zeta-Sizer), and structural morphology was examined by SEM and TEM. The drug encapsulation and loading were determined by the indirect method using HPLC. Physicochemical characterizations were performed by NMR, TGA, FTIR, and PXRD. In vitro release was conducted through a dialysis membrane in PBS (pH 7.4) using modified Vertical Franz diffusion cells. The mucoadhesion ability of the prepared nanoparticles was tested using the particle method by evaluating the change in zeta potential. The transcorneal permeabilities of 5-FU from AMNS-FU and AMSN-CMC-FU gel formulations were estimated through excised goat cornea and compared to that of 5-FU gel formulation. Eye irritation and ocular pharmacokinetic studies from gel formulations were evaluated in rabbit eyes. The optimum formulation of AMSN-CMC-FU was found to be nanoparticles with a particle size of 249.4 nm with a polydispersity of 0.429, encapsulation efficiency of 25.8 ± 5.8%, and drug loading capacity of 5.2 ± 1.2%. NMR spectra confirmed the coating of AMSN with the CMC layer. In addition, TGA, FTIR, and PXRD confirmed the drug loading inside the AMSN-CMC. Release profiles showed 100% of the drug was released from the 5-FU gel within 4 h, while AMSN-FU gel released 20.8% of the drug and AMSN-CMC-FU gel released around 55.6% after 4 h. AMSN-CMC-FU initially exhibited a 2.45-fold increase in transcorneal flux and apparent permeation of 5-FU compared to 5-FU gel, indicating a better corneal permeation. Higher bioavailability of AMSN-FU and AMSN-CMC-FU gel formulations was found compared to 5-FU gel in the ocular pharmacokinetic study with superior pharmacokinetics parameters of AMSN-CMC-FU gel. AMSN-CMC-FU showed 1.52- and 6.14-fold higher AUC0-inf in comparison to AMSN-FU and 5-FU gel, respectively. AMSN-CMC-FU gel and AMSN-FU gel were “minimally irritating” to rabbit eyes but showed minimal eye irritation potency in comparison to the 5 FU gel. Thus, the 5-FU loaded in AMSN-CMC gel could be used as a topical formulation for the treatment of ocular cancer