Spray drying is widely used in enhancing the aqueous solubility of poorly soluble compounds. In this study, the mechanism of solubility enhancement was characterized using three model drugs-naproxen, ketoprofen and furosemide. Physical mixtures of the model drug with polyvinylpyrrolidine and spray dried composites were subjected to Fourier Transform Infrared Sprectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Powder X-ray Diffraction (XRPD). The data showed that the crystalline model drugs were converted to amorphous form upon spray drying, whereas the physical mixtures did not change their crystallinity. The effect of the amorphous forms produced by Spray drying on apparent solubility and intrinsic dissolution rate was determined. All the spray dried composites exhibited higher apparent solubility and intrinsic dissolution rate when compared to the pure drugs and their physical mixtures. The stability of the spray dried composites upon storage was also determined. The amorphous nature of the compounds in the spray dried composites were retained during 3 months storage as shown by FTIR, DSC and XRPD characterization and their apparent solubility and intrinsic dissolution rates also did not change.

Rationale: The purpose of this study is to investigate the stored stability of dilutions of neuromuscular blocking agents (NMBAs), namely cisatracurium, rocuronium, and vecuronium, for skin prick/intradermal testing. Methods: Concentrations of NMBAs were monitored by liquid chromatography-mass spectrometry (LC-MS/MS) for a period of 14 days. Dilutions of NMBAs were prepared in saline by factors of 10x, 100x, 1,000x, 10,000x, and 100,000x as sensitivity of the assay allowed. Diluted drug products were stored in a laboratory refrigerator until sampling. On sampling days, aliquots of each dilution were removed and compared to a freshly prepared set of reference dilutions. Results: The results are measured as beyond use date (BUD) defined as recovery of drug versus the reference (90-110%). Based on the LC-MS/MS data, the BUD for cisatracurium diluted to 10x and 100x is 96 hours. Higher dilutions (1,000x to100,000x) should be used immediately following preparation (within less than 24 hours). Vecuronium at 10x and 100x, also has a BUD of 96 hours, and the 1,000x dilution is stable for 24 hours. The 10,000x dilution should be used immediately. Rocuronium at 10x to 1,000x has a BUD of 48 hours, yet higher dilutions (10,000x and 100,000x) should be used immediately. Conclusions: With increasing dilution factors, the stability of these drugs in saline decreases, increasing deviation between samples and references. The most stable dilutions for each of the drugs tested were 10x and 100x. Stability of these drugs is likely compromised by hydrolysis of the ester bonds in the drug molecules.

Purpose: 5-hydroxytryptamine (5-HT) is an important neurotransmitter in depression. It is believed that α 1 and α 2 adrenoceptors mediate the 5-HT level in the brain. The mechanism is complex and not well explored. Especially in different combination treatments, the receptor systems may show varied modulation capability. Additionally, some research has suggested that α 2 heteroceptors may contribute to the time delay problem in dual depression treatment which is thought as the time needed for certain inhibition receptor to get desensitized. We hypothesized that the α 2 adrenoceptors had inhibition effect on 5-HT level in dorsal raphé nucleus (DRN), Prefrontal cortex (PFC) and Hippocampus (HP) with the dual reuptake inhibition. The present study was undertaken to explore the effect of BRL44408 (α 2 receptor antagonist) on 5-HT level in rat PFC, DRN and HP under dual antidepressant with blocking the α 1 receptor. Method: Serotonin reuptake inhibitor and norepinephrine reuptake inhibitor were used to mimic the dual reuptake inhibition antidepressant. To differentiate the α 2 adrenoceptors effect from al adrenoceptors effect, prazosin, an antagonist of α 1 adrenoceptors, was added to block α 1 adrenoceptors. Using the microdialysis method, the drug combination was examined in HP area and then DRN area to explore the drug effect on time course of 5-HT release in DRN and PFC. Based on the experiment results from DRN and PFC, a mechanism-based pharmacodynamic model was developed. Result: BRL44408 increased the serotonin (5-HT) level in rat PFC, DRN and HP to different degrees with the dual reuptake inhibition (p < 0.05). The overall model reasonably captured the time course of 5-HT in both DRN and PFC with different dose schemes of BRL44408. The model predicted EC50 of BRL44408 (0.0075 µM) for the α 2 heteroreceptor which control PFC 5-HT is close to the reported value of BRL44408 for α 2 adrenorceptor (0.008 µM). However, the model predicted EC50 of BRL44408 on the α 2 heteroreceptor which control DRN 5-HT need to be explained. Simulation result from this model suggested varied modulation capability of α 2 adrenoceptors on the 5-HT in DRN and the 5-HT in PFC. Conclusion: α 2 heteroceptor play a role in regulation 5-HT level under dual reuptake inhibition. Further exploration may bring a potential target for depression treatment. The mechanism model was developed to characterize and better understand the neurotransmitter mechanisms, providing estimations of various parameters of the disease related receptor system.

The aryl hydrocarbon receptor (AhR) is a cytosolic ligand-activated transcription factor whose toxicity and carcinogenesis is mediated through various polyaromatic hydrocarbons and other environmental pollutants. The role of AhR in carcinogenesis is an area of concern due its altered levels in various tumors. AhR binds structurally diverse ligands and may elicit different responses upon ligand binding. The crystal structure of mouse AhR PAS-A domain was already obtained due to the robustness of mouse AhR in comparison to human. There is a possibility of overexpressing mouse AhR ligand binding domain in its soluble and functional form, which could be used to perform ligand binding studies. This forms the aim of this thesis. Mouse AhR ligand binding domain was constructed as mAhR aa211-384, which was purified under native conditions with the use of 6 histidine tag but soluble overexpression was not possible. Thus a solubility enhancing tag called maltose binding protein (MBP) was used for purification of mAhR aa211-384 under native conditions, which still did not yield soluble overexpression. The strategy was modified to solubilize the protein by denaturation with the use of 8M Urea, which solubilized the protein but included an issue of protein binding to column. Subsequent use of an even stronger denaturant, 6M guanidine hydrochloride, solubilized most of the protein and purified mAhR aa211-384 in huge amount. Successful refolding of mAhR aa211-384 with the help of MBP was made possible by gradual reduction of denaturant in the presence of arginine, but 6 histidine tag failed to refold the protein. The refolded protein was tested for its secondary structure by circular dichroism. Thus, mAhR aa211-384 was solubilized and purified under denaturing conditions with the help of both 6 histidine and MBP, however efficient refolding of mAhR aa211-384 was only possible with the help of MBP but not 6 histidine. The MBP-refolded mAhR aa211-384 stayed in solution even after the removal of 0.1 M arginine, thus confirming the effectiveness of MBP in protein refolding in comparison to 6 histidine tag.

Proteins are large biomolecules that have great therapeutic potential in treating many human diseases. Proteins exert higher specificity and more complicated functions; they are well endured and less inclined to evoke immune responses when compared to small molecule drugs. However, exogenous proteins when administered intravenously are prone to immune reactions. Chemical and enzymatic denaturation, and poor penetration into cells are some other challenges for clinical use of intracellular proteins. Proteins that enter cells through endocytosis will be eventually degraded in lysosomes if they do not escape the endosomal pathway before reaching lysosomes. Therefore, the development of protein delivery systems, including liposomal and/or polymeric nanoparticles would substantially facilitate the clinical use of proteins. This approach can protect the proteins from denaturation and immune reactions. Previously, our group has developed cationic lipid-coated magnesium phosphate nanoparticle (CAT-LP MgP NP) formulations to enhance the intracellular delivery of the protein, catalase. The objective of the current research is to improve the physicochemical properties of CAT-LP MgP NP. The magnesium phosphate (MgP) nanoparticles were prepared by water-in-oil micro emulsion precipitation. The cargo protein catalase was complexed with cationic liposome prepared by lipid hydration and extrusion. Then magnesium phosphate (MgP) nanoparticles were mixed with the catalase-complexed cationic liposome to form the final complexed CAT-LP MgP NP formulation. By sonication, extrusion and modification of the lipid composition, we have successfully prepared complexed CAT-LP MgP NP formulations of reduced size. The pH-sensitivity of the improved delivery system was observed at pH 6.0. Furthermore, the improved delivery system reduced the Reactive Oxygen Species (ROS) level inside EA.hy.926 cells (human umbilical vein endothelial cells) to 35% of the control, whereas the previously reported catalase formulation of our group reduced the ROS levels to 50%, indicating that the complexed formulation delivers functional catalase more efficiently into the EA.hy.926 cells. Complexed CAT-LP MgP NP with reduced size has delivered cargo protein more efficiently than encapsulated CAT-LP MgP NP.

Solubility of active pharmaceutical agents is a crucial process that determines drug absorption and ultimately its bioavailability. Many of the new therapeutically beneficial compounds discovered are lipophilic requiring various solubility enhancement strategies to improve their solubility. Among these strategies, solubility enhancement using solid dispersions is a leading method. To obtain a desirable increase in the solubility of a poorly-soluble compound, a good understanding of the molecular descriptors influencing the enhancement of solubility is essential. Therefore, the major research objective was to determine the descriptors which significantly influence the solubility enhancement by solid dispersions. After enhancing the solubility of selected poorly-soluble model compounds, a regression analysis was performed to determine the correlation of molecular descriptors of the active agent, polymer, and solvent with solubility enhancement. The partition co-efficient, hydrogen bonding and solubility parameters of polymer and water were found to influence the aqueous solubility of the poorly-soluble compounds. Aqueous solubility of a compound had an inverse relation with difference in solubility parameters of polymer and water. Similarly partition coefficient was found to be inversely related to aqueous solubility. However for an increase in hydrogen bond acceptors present in pharmaceutical agents increased their solubility, while the higher number hydrogen bond donors resulted in lower solubility. This complexity can be attributed to the contribution of hydrogen bonding in a crystal lattice and in aqueous environment. In conclusion, the contribution of partition co-efficient, solubility parameter and hydrogen bonding were found to be significant for a given set of poorly-soluble model compounds selected with a wide range of descriptors. Several models estimating aqueous solubility of compounds have been employed as screening tool in drug development process. However, all such models were developed to estimate aqueous solubility of pure active agents. Hence, the second research objective was to develop a model that could estimate aqueous solubility of Active Pharmaceutical Ingredient (API) in solubility-enhanced solid dispersions. Using multiple linear regression, a computational model was developed using the molecular descriptors of poorly-soluble compound, polymer and water. S=(2.02*HBA)-(3.37*??)-(11.56*log?P )-(0.9*HBD)+119.66 The model showed a regression (R2) value of 0.858. Upon validation, the model estimated the aqueous solubility of 79% of the compositions evaluated with within 20% variability.

Purpose. The fate of an orally administered drug is dependent on many parameters before it can reach the systemic circulation, including drug absorption and first-pass metabolism in the gut and the liver. Mammalian cells lines such as MDCK and Caco-2 are commonly employed to assess drug permeability but they lack or have low expression level of drug metabolism enzyme expression such as CYP3A4, which contributes to significant first-pass gut and liver metabolism for many drugs. Consequently, these cell lines are not sufficient to integrate metabolism when assessing drug absorption. Here, we tested MDCK and IEC-18 cells transiently over-expressing CYP3A4 as models that can simultaneously assay a compound's permeability and metabolism potential in a single experiment. Method. A recombinant adenovirus carrying the hCYP3A4 cDNA was constructed according to Stratagene's AdEasy XL Adenoviral system. This adenovirus was used to transiently transfect hCYP3A4 into MDCK and IEC-18 cells. Western blot was performed to assess the level of hCYP3A4 expression in the wild type and CYP3A4 over-expressing IEC-18 and MDCK cells. In situ metabolism and transport studies were performed with wild-types and IEC-18-3A4 or MDCK-3A4 cells. Results. The amount of CYP3A4 present in MDCK-3A4 cells was 250 times to that of wild type cells which 1/4th the amount present in human liver microsomes. The amount of CYP3A4 present in IEC-18-3A4 cells was 150 times to that of wild type cells which 1/6th the amount present in human liver microsomes. In metabolism studies, there was higher formation of metabolites in cells transfected with hCYP3A4 compared to controls. In addition, apical to basal transport studies of several drugs in IEC-18-3A4 and MDCK-3A4 showed increased appearance of metabolites compared to the wild-type cells. Conclusions. This model may be a useful to assess the extent of drug absorption into systemic circulation after oral administration.

Vitamin A metabolites and retinoids may slow the progression of breast cancer and elicit anti-neoplastic properties similar to those of omega-3 fatty acids. Studies using animal models show a decrease in the incidence, growth and metastisis of mammary tumors in the presence of specific fatty acids. This effect is also seen with use of retinoids, specifically all-trans retinoic acid (AtRA). Thus, fatty acids may also alter retinoid homeostasis in mammary carcinoma cells (MCF-7s). The potential for inter/co dependency among fatty acids and retinoids is considerable, and here it has been hypothesized that a decrease in cancer progression will occur in the presence of both compounds. MCF-7’s were seeded in a 48 well plate at 5,000 cells per well. After 24 hr, cells were treated with either 1 µM AtRA alone, fatty acids alone, or AtRA + fatty acids. Fatty acid treatments (Linoleic, and Linolenic) were administered at 2.5 uM concentrations. Each fatty acid treatment was also combined with 1 µM AtRA to determine if there is a synergistic effect on slowing cell growth. Both culture media and treatments were changed at 24 hour intervals over a 3 day trial. When compared to the controls, cells treated with 1 µM AtRA or 2.5 µM Linolenic acid both inhibited cell growth. Interestingly, when combined with Linolenic acid, AtRA treatment resulted in a significant (nearly 50%) additional growth inhibition when compared to treatment with AtRA alone. Our results suggest that AtRA and Linolenic acid have a inter/co dependency that significantly inhibits breast cancer cell growth in vitro by 73.4 % compared to control, and 49.7% compared to AtRA alone over 72 hours. We conclude that AtRA and linolenic acid have a combined effect in breast cancer cell proliferation in-vitro and their role in dietary prevention warrants further investigation.

Le manque de produits pharmaceutiques destinés à la population pédiatrique est un problème auquel sont confrontés les professionnels de la santé. Les pharmaciens doivent fréquemment se tourner vers les médicaments destinés aux adultes afin de fournir aux jeunes patients les traitements adéquats. L’utilisation de préparations magistrales pour adapter les médicaments homologués aux besoins de la population pédiatrique reste, encore à ce jour, l’option la plus souvent utilisée. Dans ce mémoire, nous proposons le développement et l’évaluation de nouvelles formulations pharmaceutiques destinées à la population pédiatrique afin de bonifier les options thérapeutiques mises à la disposition des professionnels de la santé. De plus, nous avons exploré l’utilisation de nouvelles techniques spécialisées pour surmonter des défis analytiques rencontrés, ultimement dans le but de déterminer en toute confiance la stabilité et la sécurité de ces nouvelles formulations. La première étude visait à évaluer la stabilité de préparations magistrales de chlorhydrate de clonidine (20 µg/mL) préparées avec des comprimés dans le véhicule commercial Ora-Blend. Les formulations embouteillées ont été conservées à 25°C/60% RH pendant 90 jours. Les défis analytiques rencontrés lors de l’analyse de la stabilité chimique ont été surmontés par l’implémentation d'une nouvelle méthode d'extraction en phase solide, ayant permis d’optimiser la quantification du chlorhydrate de clonidine, se retrouvant qu’en très faible quantité dans les formulations orales. L'absence d'instabilités physiques, évaluée par des mesures qualitatives et quantitatives, et l’absence d'instabilités chimiques, mise en évidence par une méthode HPLC-UV indicatrice de stabilité, confirment qu’accorder une date de péremption de 90 jours à ces préparations magistrales serait approprié. La deuxième étude portait sur l’évaluation de la stabilité de préparations magistrales d’hydroxyurée (100 mg/mL) dans l’Ora-Blend. Dans le cadre de cette étude, différentes méthodes de préparation (mortier, mélangeur, QuartetRx) et différentes sources de principe actifs (poudre, contenu des capsules, capsules entières) ont été étudiées. Toutes les formulations ont été conservées à 25°C pendant 90 jours dans des bouteilles et 14 jours dans des seringues orales. Le développement d'une méthode HPLC indicatrice de stabilité impliquant la dérivation de l’hydroxyurée par le xanthydrol aura permis la rétention l’hydroxyurée sur une colonne à phase inverse de type C18. Plus de 90.0 % de la concentration initiale d’hydroxyurée a été conservé tout au long de l’étude, et ce, pour toutes les conditions testées. L’évaluation visuelle des préparations n’a révélé aucun changement au cours de l’étude de stabilité. Des changements de pH allant jusqu'à 1.6 unités ont toutefois été observés après 90 jours d’entreposage et ont mis en lumière une voie de dégradation de l’hydroxyurée, générant ultimement l’ion ammonium. Ce dernier a été quantifié et les concentrations mesurées, définies comme acceptables. Les résultats ont montré que toutes les formulations d’hydroxyurée étudiées sont demeurées stables jusqu’à 90 jours à 25°C. Pour terminer, une étude exploratoire ayant pour but d’évaluer des comprimés à croquer à saveur d’érable a été réalisée. Le sucre d’érable et un arôme naturel d’érable ont été ajoutés à la composition des comprimés afin d’obtenir une saveur suffisamment prononcée pour masquer le goût amer de l’acétaminophène. Une étude de stabilité préliminaire, impliquant une période de 30 jours d’entreposage dans des conditions de stabilité accélérées (40°C/75%RH), aura permis d’explorer les propriétés physico-chimiques de cette nouvelle formulation, de soulever les défis potentiels et de générer des hypothèses en lien avec l’augmentation de dureté observée après seulement 14 jours d’entreposage. Les résultats de cette étude préliminaire serviront de point de départ pour le futur développement de produits pharmaceutiques à la saveur du Québec. Les techniques utilisées et les études réalisées dans le cadre de ce projet de maîtrise auront permis de générer des résultats robustes qui pourront être utilisés par les professionnels de la santé. Ces informations seront pertinentes à la pratique pharmaceutique et permettront d’offrir à la population pédiatrique des nouvelles options de traitement sécuritaires et efficaces. / The lack of pharmaceuticals intended to the pediatric population is an issue facing healthcare professionals. Pharmacists must frequently resort to adult treatments to provide adequate treatment to young patients. The use of compounding to adapt commercial drugs to the needs of the pediatric population is still, to this day, the most considered option. In this master’s thesis, we propose the development and evaluation of new medicinal preparations for pediatrics in order to improve and diversify the therapeutic options available to healthcare professionals. In addition, we have explored the use of new specialized techniques to overcome analytical challenges encountered, with the goal of confidently determining the stability and safety of these new formulations. The first study aimed to assess the stability of compound preparations of clonidine hydrochloride (20 µg / mL) prepared with tablets in the commercial vehicle Ora-Blend. Bottled formulations were stored at 25°C/60% RH for 90 days. The analytical challenges encountered during the analysis of chemical stability were overcome by the implementation of a new method of solid phase extraction, which allowed to optimize the quantification of clonidine hydrochloride, present in very small amount in oral formulations. The absence of physical instabilities, assessed by qualitative and quantitative measurements, and the absence of chemical instabilities, as demonstrated by a stability indicating HPLC-UV method, confirm that it would be appropriate to grant a 90-day expiration date to these compounded oral liquids. The second study evaluated the stability of compound preparations of hydroxyurea (100 mg / mL) in Ora-Blend. In this study, different preparation methods (mortar, mixer, QuartetRx) and different sources of hydroxyurea (powder, content of capsules, whole capsules) were studied. All formulations were stored at 25°C for 90 days in bottles and 14 days in oral syringes. The development of a stability indicating HPLC method involving the derivatization of hydroxyurea by xanthydrol will have enabled hydroxyurea retention on a C18 type reverse phase column. Over 90.0% of the initial hydroxyurea concentration was recovered throughout the study under all conditions tested. Visual evaluation of the preparations did not reveal any changes during the stability study. Changes in pH of up to 1.6 units were observed after 90 days of storage and revealed a degradation pathway for hydroxyurea, ultimately generating ammonium ion. The latter was quantified, and the measured concentrations defined as acceptable. The results showed that all hydroxyurea formulations studied were stable for up to 90 days at 25°C. Finally, an exploratory study to evaluate maple flavored chewable tablets was carried out. Maple sugar and a natural maple flavor have been added to the composition of the tablets to achieve a flavor strong enough to mask the bitter taste of acetaminophen. The pre-stability study, involving a period of 30 days of storage under accelerated stability conditions (40°C/75% RH), will have made it possible to explore the physicochemical properties of this new formulation, to raise the potential challenges and generate hypotheses related to the increase in hardness observed after only 14 days of storage. The results of this preliminary study will serve as a starting point for the future development of pharmaceutical products with a Quebec flavor. The techniques used and the studies performed will have generated robust results that could help healthcare professionals in their practice.

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