Dissertation (Ph.D.)--University of Michigan

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Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression.

The thesis work entails a bench-to-bedside translational research approach to the development of pediatric fixed dose combination of zidovudine/lamivudine/nevirapine (60/30/50mg) orally disintegrating tablets. A simple and cost-effective, direct compression method was used. Preformulation studies that included analytical and bio-analytical assay development, excipient selection and characterization of drug-excipient interaction for initial formulation were conducted. Response surface methodology was utilized to optimize the formulation in terms of disintegration time and crushing strength. Stable ODT tablet was developed with desired friability (< 1%), reasonable crushing strength, disintegration time (< 30sec) and other quality attributes such as potency and dissolution. An open label randomized two-way cross-over bioequivalence of the product (with approved IRBs), conducted in 24 healthy adult volunteers, indicated the product to be bioequivalent with the innovators. 90% C.I of the point estimates of PK parameters evaluated were in the range of 80-125% as specified by FDA. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmaceutics / MS; / Thesis;

Gels are popular dosage forms.  This topical dosage form may be advantageous compared to oral or parenteral dosage forms. Favorable rheological or bioadhesive properties of gels might provide extended contact times at the site of administration compared to aqueous solutions. However, due to the high water content of gels, these are usually quickly emptied of the drug substance. One way of prolonging the drug release from gels is to contain the drug substance in catanionic aggregates in the gel. These aggregates are formed in solutions of oppositely charged surfactants and a drug can be used instead of one of the surfactants.   In this thesis catanionic aggregates composed of drug substances and oppositely charged surfactants were studied and the possibility to use these aggregates for the purpose of prolonged drug release was investigated. The formation of catanionic aggregates when using drugs was found to be a common occurrence in addition to which, the oppositely charged surfactant can be varied and surfactants of natural origin with a low toxicity were used. Most combinations tested rendered either vesicles or elongated micelles. When the catanionic aggregates were contained in gels the drug release was substantially prolonged. The apparent diffusion coefficients were lowered 10-100 times compared to the reference gels. When gels with catanionic vesicles with substantial prolonged drug release were applied to skin the penetration rate was lowered extensively. No morphological differences were observed between skin samples that had been exposed to formulations containing catanionic aggregates and skin samples exposed to saline solution, air or formulations containing only the drug. Both conventional, covalently linked pre-formed gels and physical gels, where the catanionic vesicles form the cross-links upon interaction with the polymer, can be used for these purposes. When the effect of drug release on aggregate structure was studied, it was shown that vesicles are present in both conventional and physical gels throughout the drug release process.   This thesis shows that catanionic aggregates contained in gels can present an advantageous formulation strategy to prolong the drug release, thereby improving the efficiency of gel formulations.

Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression.

Islet transplantation holds vast potential as a treatment for type 1 diabetes mellitus and provides recipients with short term insulin independence. A major limitation of this treatment is the lack of donor beta (β) cells available for transplantation. Significant progress has been made with stem cell differentiation protocols; current methods have generated cell populations which possess a functioning β cell phenotype. However, these cells are not suitable for clinical transplantation. Two dimensional cell culture systems do not accurately mimic the complexity of the in vivo pancreatic environment, reducing the effectiveness of current β-cell differentiation protocols. The paradigm shift into three dimensional tissue culture provides an attractive area of investigation, and the use of three dimensional culture methods has improved growth in a variety of cell types ex vivo. The mass culture of β cell analogues on a 3D biomimetic environment is now necessary, and may offer a new platform on which an alternative source of transplantable cell populations can be differentiated and cultured successfully. This thesis aims to develop and condition BioVyon™, a high density polyethylene (HDPE) based biomaterial, for use as a mass β cell cultivation system In order to achieve this, a number of objectives will need to be met: (I) Complete characterisation and assessment of all properties of Biovyon™ that have a direct influence on cell culture, (ii) modification of the BioVyon™ surface chemistry to promote cell adhesion and growth, (iii) absorbance of proteins to mimic the pancreatic environment and aid proliferation of the Min-6 cell line, (iv) assessment of the Min-6 cell line phenotype after extended period of culture on the modified BioVyon ™ environment. Scanning electron microscopy and Atomic Force Microscopy were used to characterise the surface of the HDPE material post plasma etching. Advancing and receding (ARCA) and Fourier Transform Infrared Spectroscopy were used to analyse the elemental changes to the polymer surface. The HDPE biomaterial was conditioned using plasma etching, subsequent adhesion and growth of Min-6 cells was quantified using a lactate dehydrogenase assay. Min-6 populations were seeded at a density of lxl0/6i on BioVyon™ and tissue culture plastic of comparable surface area, and analysed after extended periods of growth. An insulin EUSA was used to quantify insulin released by populations of β cells at different time points witin the BioVyon™ in response to fluctuating glucose concentrations. The results obtained in this thesis indicate that BioVyon™ offers an appropriate structural environment for cell culture. Pore size and frit dimensions allow for cell infiltration and the effective diffusion of oxygen and nutrients. Plasma etching incorporated oxygen groups and a novel surface topography that improved cell adhesion and growth. β-cell phenotype was protected and sustained in cell populations cultured within the BioVyon™ environment. In conclusion, BioVyon™ can be conditioned to function as an effective 30 cell culture system. Modification of the surface chemistry has enabled BioVyonTl • to harbour and sustain large populations of the Min-6 cell line. The protected β-cell phenotype in the Min-6 populations suggests BioVyon™ could hold potential as a stem cell culture and differentiation platform.

Complement activation-related pseudoallergy (CARPA) is a term refers to those hypersensitivity reactions where the allergen can activate complement. This study examined the effect of nanomedicines and polyethylene glycol on complement activation. Single-walled carbon nanotubes, which have many medical applications as drug delivery systems, activated human complement independently of the C1q-dependent classical and the alternative pathways, as reflected by a significant rise in serum levels of S-protein bound form of terminal complex (SC5b-9) and C4d. This study also shows that polyethylene glycol (PEG) with different molecular weights was capable of activating complement. It was found that molecular weight of PEG plays an important role in activating both calcium sensitive and alternative pathways of the human complement system, with relatively higher molecular weight polymer resulting in more activation to a certain extent.

Purpose: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients have shown to enhance bioavailability of several reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of specific monoglycerides on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. Methods: A preliminary study was first conducted to determine the effect of Peceol®, a mono- and di-glyceride mixture, on MPR2 efflux activity. Then, the 24- hour non-cytotoxic ranges of specific monoglycerides (1-monopalmitin, 1- monostearin and 1-monoolein) were determined using MTS and LDH assays in Caco-2 cells. Then, the effects of chosen monoglycerides on the functional activity of MRP2 were assessed via rhodamine 123 (Rh123) accumulation and estradiol 17 β-D-glucuronide(E₂17βG) bidirectional transport studies. The dose responses of Rh123 accumulation with each monoglyceride treatment were also determined. Lastly, Western blotting was used to probe the monoglycerides effect on MRP2 protein expression. Results: In the preliminary study, significant increase in Rh123 accumulation and decrease in E₂17βG efflux ratio were observed in Peceol® treated cells. The non-cytotoxic concentration ranges for 1-monopalmitin, 1-monostearin and 1- monoolein were within 1 mM, 1 mM and 500 μM, respectively. Cells treated with 1 mM 1-monoplamitin, 1 mM 1-monostearin, 500 μM 1-monoolein and 50 μM MK571 (a MRP2 inhibitor) resulted in significant increases in Rh123 accumulation and decreases in E₂17βG efflux ratio compared to the control (medium treated only). The three monoglycerides did not show Rh123 accumulation in a dose-responsive manner. MRP2 protein expressions in 1- monopalmitin and 1-monoolein treated cells were decreased by 19% and 35%, respectively; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. Conclusions: These findings suggested that 1-monoolein, 1-monostearin and 1- monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1- monoolein treatment could be partially explained by the non-specific down regulation of MRP2 protein expression. / Pharmaceutical Sciences, Faculty of / Graduate