▼ Sulfonation is an important reaction in regulating the biological activities of a variety of endogenous and environmental compounds and is catalyzed by cytosolic sulfotransferases that use PAPS (3'-phosphoadenosine-5'-phosphosulfate) as the sulfonate (SO3-) donor. SULTs are present in mammals and other vertebrates and play an important role in the detoxification of xenobiotics especially environmental estrogens. SULTs are also involved in the biotransformation of endogenous compounds (hormones, steroids) which might be a mechanism for maintaining the homeostasis of these compounds in-vivo. In this study we have attempted to assess the role of the hydroxysteroid sulfotransferase SULT2 ST2 in zebrafish (Danio rerio) development by knocking down the expression of the enzyme using a morpholino. Zebrafish embryos were microinjected with a morpholino which had a sequence complementary to the sequence of the SULT2 ST2 gene. The injections were done when the embryos were in the 1-4 cell stage with three concentrations of the morpholino: 0.5 ng/nL, 1.0 ng/nL and 2.0 ng/nL. The embryos were then observed for survival rates and abnormal phenotypes up to 144 hours post fertilization (hpf). The phenotypes observed were cardiovascular abnormalities such as cardiac edema and irregular heartbeat, abdominal edema, lordosis, notochord deformities, tail deformities and very few cases of craniofacial malformations. At the highest concentration of the morpholino, almost all knockdowns displayed mild to severe cardiac edema which was the specific phenotype at that concentration after the hatching period (48-55 hpf). The extent of knockdown was also determined by western blot experiment and it was found that the knockdown was partial and there was some enzyme present at measurable levels in the embryos. In conclusion, morpholino knockdown of the SULT2 ST2 gene in zebrafish embryos caused several non-specific phenotypes. At the 2.0 ng/nL concentration mild to severe cardiac edema was observed in the most of the knockdowns after hatching which might indicate that SULT2 ST2 has a role to play in the cardiac development of zebrafish that involves maintaining steroid hormone homeostasis. Advisors/Committee Members: Williams, Frederick.

▼ A considerable number of drugs on the market have been shown to have low bioavailability and solubility due to poor dissolution in the gastrointestinal tract. Different approaches have been attempted in the past to enhance the dissolution and oral absorption of drugs. Among these approaches which have been used includes binary eutectic mixture formation between drugs and hydrophilic carriers. A binary eutectic mixture is a mixture of two constituents that solidifies simultaneously when cooled from the liquid state, without changing the composition. In binary mixtures, the poorly soluble drug is dispersed in a hydrophilic carrier resulting in a reduction of particle size which increases the dissolution rate, thus changing the biopharmaceutical properties. In this study, the dissolution rates for tolbutamide and haloperidol in the presence of hydrophilic carriers were investigated using differential scanning calorimetry (DSC), x-ray powder diffraction (PXRD), scanning electron microscope (SEM) and dissolution testing. The physical mixture method was used for the preparation of the eutectic mixtures. A eutectic mixture was formed between 80% tolbutamide and 20% nicotinamide, 55% haloperidol and 45% aminophylline and 80% tolbutamide and 20% niacin. The PXRD results show that the eutectic mixtures are crystalline, stable with no interaction among the constituents. The SEM results show that there is a major particle size reduction which in turn enhances the dissolution rate of the drug. Dissolution studies revealed that significant increases in drug solubility are seen in the eutectic mixtures. Advisors/Committee Members: Alexander, Kenneth.

▼ Eudragit® nanoparticles appear to be a suitable inert carrier for ophthalmic drug delivery due to their capability to form nanodispersion with smaller particle size, positive surface charge, good stability, biocompatibility and absence of any irritant effect on the cornea, iris, and conjunctiva. Sulfacetamide loaded polymeric nanosuspension was prepared from an inert polymer resin (Eudragit® RL 100) with the aim of improving the availability of sulfacetamide at the intraocular level and thereby reducing the frequency of dosing for bacterial infections in the eye. Nanosuspensions were prepared by the solvent displacement method using acetone and 1% (w/v) Pluronic® F108 solution. Drug to polymer ratio was chosen at four levels: 10/100 (B1), 20/100 (B2), 30/100 (B3), 40/100 (B4) (by weight). Characterization of nanosupension was performed by measuring particle size, zeta potential, Fourier Transform infrared spectra (FTIR), Differential Scanning Calorimetry (DSC), Powder X-Ray Diffraction (PXRD), drug entrapment efficiency and in vitro release. In addition, freeze drying, redispersibility and short term stability study at room temperature and at 40C were performed. Spherical, uniform particles (size below 500 nm) with polydispersity index range of 0.414 to 0.67 and positive zeta potential were obtained. Positive surface charge can allow a longer residence time of nanoparticles on the cornea surface, with a consequence slower drug release and higher drug concentrations in the aqueous humor, compared to classical eye drop. No significant drug polymer interaction was observed in the solid state characterization of freeze dried nanosuspension using DSC, PXRD, FTIR. Drug entrapment efficiency was found to be in the range of about 28% to about 35%. In order to increase drug entrapment efficiency, selected batch was chosen to study the effect of changing polymer content, pH of external media and incorporation of polymethyl methacrylate (PMMA) on drug entrapment efficiency. Changing the external phase pH and incorporation of PMMA significantly increased drug entrapment efficiency of nanoparticles. No significant change in average particle size was observed after storage at room temperature and at 40C. Freeze dried nanosuspesnions were easily redispersed after manual hand shaking. Both batch of B3 containing 5% sucrose and 5% mannitol as cryoprotectant exhibited good redispersibility in water. The results indicate that formulation of Sulfacetamide in Eudragit® RL 100 nanosuspension could be utilized as potential delivery system for treating ocular bacterial infections. Advisors/Committee Members: Alexander, Kenneth.

▼ Cytosolic sulfotransferases are phase II drug metabolizing enzymes responsible for carrying out a sulfonation reaction which adds a sulfonyl group to a hydroxyl or amino group of its substrate. These enzymes are believed to be involved with the regulation of endogenous hormones such as neurosteroids as well as xenobiotics. Considering the increasing rates of environmental estrogen exposures, it is important to understand the role that sulfotransferases may play in the early stages of vertebrate development. SULT2 ST1 is a sulfotransferase identified in zebrafish which resembles the SULT2 A1 gene in humans. To establish a link between sulfotransferase expression and developmental toxicity, the SULT2 ST1 gene was knocked down in the zebrafish model. This was accomplished by microinjection of translation blocking morpholino oligonucleotides into <4 cell stage zebrafish embryos. The phenotypic changes observed in the SULT2 ST1 knockdown fish indicate that the absence of the enzyme may be responsible for underdeveloped fin and systemic failure in embryo development phenotypes. Western Blot was used to confirm a reduction in expressed protein in knockdown animals. Advisors/Committee Members: Williams, Frederick.

▼ Sulfation is one of the important Phase II reactions. We identified and characterized two novel zebrafish sulfotransferase SULT1 ST9 and SULT3 ST4. SULT1 ST9 showed strong sulfating activity toward substrates such as acetaminophen and 3 3,3',5-triiodo-L-thyronine. SULT3 ST4 exhibited strong sulfating activity towards substrate DHEA, and weaker activity toward mestranol. pH dependency assays and kinetics assays were used to characterized two enzymes’ interactions with these substrates. Acetaminophen and dextromethorphan are widely used in over-the-counter cough and cold medications. Their efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of acetaminophen and dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis (curving body trunk), non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Moreover, bradycardia (30-70 heart beats/min vs. ~130 heart beats/min for normal control) was observed for the embryos/larvae treated with elevated concentrations of dextromethorphan. Another three sets of zebrafish embryos/larvae were exposed to acetaminophen at 1 hpf, 24 hpf, and 48 hpf, respectively, during embryonic/larval development. Compared with the 24 and 48 hpf sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. .With increasing concentrations of acetaminophen, there was a significant dose-dependent morphological change in the developing embryos with regard to the development of the eyes and swim bladder, and distribution of pigment. There was no noticeable difference among larvae of the 1, 24 and 48 hpf sets in terms of these morphological changes. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, was due to the developmental expression of the Phase II enzymes sulfotransferases involved in the metabolism of dextromethorphan/ acetaminophen warrants further investigation. Advisors/Committee Members: Liu, Ming-Cheh.

▼ The purpose of this research was to formulate pharmaceutically relevant microemulsion systems using dioctyl sodium sulfosuccinate (DOSS) as the surfactant. Visually clear microemulsions were identified by titrating mixtures of DOSS and oil with water. The maximum amount of water incorporated in surfactant/oil mixtures was plotted in a ternary phase diagram. Based on the information from phase diagrams DOSS/Ethyl oleate (EO) and DOSS/Crodamol PMP (PMP) mixtures were selected for further studies. Microemulsions were characterized using polarized light microscopy, electrical conductivity, rheology, and dynamic light scattering (DLS). The effects of pH and the addition of acyclovir on the microemulsion-forming compositions were investigated. Anticipating an ophthalmic or parenteral delivery method for the formulations, an aseptic filtration method for sterilization was developed using membrane filtration. This method was validated via filtration and direct inoculation by plating on blood agar and using E. coli as the positive control. The safety of the formulations on NIH 3T3 cells was studied using a neutral red assay and validated using a Bradford protein assay. Dissolution studies were performed on microemulsions containing methylene blue to evaluate the drug release profile from the microemulsion system. A colorimetric estimation method was used to determine the amount of dye released from the formulation over a 72-hour period. DOSS/EO and DOSS/PMP mixtures were able to emulsify a maximum of 13.7% and 29.7% RO water. No birefringence was observed in the microemulsion formulations. Low electrical conductivity values of approximately 0.05 μS/cm indicated the existence of water-in-oil microemulsions. The conductivity studies demonstrated a “percolation phenomenon” in the formulations when the concentration of water exceeded a threshold value. DOSS/EO microemulsions exhibited Newtonian flow and viscosities of approximately 9 CP. Particle sizes for these DOSS/EO/water and DOSS/PMP/water systems were less than 30 nm in size (mean volume weighted diameter) as determined by DLS. The pH of the buffer influenced the total quantity of buffer that could be incorporated into microemulsion systems. Formulations filtered aseptically were free of bacteria when gram-stained and visualized under a microscope. The filtration method of sterilization was validated by the absence of microbial growth on blood agar plates over a 14-day period. All DOSS/EO microemulsion formulations showed no toxicity to 3T3 cells, except for samples with concentration of 1:15 and Wo values of 10 and 15. Acyclovir showed little effect on microemulsion formulation as similar percentages of acyclovir solution were incorporated into the DOSS/EO and DOSS/PMP mixtures as RO water. In vitro dye release studies demonstrate controlled release of the model drug over a 72-hour time period. Stable microemulsion formulations were prepared using DOSS/EO and DOSS/PMP mixtures. Development and validation of an aseptic filtration method of sterilization shows potential for these formulations to be used as parenteral or ophthalmic preparations. Successful incorporation of an anti-viral drug into the formulations containing pharmaceutically relevant components also shows potential for various drug delivery applications. Advisors/Committee Members: Nesamony, Jerry.