Hu, Ming2020-01-032020-01-03December 22017-12December 2https://hdl.handle.net/10657/5688Statement of the Problem: Sanfilippo syndrome or mucopolysaccharidosis type III (MPS III), a type of lysosomal storage disease, is a rare genetic disorder inherited in an autosomal recessive manner. Individuals affected by this disease lack the ability to produce one of the four enzymes responsible for the lysosomal degradation of heparan sulfate (HS). Heparan sulfate is thus accumulated in virtually every cell of the body leading to their progressive damage. The central nervous system, in particular, is affected by the accumulation of heparan sulfate, causing severe neurological impairments such as neurodegeneration and neuroinflammation. Currently, no effective therapy is available for treating MPS III. Genistein, a dietary soy isoflavone, has been observed to effectively reduce the production and reduce or prevent further accumulation of heparan sulfate in lysosomes in vitro as well as in vivo. However, in vivo reduction of heparan sulfate was seen only in the peripheral tissues but not observed in the CNS of MPS III mouse model. Low oral bioavailability, extensive metabolism, enterohepatic recycling and efflux transporters are few factors that are thought to be responsible for poor penetration of genistein into the brain. We aim to develop strategies to increase genistein concentration in the CNS, decrease the level of heparan sulfate in the CNS and thus effectively treat MPS III. Towards our goals, two major specific aims were proposed: (1) To improve accumulation of genistein in brain and (2) To evaluate the efficacy of genistein formulation. Methods: Genistein nanosuspensions were prepared by wet milling technique and characterized in order to select the most stable and uniform formulation with minimal particle size. Transcellular transport studies were carried out using Caco-2 monolayer to compare transport and permeability of unformulated genistein with genistein nanosuspension at a concentration of 200 M. Pharmacokinetic studies were carried out on 5 groups of FVB mice (N=4) for different oral doses of unformulated genistein (5 and 50 mg/kg) and genistein nanosuspension (5, 50 and 200 mg/kg). Blood samples were analyzed using a previously validated LC-MS/MS method. Single oral dose and multiple oral dose brain distribution studies were carried out in 4 groups of FVB mice and 3 groups of C57BL mice, respectively, at 200 mg/kg. Genistein concentration in brain samples were quantified by our developed and validated LC-MS/MS assay. A highly specific, sensitive and reproducible UHPLC-MS/MS method for rapid detection and analysis of heparan sulfate in murine brain tissue was also developed with successful ability to quantify HS levels in MPS IIIB mice. Results: We were able to prepare a stable and uniform genistein nanosuspension, which was able to enhance the oral absorption and increase the brain uptake of genistein. The brain distribution of the formulation was evaluated using the UPLC-MS/MS method we developed and validated for quantification of genistein in mouse brain matrix. Consecutive once a day dosing of nanosuspension resulted in higher distribution of genistein in mouse brain. The heparan sulfate polysaccharide was chemically derivatized to its disaccharide form which was quantified using UPLC-MS/MS. The measurements of HS were normalized to a spiked deuterated HS internal standard. Chromatographic separation of the derivatized products were achieved on a Biphenyl column using 10 mM ammonium acetate in water and 10 mM ammonium acetate in 100% methanol as mobile phases with a gradient elution. The assay was determined to be linear over a concentration range of 9.765625 - 625 g/mL with 9.765625 g/mL as the LLOQ. The intra- and inter-day accuracy and precision were determined to be within 20% in brain homogenate. HS was also tested for stability under various storage conditions such as bench-top stability (at 25C for 4 hours), three freeze-thaw cycles stability and auto-sampler stability (at 10C for 24 hours) with stability values within 20% range of nominal concentrations. Conclusions: We believe that our genistein formulation would be effective in treating Sanfilippo syndrome. Additionally, our strategy would find application in enhancing the CNS delivery of drugs which can be used to treat other CNS conditions whose potential has been hampered by the blood brain barrier. A straightforward, highly specific, sensitive and effective UPLC-MS/MS method for quantifying heparan sulfate in the brain matrix was devised. The validated assay was further applied for comparison and quantification of HS in brain tissues of wild type and MPS IIIB mice.application/pdfengThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).Mucopolysaccharidosis type IIIGenisteinNanosuspensionsCNSBrainHeparan sulfateLC-MS2020-01-03Thesisborn digital