published_or_final_version / Nursing Studies / Master / Master of Nursing

The purpose of this project was to fabricate biodegradable ophthalmic inserts for controlled delivery of pilocarpine and evaluate them by both in-vitro and in-vivo studies. Emphasis was placed on the use of an inexpensive material as a drug carrier and on the ease of fabrication of the device. Based on these criteria, absorbable gelatin was selected to fabricate a matrix system. Absorbable gelatin can be obtained by either thermal treatment or chemical crosslinking of gelatin. In the first part of this project, we fabricated an insert using Gelfoamᴿ, an absorbable gelatin sponge obtained by thermal treatment. A prolonged in-vitro release of pilocarpine from the device was achieved through pharmaceutical modification by embedding a retardant in the pores. The devices impregnated with polyethylene glycol monostearate (PMS) and cetyl esters wax (CEW) were found to be most effective. The in-vivo evaluation of the devices indicated that pharmaceutical modification of Gelfoamᴿ is an effective means of improving the biological activity of pilocarpine without altering the biodegradability of the biopolymer backbone. The CEW device produces a substantial improvement in drug bioavailability and an increase in the duration of biological effect over that from the two commercial formulations, the eyedrop and the gel. In the second part of the project, we fabricated absorbable gelatin inserts through chemical crosslinking of gelatin. The effect of selected fabrication variables on profiles of the in-vitro release of pilocarpine and the dynamic water uptake by the crosslinked gelatin devices was investigated. These results were further substantiated by the measurement of the degree of crosslinking of gelatin. The in-vivo study indicated that the modification of the structure of gelatin by crosslinking is another simple and effective way of improving bioavailability and extending the duration of effect of pilocarpine incorporated in the biopolymeric device. In addition, altering the degree of crosslinking of gelatin allows a variation of the biodegradation time of the polymer.

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The effect of triacetin in combination with common cosolvents on the solubility of phenytoin and diazepam was studied. The cosolvents were PEG 400 and propylene glycol. In addition, the data were used to test the following model: UNFORMATTED EQUATION FOLLOWS: log Sᵈ(c,p,w) = log Sᵈ(w) + f(c)σᵈ(c) + [Sᵖ(w)10(f(c)σᵖ(c))/D(p)] σᵈ(p). UNFORMATTED EQUATION ENDS. The term on the left side of the equation is the solubility of a drug in the ternary system. This is related to the aqueous solubility of the drug, the solubility of the drug in a completely miscible organic solvent (CMOS), and the solubility of the partially miscible organic solvent (PMOS). This model was proposed by Gupta et al. (1989) and predicts the solubility of a ternary system composed of a CMOS and PMOS. The results indicate the triacetin does increase the solubility of the two poorly water-soluble drugs. There is good correlation between the observed and predicted increase in the solubility of the drugs.

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In Indiana, Ixodes scapularis is an important tick in public health because it feeds on a variety of hosts including humans, and transmits Borrelia burgdorferi (Lyme disease), Anaplasma phagocytophilum (human granulocytic ehrlichiosis), and Babesia microti (babesiosis). Symbiotic, non-pathogenic Rickettsia found in Ixodes scapularis may play a role in excluding pathogenic species of Rickettsia from being transovarially transmitted. In order to investigate this idea further in Indiana, a total of 378 adult I. scapularis from 4 different counties (Jasper, Pulaski, Newton and Starke) were tested by polymerase chain reaction analysis (PCR) for the presence of Rickettsia sp. Four positive samples from the PCR (using Rocky Mountain spotted fever group specific primers to target the rOmpA gene; Rr190.70p and RH 90.602n) reactions were sequenced to verify identity. These four samples matched closest to the reference number AB002268 from GenBank which describes, I. scapularis endosymbiont DNA for rOmpA. A total of 62 engorged females were tested; 53 (85.5%) harbored the rickettsial symbiont. A total of 41 questing females were tested; 33 (80.5%) were positive. Of the 249 males tested, 14 (5.6%) were positive. A restriction digestion on some of the positive samples revealed that the 1 scapularis symbiont was different from R. montana and R. rickettsii. The second goal of this study was to identify the presence of B. microti. In I. scapularis ticks, this would be the first time this pathogen was identified in Indiana. To accomplish this goal 106, ticks were tested using the primers Babl and Bab4, which target the 18S rRNA gene specific for B. microti. Three tick samples were found to harbor B. microti as determined by sequencing. However, sequencing of amplification band in the negative control also yielded B. microti. Thus, the presence of B. microti in Indiana ticks could not be confirmed. A negative control was also sequenced and was identified as Babesia microti indicating that there was a contamination so it is not possible to conclude that B. microti was found in Indiana ticks. / Department of Physiology and Health Science

The multidrug resistance associated proteins (MRP(human)Mrp (rat) are ATP-dependent transporters responsible for the efflux of a wide range of substrates, including endogenous compounds e.g. bilirubin, drug metabolites e.g. paracetamol glucuronide and fluorescent dyes e.g. 5 (and 6)-carboxy-2’,7’-dichlorofluorescein (CDF). Mrp1-6 (<i>abccl-6)</i> are expressed in rat liver, with Mrp2 being expressed at the highest level. Isolation of hepatocytes by <i>in situ</i> collagenase perfusion causes bile canalicular disruption and internalisation of Mrp2. Cells cultured in a sandwich configuration of Matrigel or collagen (Type 1) showed bile canalicular reformation at different days in cell culture, depending on the extracellular matrix and time of overlay. We have developed a method for quantifying Mrp-mediated efflux in hepatocytes cultured in a sandwich configuration of collagen (Type 1). This method is unique in its ability to distinguish between sinusoidal efflux, canalicular efflux and diffusion in intact hepatocytes. Alternative <i>in vitro</i> models of Mrp2-mediated efflux include the vesicular (direct and indirect methods) and the ATPase assays. We have used these assays to identify atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin as substrates of human and rat MRP2/Mrp2. A close correlation was seen between the kinetic parameters of transport of the Mrp2 substrate; CDF determined in sandwich cultured rat hepatocytes using the method above (Km = 3.5 – 9.9 μM), vesicle preparations (Km = 37.9 μM) and membrane preparations (Km = 18.7 μM). We also present data to implicate the nuclear receptors, PXR, CAR and FXR in the regulation of <i>abcc2</i> and <i>abcc3</i> and PXR and CAR in <i>abcc1</i> gene regulation. <i>Abcc2 </i>and abcc5<i> </i>are also up-regulated in response to a toxic insult to the cell, probably via Nrf2 activation, suggesting a role in cell defence.