Browsing by Subject "Experimental & Clinical Pharmacology"

Now showing 1 - 3 of 3
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    Development of an Analytical Assay for the Determination of Vinpocetine’s Primary Metabolite, Apovincaminic Acid, in Human Plasma
    (2012-04-18) Gao, Robert; Paz, Matt
    Vinpocetine (VP) is a derivative obtained from (+) vincamine, and is commonly extracted from periwinkle. It has been shown to alleviate risks associated with stroke by enhancing cerebral blood flow. VP is also useful for the treatment of epilepsy and for the prevention of dementia. Current research is examining whether VP’s therapeutic effect is due to its active metabolite, AVA.
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    Development of intravenous topiramate for neuroprotection and seizure control in neonates.
    (2011-04) Clark, Annie Marie
    Hypoxic-ischemic brain injury in newborns is a significant medical problem with a high mortality rate, grave neurological sequelae including impaired cognition, neonatal seizures, and serious treatment-related adverse effects that can cause further brain injury. A safer, more effective treatment for neonatal seizures combined with the potential for neuroprotection would represent a significant advancement in the care of babies with hypoxic-ischemic brain injury. Intravenous topiramate holds the promise of controlling seizures and providing neuroprotection in newborn babies, but its safety and dosing must first be established in adults and, possibly, older infants and children. When designing a new treatment option for neonatal seizures it is important to understand the developmental differences in the pathophysiology of seizures in the immature brain compared to the adult brain. Developmental age-specific mechanisms exist that alter the generation of seizures, the effect of seizures on the brain, and the effectiveness and impact of antiseizure therapy. Differences in the expression and activity of excitatory and inhibitory pathways in the developing brain may explain why many of the traditional antiepileptic drugs used to treat neonatal seizures are ineffective. The research presented in my dissertation is focused on defining the safety and pharmacokinetics of intravenous topiramate. Topiramate is an antiepileptic drug used in adults and children to treat epilepsy. Recent research has shown topiramate is highly effective in controlling seizures and is neuroprotective in newborn laboratory animals in models of status epilepticus and cerebral ischemia. The proven safety and effectiveness of topiramate for seizures in older children and adults together with substantial laboratory evidence showing benefit in models of hypoxic-ischemic encephalopathy strongly suggest that topiramate would be useful in the treatment of neonatal seizures and in addition might provide neuroprotection resulting from hypoxic-ischemic insult. The studies included in this thesis include an animal study, a Phase I study in patients with epilepsy and migraines, and a healthy volunteer pharmacokinetic and safety study. The goal of the animal study was to determine plasma topiramate concentrations in rat pups given doses previously shown to result in neuroprotective effects. In that study, the neuroprotective dose produced concentrations slightly above the proposed therapeutic range (5 to 20 µg/mL), while the non-neuroprotective dose produced concentrations approximately twice as high as the therapeutic range for topiramate when used to treat epilepsy. Results from this study now provide target concentrations for future neuroprotection studies. Results for the two human studies included in this thesis provide previously unreported information about topiramate. In adults, topiramate plasma concentrations attained by intravenous infusion were very similar to oral administration. The determination that the oral absorption is approximately 100% indicates patients can be given the same dose when switched from intravenous to oral, or vice versa. The studies also revealed an extended elimination half-life of topiramate indicating it can be given once or twice daily in some patients while maintaining targeted plasma concentrations. Intravenous infusion of doses of 25 mg to 100 mg over 10 to 15 minutes appears to be safe. No serious adverse events were reported by subjects following intravenous or oral administration of topiramate. Subjects reported no local discomfort due to administration of the intravenous formulation. Reported side effects were generally mild and resolved by 4 hours regardless of route of medication. Topiramate is known to have cognitive and neurological adverse events. Onset of cognitive adverse events and ataxia occurred early post-infusion, demonstrating the intravenous infusion may have quick penetration into the brain. For the treatment of neonatal seizures, in which a fast onset of action is required, rapid penetration into the brain is beneficial. Ideally, a rapid reduction of the duration and frequency of seizures should minimize the long-term neurodevelopmental adverse outcomes that occur after neonatal seizures. These studies provide pharmacokinetic and safety data needed to begin studies in younger patients. Results from these studies will inform the design of subsequent studies, including controlled clinical trials intended to determine the efficacy and safety of intravenous topiramate for neuroprotection and seizure control in neonates.
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    Staphylococcal toxic shock syndrome Toxin-1 interactions with human vaginal epithelial cells and novel therapeutics.
    (2011-01) Schaefers, Matthew Michael
    Staphylococcus aureus is a significant human pathogen that causes a wide range of diseases from skin and soft tissue infections, pneumonia, osteomyelitis, to toxic shock syndrome (TSS). S. aureus initiates infections at skin and mucosal surfaces by producing a multitude of virulence factors, including superantigens (SAgs). SAgs are exotoxins that enhance the ability of S. aureus to cause infection by dysregulation of the host’s immune system. Numerous Staphylococcal SAgs have been identified including the staphylococcal enterotoxins (SEA- SEU) and Toxic Shock Syndrome Toxin-1 (TSST-1). SAgs cause a wide range of diseases such as food-poisoning, atopic dermatitis, and TSS by relatively unknown mechanisms of action on mucosal surfaces. The ability of SAgs to non-specifically cross-link T-cells and antigen presenting cells (APC), which results in a cytokine storm and toxic shock syndrome have been studied extensively. However, interactions of SAgs with mucosal surfaces remain poorly understood. TSST-1, the most common cause of menstrual TSS (mTSS), induces proinflammatory cytokines from vaginal epithelial cells These proinflammatory effects are hypothesized to contribute to the progression of mTSS by disrupting the permeability barrier of the vaginal mucosa directly and by causing a migration of neutrophils, macrophages and lymphocytes to the site of infection. The aims of this thesis were to 1) characterize TSST-1’s mechanism of action on human vaginal epithelial cells (HVEC), leading to the induction of proinflammatory cytokines, including the identification of the TSST-1 HVEC receptor and residues of TSST-1 critical for HVEC interactions, and to 2) evaluate curcumin, an anti-inflammatory compound, as an anti-TSS mucosal therapeutic. HVEC were exposed to TSST-1, and cytokine expression levels were determined by real-time reverse transcription polymerase chain reaction (PCR) and multiplex cytokine assay. IL-6, IL-8, MIP-3α, and TNF- α transcripts were up-regulated (1.5- to 12-fold), with corresponding increases in protein expression. TSST-1 activated an NF-κB luciferase reporter in HVEC, suggesting that NF-κB is a downstream target of TSST-1 signaling. Previous studies have suggested that major histocompatibility complex class II molecules (MHC II) could serve as the epithelial receptors for SAgs, and when activated, induce cytokines. Flow cytometry and Western blotting of HVEC did not detect MHC II molecules. These data suggest that MHC II is not the HVEC surface receptor responsible for induction of cytokines by TSST-1 and another undefined SAg epithelial receptor present on the surface of HVEC is implicated. A dodecapeptide region (TSST-1 amino acids F119 to D130) that is relatively conserved among SAgs has been implicated in SAg penetration of the epithelium. Single amino acid mutations were constructed in TSST-1 amino acids D120 to D130. All mutants maintained superantigenicity similar to wild type toxin. TSST-1 mutants induced IL-8 from HVEC; however, three toxin mutants (S127A, T128A, and D130A) induced lower levels of IL-8 compared to wild type TSST-1. These toxin mutants were administered intravenously to rabbits and all three were 100% lethal. When administered vaginally to rabbits, D130A toxin was nonlethal, while wild type TSST-1 was 100% lethal. Residue D130 may contribute to toxin binding to an epithelial receptor that allows it to penetrate the vaginal mucosa, induce cytokines/chemokines from epithelial cells, and cause TSS. CD40 was explored as a candidate HVEC receptor for TSST-1, based on a previous study which described synergistic activity of CD40 with MHC II for TSST-1 to induce cytokines from monocytes. HVEC (by flow cytometry) and ex vivo human ectocervical tissue (by immunohistochemistry) both expressed CD40. The biological role of CD40 in TSST-1-induced cytokine production was evaluated by reducing CD40 expression in HVEC by stable transfection with small hairpin RNA (shRNA) plasmids by 60% on the protein level. Surprisingly, CD40 shRNA-expressing HVEC produced more IL-8 in response to TSST-1 compared to irrelevant shRNA expressing HVEC. TSST-1 also bound to HVEC expressing shRNA more than HVEC expressing irrelevant shRNA. These data suggest that CD40 is not the HVEC receptor for TSST-1 that is responsible for induction of proinflammatory cytokines. Curcumin, a component of the spice turmeric, is a compound that has been used in traditional medicine therapies for 4,000 years. Curcumin was evaluated as a potential anti-TSST-1 agent by targeting the host mucosal response to TSST-1 and S. aureus. Curcumin inhibited S. aureus exoprotein- or live S. aureus-induced IL-8 production in an ex vivo porcine vaginal tissue model. The importance of TSST-1-induced inflammation of the vaginal mucosa in TSS disease progression was tested by using curcumin to prevent TSS in a rabbit vaginal model. Curcumin co-administrated with TSST-1 intravaginally significantly reduced lethality of TSST-1 by 60% (5 of 8 rabbits survived whereas 0 of 8 rabbits survived in TSST-1 controls, p <0.05). In addition, TNF-α was undetectable from serum or vaginal tissue of curcumin-treated rabbits that survived. These data demonstrate the importance of local inflammation in the progression of TSS, and curcumin as a potential anti-TSS agent. These studies describe the importance of TSST-1 interactions with vaginal epithelial cells in disease progression to TSS. The mechanism of TSST-1 activation of proinflammatory cytokines was determined to be through the NF-κB pathway with TSST-1 amino acid D130 being critical for induction of cytokines from epithelial cells. However, the NF-κB pathway was not induced by TSST-1 binding to MHC II molecules or CD40 suggesting the role of an unidentified epithelial receptor. These studies also determined that curcumin inhibits S. aureus exoprotein-induced cytokine response in ex vivo vaginal porcine tissue; and when administered intravaginally, curcumin partially prevents TSS in a rabbit model of TSS, demonstrating the importance of local inflammation caused by TSST-1 in progression of TSS. These studies were conducted to develop novel therapies for the prevention and treatment of superantigen-mediated diseases.