Class of 2005 Abstract / Objectives: First, to evaluate five recent gentamicin dosing protocols that are specific for neonates and determine how frequently each protocol yields desirable peak and trough concentrations. Second, to make our evaluation more robust, we included AUC as one of the pharmacokinetic parameters and compared it to traditional parameters. Finally, to evaluate a fixed dosing protocol (3 mg/kg Q24-hours) that is currently being used at one Arizona hospital (UMC). Methods: This retrospective evaluation involved datasets from three independent sources. Dataset 1 was from a previously published study, while datasets 2 and 3 were derived for this study. Datasets 1 and 2 were pooled to evaluate the five dosing protocols, while dataset 3 was used to evaluate the fixed dosing protocol used at UMC. For all subjects, demographic and laboratory data was obtained from hospital databases or charts. The data collected was used to construct pharmacokinetic values, which in turn were used in simulations with the five protocols. Dataset 3 was evaluated as a whole for frequency of desired peaks and troughs, then for subsets based on weight, gestational age, and Apgar scores. Results: Of the five evaluated, the Avent protocol yielded the fewest potentially toxic troughs. The Murphy-Carter protocol stood out in that it was the easiest to use, most universally applicable, and it yielded only slightly fewer desired troughs then the Avent protocol. AUC values proved to be a novel and exceptionally useful tool in evaluating the dosing protocols. The fixed dosing protocol used at UMC was shown to consistently produce favorable trough concentrations as a whole as well as in our subset analyses. Implications: The multitude of dosing protocols that have been offered can create confusion among health care professionals and lead to discrepancies in dosing. The primary goal of any of these protocols is to minimize the risk of toxicity while avoiding subtherapeutic doses. A dosing protocol that can consistently meet these criterion, yet offer simplicity and wide applicability, then we can come that much closer to a universal standard.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Aneta Pražáková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of selected biomarkers of nephrotoxicity in urine and Plasma The discovery and development of novel biomarkers, that can be used for diagnosis of kidney damage earlier and more accurately, are needed for the effective prediction of drug-induced nephrotoxicity. Mechanisms of drug-induced nephrotoxicity include changes in glomerular hemodynamics, tubular cell cytotoxicity, inflammation, crystalline nephropathy, etc. Detection at initial stage of damage using sensitive and specific biomarkers belongs between one of the most important strategies in the treatment of acute kidney injury and renal failure. Although some these biomarkers do not show specificity and sensitivity, several promising biomarker candidates have been established recently to evaluate nephrotoxicity, e.g. selected KIM-1, cystatin C and NGAL. The advantages of these biomarkers compared to traditionally used biomarkers are higher sensitivity, specificity, just mentioned early diagnosis and non-invasiveness (the possibility of determination levels of the biomarkers from blood or urine). The aim of this diploma thesis was to determine...

Aim: Microbial biofilm matrix contains polysaccharides and proteins and can require extracellular nucleic acids for initial formation. Experiments were designed to identify infectious pathogens in human aneurysms and to characterize biofilm formed by calcified human arterial-derived nanoparticles. Materials & method: A total of 26 different microbial pathogens were isolated from 48 inflammatory aneurysms. Consistent amounts (0.49 McFarland units) of nanoparticles derived from similar tissue were seeded into 24-well plates and cultured for 21 days in the absence (control) or presence of RNase, tetracycline or gentamicin. Results: Control biofilm developed within 14 days, as detected by concanavalin A and BacLight™ Green staining. The formation of biofilm in wells treated with RNase was not different from the control; however, gentamicin partially inhibited and tetracycline completely inhibited biofilm formation. Therefore, nanoparticle biofilm retains some characteristics of conventional bacterial biofilm and requires protein-calcium interactions, although extracellular RNA is not required. Conclusion: This model system may also allow study of nanosized vesicles derived from donor tissue, including any microbes present, and could provide a useful tool for in vitro investigation of nanoparticle biofilm formation.

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Neuronal networks, derived from mouse embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate effects of gentamicin pretreatment on pharmacological response to the L-type calcium channel blocker, verapamil. Gentamicin is a broad spectrum antibiotic used to control bacterial contamination in cell culture. The addition of gentamicin directly to medium affects the pharmacological and morphological properties of the cells in culture. A reproducible dose response curve to verapamil from untreated cultures was established and the mean EC50 was calculated to be 1.5 ± 0.5 μM (n=10). 40 μM bicuculline was added to some cell cultures to stabilize activity and verapamil dose response curves were performed in presence of bicuculline, EC50 1.4 ± 0.1 μM (n=9). Statistical analysis showed no significant difference in verapamil EC50s values obtained in presence of bicuculline and hence the data was combined and a standard verapamil EC50 was calculated as 1.4 ± 0.13 μM (n=19). This EC50 was then used to compare verapamil EC50s obtained from neuronal cell cultures with chronic and acute exposures to gentamicin. FC cultures (21- 38 days old) were found to be stable in presence of 2300 μM gentamicin. The recommended concentration of gentamicin for contamination control is 5uL /1 ml medium (108 μM). At this concentration, the verapamil EC50 shifted from 1.4 ± 0.13 μM to 0.9 ± 0.2 μM. Given the limited data points and only two complete CRCs, statistical comparison was not feasible. However, there is a definite trend that shows sensitization of cells to verapamil in presence of gentamicin. The cultures exposed to 108 μM gentamicin for 5 days after seeding showed loss of adhesion and no data could be collected for pharmacological analysis. To conclude, acute gentamicin exposure of neuronal cell cultures causes increased sensitivity to verapamil and chronic or long term exposure to gentamicin may cause loss of adhesion of the cell culture by affecting the glial growth. The effect of chronic exposure to gentamicin on pharmacological responses to verapamil remains inconclusive.

Indiana University-Purdue University Indianapolis (IUPUI) / At the present time, gentamicin is used in the treatment of both Gram-negative and Gram-positive bacterial infections. However, the poorly understood side effect of nephrotoxicity is a serious problem and is one of the dose-limiting factors in the use of gentamicin. In our model system, Saccharomyces cerevisiae, which is relatively resistant to gentamicin, at least 20 genes are required for gentamicin resistance. Inspection of the physical and genetic interactions of the gentamicin sensitive mutants reveals a network centered on the ARF pathway which plays a key role in the regulation of retrograde trafficking. Our studies show that arf1ts arf1Δ arf2Δ cells, gea1ts gea1Δ gea2Δ cells, and gcs1ts gcs1Δ glo3Δ cells are all hypersensitive to gentamicin which indicates that impaired Arf1 function causes yeast cells to become hypersensitive to gentamicin. As evidence, cellular CPY trafficking and processing are blocked by the presence of gentamicin in some of these mutants. Interestingly, gentamicin can directly affect the level of the GTP-bound form of Arf1 in a cell growth phase-dependent manner; even though total Arf1 levels in S. cerevisiae are not affected. As predicted, we also find that gentamicin-bound resin can enrich both yeast Arf1-TAP protein and rat Arf1 protein in vitro. With the help of mass spectrometry, we also generated a gentamicin-binding protein list. Gentamicin hypersensitivity is also observed in S. cerevisiae double deletion strains that lack both ARF1 and ARF2 but are kept alive by the presence of hARF4 or bARF1. Increased -1 programmed ribosomal frameshifting efficiency is also observed in cells treated with gentamicin. Finally, a comparison of a gentamicin mixture and four of the gentamicin congeners reveals that gentamicin C1 is less toxic than other gentamicin congeners or the gentamicin total mixture.

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Gentamicin C complex is a mixture of five structurally similar aminoglycoside antibiotics, gentamicins C1, C1a, C2, C2a, and C2b, produced by the actinomycete bacterium Micromonospora echinospora. It is established in clinical use and despite significant toxicity remains valuable to treat severe Gram-negative bacterial infections. There is a pressing need to develop novel versions of such antibiotics to combat the rise of resistance among pathogens. Engineering of the pathway requires a detailed knowledge of the genes, enzymes, and intermediates involved. The final steps of gentamicin biosynthesis begin at gentamicin X2, the last common intermediate of the C complex. 6'-C-Methylation generates two branches, with analogous reactions happening in both. Candidate genes and enzymes for the steps from the first 6'-C-methylated intermediate, G418, to an aminated metabolite JI-20B have already been described, but none for the subsequent loss of two hydroxyl groups from Ring II, or the N-methylation that then occurs. A novel separation method using dynamic countercurrent chromatography was successfully applied to the difficult purification of gentamicin metabolites. The results described here allowed a detailed mechanism to be proposed for almost the entire pathway from G418 to the C complex, and by analogy for the unbranched pathway, too. The last step of the pathway is 6'-N-methylation of gentamicins C1a and C2. Genome mining and cell-free assays were used by the group of Professor Yuhui Sun (Wuhan University) to identify genL, a methyltransferase gene encoded elsewhere on the M. echinospora genome and capable of this catalysis. Here, in vitro reactions with recombinant GenL confirmed its function, and its kinetic parameters were measured with its substrates. The full mechanistic pathway for the late stages of gentamicin C complex biosynthesis has therefore now been elucidated.