Class of 2013 Abstract / Specific Aims: The objective of this study was to compare the use of amikacin 1 year before the national drug shortage and 1 year during drug shortage in order to evaluate the impact of the drug shortage on prescribing amikacin at an academic medical center. Methods: All patients admitted to an academic medical center between January 1, 2008 to December 31, 2008 before the shortage and January 1, 2011 to December 1, 2011 during the shortage who were prescribed amikacin were evaluated. Data collected included demographic information, type of infection, aminoglycoside therapy prescribed, laboratory data, culture and susceptibility data, therapy outcomes, and potential complications of aminoglycoside therapy. Appropriateness of amikacin therapy was based on each subject’s clinical condition, culture and susceptibility results, and availability of an alternative antibiotic agent. The use of amikacin was considered inappropriate in subjects with Gram-negative organisms that had either tobramycin or gentamicin minimum inhibitory concentrations of less than or equal to 2 mcg/mL. Main Results: A total of 11 subjects in 2008 and 17 subjects in 2011 who were prescribed amikacin were evaluated. The median and range duration of amikacin therapy was 2.2 days and 0-17 days in 2008. In 2011, the median and range duration of amikacin therapy was 4.6 days and 0-38 days. In 2008 and 2011, 27% and 47% were subjects with cystic fibrosis and/or a history of solid organ transplant, respectively. In 2008 73% of amikacin orders were classified as appropriate while 59% of amikacin orders were classified as appropriate in 2011. Ototoxicity was reported in one subject who received amikacin in both 2008 and 2011. Conclusion: Despite restrictions for ordering amikacin implemented during a nationwide shortage, the percentage of appropriate orders for amikacin was lower during the shortage compared to before the shortage at an academic medical center.

No description available.

The use of aminoglycosides in the treatment of the febrile neutropaenic patient with haematological disease is difficult and often suboptimal. This study reviews the available literature to establish therapeutic guidelines in this population and then reports the use of a Bayesian statistics based predictive model to implement and manage therapy in 10 patients. A review of the literature on aminoglycoside Pharmacology and clinical use is essential to determine therapeutic guidelines for this population. Aminoglycosides are amino sugars in glycosidic linkage and are polycations at physiological PH. The antibiotic effect is mediated through inhibition of protein synthesis and disruption of cell membrane integrity. Principal use is in treatment of Gram negative infection although aminoglycosides have activity against some Gram positive organisms including staphylococci. Aminoglycosides are inactive against anaerobes. Acquired resistance is mediated by bacterial enzymatic drug metabolism. Aminoglycosides are nephro- and ototoxic, this is the major constraint in clinical use.

No description available.

Le développement de nouveaux antibiotiques est un enjeu majeur de santé publique. Etant donné, le fort potentiel des aminoglycosides en tant qu’antibiotique, ces composés ont attisé l’intérêt de plusieurs groupes de recherche. Cependant, leur usage est encore très limité, malgré leur ancienneté, du fait de leur toxicité et du développement toujours croissant des mécanismes de résistances aux aminoglycosides. Afin de mieux appréhender les problèmes inhérents à leur utilisation, il est crucial de mieux comprendre leur action sur les différentes cibles cellulaires, et d’étudier leur interaction avec leur cible moléculaire (ARN et protéine). En plus de leur pouvoir antibiotique, les aminoglycosides sont également des ligands universels pour des ARN, capables d’interagir spécifiquement avec notamment les ARN du VIH-1 suivants : DIS, TAR, RRE. L’élaboration d’aminoglycosides modifiés présente un énorme avantage car le domaine d’application, et en conséquence les retombées, sont grandes. Néanmoins, la complexité structurale de ces molécules est un frein majeur, la fonctionnalisation chimiosélective est indispensable mais malheureusement peu décrite dans la littérature. Dans le cadre de ce travail, nous avons développé deux types d’approches pour cibler le DIS et/ou le site A du ribosome bactérien. La première originale, mais risquée se base sur le concept de click in situ. La seconde approche est traditionnelle et est basée sur la fonctionnalisation sélective de certaines positions clés des aminoglycosides. / The development of new antibiotics is a major public health issue. Given the high potential of aminoglycosides as antibiotics, these compounds have aroused great interest in many research groups. However, despite their maturity, their use is still limited because of their toxicity and the increasing development of resistance mechanisms to aminoglycosides. To better understand the problems inherent to their use, it is crucial to understand their action a cellular level, and to study the interactions with their molecular targets (RNA and protein). In addition to their antibiotic power, aminoglycosides are also universal ligands for several RNAs, capable of specific interactions with RNAs of HIV-1: DIS, TAR and RRE. The elaboration of modified aminoglycosides presents a huge advantage because the domain of application, and therefore the benefits, are important. Nevertheless, the structural complexity of these molecules is a major constraint, chemoselective functionalization is essential but unfortunately poorly described in the literature.In this work, we developed two approaches to target the DIS and/or the A site of the bacterialribosome. The first one, unique but challenging is based on the concept of in situ click chemistry. The second approach is conventional and is based on the selective functionalization of some keypositions of aminoglycosides.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Aminoglycoside antibiotics are losing their bactericidal efficacy due to the spread of enzymes that catalyze a covalent modification to them. A common property of many of these aminoglycoside modifying enzymes (AGMEs) is the capacity to modify multiple diverse aminoglycosides thus conferring resistance to these drugs among several pathogenic bacterial species. To gain a better understanding of the protein-antibiotic interactions responsible for resistance and the promiscuous nature of AGMEs, a variety of biophysical techniques including nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), steady state kinetics, intrinsic tryptophan fluorescence, and computational modeling are employed in this work. Results and discussion presented herein are divided into two parts. In Part I, a detailed thermodynamic and kinetic characterization of the association between the aminoglycoside acetyltransferase-(3)-IIIb (AAC) and several antibiotics and/or coenzyme(s) provides insight into the global properties of the protein. AAC is shown to have a broad substrate range where antibiotic interaction occurs with a favorable enthalpy and unfavorable entropy. When coenzyme A (the non-catalytic form of the acetyl donor, acetyl coenzyme A) is present, enthalpy becomes more favored, entropy more disfavored, and antibiotic affinity significantly increases. AAC shows preference for antibiotics with amine groups at the 2′ and 6′ positions and to those possessing four or more pseudo-saccharide rings. These and other data lay the foundation for understanding AAC and lead into the next discussion wherein the source of promiscuity of AGMEs is explored in Part II. The aminoglycoside phosphotransferase-(3′)-IIIa (APH), a representative from the phosphotransferase family of AGMEs, has been well characterized previously. However, none of the data presented to date provides rationale for its promiscuity. In this work, NMR derived hydrogen-deuterium exchange experiments reveal that APH maneuvers its entire structure to accommodate diverse antibiotics. Furthermore, presence of an antibiotic creates a more stable APH conformation while coenzyme induces an antibiotic dependent increase in the flexibility of APH. For comparison, a computationally derived homology model of AAC predicts that its promiscuous nature may be due to a large flexible loop. Taken together, APH and AAC, two structurally and functionally diverse proteins, utilize different aspects of structural flexibility to facilitate a broad substrate repertoire that is key to bacterial survival.

Reading of messenger RNA (mRNA) by aminoacyl-tRNAs (aa-tRNAs) on the ribosomes in the bacterial cell occurs with high accuracy. It follows from the physical chemistry of enzymatic reactions that there must be a trade-off between rate and accuracy of initial tRNA selection in protein synthesis: when the current accuracy, the A-value, approaches its maximal possible value, the d-value, the kinetic efficiency of the reaction approaches zero. We have used an in vitro system for mRNA translation with purified E. coli components to estimate the d- and A-values by which aa-tRNAs discriminate between their cognate and near cognate codons displayed in the ribosomal A site. In the case of tRNALys, we verified the prediction of a linear trade-off between kinetic efficiency of cognate codon reading and the accuracy of codon selection. These experiments have been extended to a larger set of tRNAs, including tRNAPhe, tRNAGlu, tRNAHis, tRNACys, tRNAAsp and tRNATyr, and linear efficiency-accuracy trade-off was observed in all cases. Similar to tRNALys, tRNAPhe discriminated with higher accuracy against a particular mismatch in the second than in the first codon position. Remarkably high d-values were observed for tRNAGlu discrimination against a C-C mismatch in the first codon position (70 000) and for tRNAPhe discrimination against an A-G mismatch in the second codon position (79 000). At the same time, we have found a remarkably small d-value (200) for tRNAGlu misreading G in the middle position of the codon (U-G mismatch). Aminoglycoside antibiotics induce large codon reading errors by tRNAs. We have studied the mechanism of aminoglycoside action and found that the drug stabilized aminoacyl-tRNA in a codon selective in relation to a codon non-selective state. This greatly enhanced the probability of near cognate aminoacyl-tRNAs to successfully transcend the initial selection step of the translating ribosome. We showed that Mg2+ ions, in contrast, favour codon non-selective states and thus induce errors in a principally different way than aminoglycosides.  We also designed experiments to estimate the overall accuracy of peptide bond formation with, including initial selection accuracy and proofreading of tRNAs after GTP hydrolysis on EF-Tu. Our experiments have now made it possible to calibrate the accuracy of tRNA selection in the test tube to that in the living cells. We will now also be able to investigate the degree to which the accuracy of tRNA selection has been optimized for maximal fitness.

Clinical ototoxicity - permanent hearing loss caused by medications - is estimated to affect millions of patients annually. Two classes of drug are largely to blame: platinum-based chemotherapeutics, primarily cisplatin, and aminoglycoside antibiotics. Development of methods to prevent ototoxicity depends upon an understanding of its mechanisms and may benefit from an understanding of native protective pathways of the inner ear. As the mechanisms behind cisplatin ototoxicity remain unclear, I first sought, and herein report, a refined mouse model of cisplatin ototoxicity which will allow for further in vivo investigation of cisplatin ototoxicity and potential methods for its prevention. This low-dose, multi-cycle model was found to accurately reproduce cisplatin ototoxicity as it has been described clinically and histopathologically. I then used this mouse model of cisplatin ototoxicity to investigate cisplatin pharmacokinetics in the cochlea and their role in driving cisplatin ototoxicity. Cisplatin was found to be retained within the cochlea for months following its administration. This initial finding in mice was extended to cochlear tissue samples from deceased human patients. Analysis of intra-cochlear cisplatin distribution in murine and human tissue identified the stria vascularis region of the cochlea as a promising target for intervention. With the nature of aminoglycoside ototoxicity better understood, I investigated a native inner ear protective pathway which could be leveraged to promote sensory hair cell survival. The improved hair cell survival that has previously been demonstrated as a result of heat stress was found to be mediated by cell-cell communication via extracellular vesicles. Further, hair cell protection against aminoglycosides could be reproduced through the application of exogenous, non-inner ear-derived extracellular vesicles. In sum, these data provide new insight into mechanisms of ototoxicity and details of cellular pathways which can help protect against it.

D-methionine (D-met) has demonstrated otoprotection from noise-, aminoglycoside-, and cisplatin-induced hearing loss in animal studies. As a result, D-met is currently progressing through translational "bench to bedside" research. However, D-met's exact otoprotective mechanisms have not been fully elucidated. This study investigated relationships between dose- and time-dependent D-met otoprotection from noise- and aminoglycoside-induced hearing loss. Further, the study correlated protective D-met dose to endogenous antioxidant enzyme activity and lipid peroxidation. Specific aim 1 tested D-met dose response protection by auditory brainstem response (ABR) threshold shift analysis and outer hair cell (OHC) quantification. D-met doses ranging from 25-200 mg/kg/dose were administered to chinchillas every 12 hours five times each before and after steady state noise exposure totaling 10 D-met doses. Results demonstrated optimal, sub-optimal, and supra-optimal bi-phasic D-met otoprotective dose response. Optimal D-met protection from steady state noise occurred at the 50 mg/kg/dose level. OHC quantification confirmed electrophysiological assessment. Specific aim 2 measured D-met rescue protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five intraperitoneal (ip) D-met injections were administered every 12 hours beginning 3, 5, 7, 9, 12, 18, 24, 36, or 48 hours after steady state noise exposure. Results measured full D-met protection when administration began as late as 24 hours after noise secession. Significant partial protection was also measured for the 36 hour delay. OHC quantification confirmed electrophysiological assessment. Specific aim 3 measured D-met preloading protection from steady state noise exposure by ABR threshold shift analysis and OHC quantification. Five ip D-met injections were administered every 12 hours beginning 2, 2.5, or 3 days prior to steady state noise exposure. Results measured significant D-met protection when administration ended as early as 24 hours prior to noise exposure. OHC quantification confirmed electrophysiological assessment. Specific aim 4 tested dose-dependent D-met influence on antioxidant enzyme activity and oxidative stress in steady state noise-exposed chinchillas. One ip D-met injection, ranging from 25 to 200 mg/kg/dose, was administered every 12 hours beginning 2 days prior to steady state noise exposure for a total of 5 injections. Two hours post-noise exposure, animals were sacrificed and serum, liver, and cochleae were collected for endogenous antioxidant analysis. Glutaredoxin 2 (Grx2) was also analyzed 21 days post-noise exposure. Lower D-met doses (25 and 50 mg/kg/dose) increased superoxide dismutase and catalase activity. Glutathione reductase and glutathione peroxidase activities significantly increased with D-met doses but only at high concentrations (200 mg/kg/dose). At 21 days post-noise, Grx2 activity was significantly decreased in liver but greatly increased in the cochlea with high D-met doses (200 mg/kg/dose). The endogenous enzyme studies suggest optimal protective D-met dose determined in specific aims 1 through 3 may be secondary to increased superoxide dismutase and catalase activity which may result from D-met's free radical scavenging characteristics. Glutathione pathway activity increased only with high D-met doses that resulted in less optimal protection in specific aim 1. Thus, D-met-induced glutathione pathway enhancement may be a compensatory or saturation mechanism rather than the primary protective mechanism. Further, the extended pre-loading and rescue protection may be a result of significantly increased s-glutathionylation activity in the cochlea. Specific aim 5 tested D-met protection from impulse noise exposures. D-met dose response, rescue, and antioxidant enzyme assay protocols, similar to those in specific aims 1, 3, and 4 in steady state animals, were performed on impulse noise-exposed chinchillas. D-met provided dose- and time-dependent optimal protection from impulse noise similar to the steady-state noise studies. Optimal D-met protection was measured at the 100 mg/kg/dose level with complete rescue protection as late as 24 hours post-noise exposure. Endogenous enzyme activity measures demonstrated significant superoxide dismutase, catalase, and glutathione peroxidase activity increases which correlated with optimal D-met protective dose (100 mg/kg/dose) and catalase and superoxide dismutase activity decreases at the higher doses (200 mg/kg/dose). Specific aim 6 tested dose-dependent D-met protection from tobramycin, amikacin, kanamycin, and gentamicin aminoglycoside antibiotics. Guinea pig animal models were normalized to achieve a 30-40 dB ABR threshold shift with the lowest possible aminoglycoside dose. D-met and the aforementioned single aminoglycoside were administered for 21, 28, 23, or 14 days, respectively. ABRs were collected and assessed at baseline, 2, 4, and 6 weeks after drug administration initiation. After the 6-week ABR data collection, cochleae were collected and prepared for OHC quantification. ABR threshold shifts and OHC quantifications demonstrate significant bi-phasic D-met-induced protection from each aminoglycoside type with different D-met doses. OHC quantification confirmed electrophysiological assessment. This study identified optimal protective D-met dose for aminoglycoside- and noise- induced ototoxicity. It also identified optimal protective D-met dose timing for steady state and impulse noise-induced hearing loss. Further, this study has identified dose-dependent D-met-induced endogenous antioxidant changes and Grx2 enhancement, and therefore s-glutathionylation, as a potential mechanism for D-met protection. Thus, dose- and time-dependent D-met protection influences endogenous antioxidant activity, but exact optimal D-met protection will continue to warrant further investigation.