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Class of 2012 Abstract / Objectives: The primary objective of this study was to evaluate the appropriateness of when aminoglycoside serum concentrations are obtained and assess whether the timing and techniques used in obtaining aminoglycoside serum concentrations are appropriate. Additionally, pharmacists’ interpretation of aminoglycoside serum concentrations and the appropriateness of intervention in response to these results were assessed. Methods: This descriptive retrospective study to evaluate the appropriateness of aminoglycoside monitoring at an academic medical center has been approved by the Institutional Review Board. Patients over the age of 46 weeks gestational age admitted to an academic medical center between February 1, 2010 to February 1, 2011 who were prescribed intravenous aminoglycoside therapy were included in this study. Patients with therapy duration of less than 72 hours without at least one aminoglycoside level were excluded. The time of aminoglycoside concentrations in relation to time of aminoglycoside administration along with calculated pharmacokinetic parameters and therapy recommendations documented in clinical notes were also recorded. Appropriateness of aminoglycoside monitoring and documentation were determined by use of expert opinion and pharmacokinetic guidelines. Results: Timing of aminoglycoside serum concentrations and subsequent clinical assessments were evaluated in 103 subjects. The median (range) age was 28 (0.2 – 88) years. The initial aminoglycoside prescribed in 12%, 40%, and 48% of subjects was amikacin, gentamicin, and tobramycin, respectively. A total of 314 aminoglycoside concentrations were obtained: 41 amikacin, 129 gentamicin, and 144 tobramycin. At least one clinical pharmacokinetic assessment of aminoglycoside concentration(s) was written for 91 subjects (88%). The aminoglycoside indication, actual time of aminoglycoside dose administration, estimated renal function, and both goal peak/trough aminoglycoside concentrations were documented in at least one aminoglycoside clinical note for each of these 91 subjects at a rate of 95%, 80%, 89%, and 51%, respectively. Calculated peak, trough, estimated volume of distribution, and estimated half-life or ke were documented in 53 subjects. Conclusions: Aminoglycoside serum concentration monitoring can be used to maximize therapeutic outcomes while minimizing toxicity. However, errors in obtaining and evaluating serum drug levels can arise that may affect patient outcomes. For monitoring to be effective, the timing of serum concentration orders, the process of obtaining serum concentration samples, and the interpretation of data including pharmacokinetic calculations should be accurate.

Class of 2012 Abstract / Objectives: The primary objective of this study was to evaluate the appropriateness of when aminoglycoside serum concentrations are obtained and assess whether the timing and techniques used in obtaining aminoglycoside serum concentrations are appropriate. Additionally, pharmacists’ interpretation of aminoglycoside serum concentrations and the appropriateness of intervention in response to these results were assessed. Methods: This descriptive retrospective study to evaluate the appropriateness of aminoglycoside monitoring at an academic medical center has been approved by the Institutional Review Board. Patients over the age of 46 weeks gestational age admitted to an academic medical center between February 1, 2010 to February 1, 2011 who were prescribed intravenous aminoglycoside therapy were included in this study. Patients with therapy duration of less than 72 hours without at least one aminoglycoside level were excluded. The time of aminoglycoside concentrations in relation to time of aminoglycoside administration along with calculated pharmacokinetic parameters and therapy recommendations documented in clinical notes were also recorded. Appropriateness of aminoglycoside monitoring and documentation were determined by use of expert opinion and pharmacokinetic guidelines. Results: Timing of aminoglycoside serum concentrations and subsequent clinical assessments were evaluated in 103 subjects. The median (range) age was 28 (0.2 – 88) years. The initial aminoglycoside prescribed in 12%, 40%, and 48% of subjects was amikacin, gentamicin, and tobramycin, respectively. A total of 314 aminoglycoside concentrations were obtained: 41 amikacin, 129 gentamicin, and 144 tobramycin. At least one clinical pharmacokinetic assessment of aminoglycoside concentration(s) was written for 91 subjects (88%). The aminoglycoside indication, actual time of aminoglycoside dose administration, estimated renal function, and both goal peak/trough aminoglycoside concentrations were documented in at least one aminoglycoside clinical note for each of these 91 subjects at a rate of 95%, 80%, 89%, and 51%, respectively. Calculated peak, trough, estimated volume of distribution, and estimated half-life or ke were documented in 53 subjects. Conclusions: Aminoglycoside serum concentration monitoring can be used to maximize therapeutic outcomes while minimizing toxicity. However, errors in obtaining and evaluating serum drug levels can arise that may affect patient outcomes. For monitoring to be effective, the timing of serum concentration orders, the process of obtaining serum concentration samples, and the interpretation of data including pharmacokinetic calculations should be accurate.

Class of 2005 Abstract / Objectives: To determine the pharmacokinetic parameters of a patient population from drug concentration measurements records created by the pharmacokinetic service at a regional hospital in South Carolina, to predict peak and trough concentrations from three large dose-extended interval (LDEI) protocols to determine which method produce the highest percentage of concentrations that fall in the desired ranges, and to compare pharmacokinetic parameters of overweight and normal weight patients. Methods: This was a descriptive, retrospective study that used clinical data from 121 of 208 patient data forms. The collected data included patient age, gender, weight, height, serum creatinine (Scr), measured serum peak and trough concentrations, time of dosing, dose and dosing interval. These were used to determine individual pharmacokinetic parameters and predict peak and trough concentrations from three LDEI dosing protocols. Results: Method II produced the highest percentage of patients with peaks and troughs falling into the target range (95.9%). The Hartford method produced the highest percentage (79.3%) of patients achieving peak concentrations >20mg/L. All three methods achieved low troughs of <2mg/L, <1mg/L, <0.5mg/L, and <0.1mg/L at least 95%, 80%, 70%, and 50% of the time, respectively. No statistical significance was found between the group having actual body weight/ideal body weight ratio (ABW/IBW) greater than 1.2 and another group having ABW/IBW <1.2 for ABW, volume of distribution (V), elimination half-life (T1/2) and aminoglycoside clearance (Clag). Also, when overweight patients were excluded, a higher correlation between elimination rate constant (k) and creatinine clearance (CrCl) was found than when all patients were combined. In other words, as k increases, CrCl increases. Implications: Even though Method II produced the greatest percentage of peak and trough concentrations within its stated target range, the Hartford method may be the best dosing protocol to use since it achieves high peak concentrations (>20mg/L) while maintaining low trough concentrations. In addition, based on our data, we can assume that overweight people affect the predicted k value. There was no statistical significance between actual and predicted pharmacokinetic characteristics in overweight patients.

Bacterial resistance to the aminoglycoside antibiotics is a major health concern because of the elimination of a therapeutic option for the treatment of nosocomial infections. Clinical resistance is commonly caused by the acquisition of genes that encode an aminoglycoside modifying enzyme. These enzymes offer a potential therapeutic target in the fight against aminoglycoside resistance. By gaining a structural understanding of these enzymes the potential is created for rational drug design. The research presented here deals with structural studies on two aminoglycoside resistance enzymes. First the initial stages of structural determination for the bifunctional Aminoglycoside 6'-N-Acetyl transferase Aminoglycoside 2''-O-Phosphotransferase (AAC(6')-APH(2")) including the optimization of the purification procedure for this enzyme. The second enzyme is the Aminoglycoside 3'-O-Phosphotransferase (APH(3')IIIa). Computational studies on this enzyme have been carried out in order to determine models for aminoglycoside binding and also to search for potential enzyme inhibitors. The molecular docking studies for both the aminoglycoside binding and inhibitor search involved the development of a number of novel methods to improve the chance of obtaining a correct model, and to aid in the analysis of the data from the docking studies. These methods have the potential to be applied in future structure based drug design / Thesis / Master of Science (MS)

The overwhelming increase in antibiotic resistant bacterial strains poses a serious public health problem, with multiply-resistant strains becoming an important cause of mortality in hospitals. The predominant mechanism of resistance to aminoglycoside antibiotics involves enzymatic modification of the drug, rendering it ineffective. The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ii (AAC(6')-Ii) in complex with its cofactor, acetyl coenzyme A (AcCoA), was determined at 2.7 Å resolution by the multiwavelength anomalous diffraction technique. The resolution of this structure was subsequently extended to 2.15 Å by molecular replacement, with no significant changes in the topology of the complex. The enzyme was found to exhibit a novel CoA-binding fold, with the cofactor bound in a cleft between the N-and C-terminal arms of the protein molecule. Although the enzyme packs as a monomer in the I4₁32 crystal form, the most probable physiological dimer of the complex was determined through analysis of a number of symmetry-related molecules. The crystal structure of the AAC(6')-Ii•AcCoA complex was compared to the structures of three members of a large superfamily of GCN5-related 𝘕-acetyltransferases (GNATs), namely yeast histone acetyltransferase HAT1, 𝘕-myristoyltransferase, and aminoglycoside acetyltransferase(3)-Ia. Despite negligible sequence similarity between these GNAT superfamily members, a distinct folding pattern is conserved in all four structures. This establishes AAC(6')-Ii as a structural homolog of enzymes with protein acetylating activity, supporting the hypothesis that the enzyme may possess another physiological function in 𝘌𝘯𝘵𝘦𝘳𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘧𝘢𝘦𝘤𝘪𝘶𝘮. / Thesis / Master of Science (MS)

Class of 2007 Abstract / Objectives: To create a nomogram that can accurately predict dosing intervals for aminoglycoside dosing in neonates based on one concentration measurement. Methods: Pooled pharmacokinetic data from previous studies were used (n=341) to create a nomogram that would accurately predict dosing intervals for aminoglycosides. The population value for volume of distribution (0.45 L/kg) was used to formulate a nomogram to select a dosing interval for neonates that would achieve a trough concentration of < 0.5 mg/L one hour prior to the next scheduled dose. The fixed dose of 4 mg/kg was used to simulate concentration elimination profiles all neonates in the study group. The data from the study group elimination profile was then compared against the nomogram and evaluated for the number of correct dosing intervals the nomogram predicted from hour 6 to 22 at 1 hour intervals. The trough concentration cutoff was < 0.5 mg/L with neonates not achieving this concentration prior to the next dose to be deemed dosed incorrectly. The nomogram was considered to have failed at any time point where the nomogram indicated an interval that would not have achieved the desired trough concentration of < 0.5 mg/L or if the interval chosen by the nomogram was longer then necessary. Results: The simulated data from the test group showed that from 15 to 21 hours post infusion 81.0 to 92.1% of neonates had the correct interval predicted by the nomogram. Greater accuracy was achieved the longer time that elapsed before a concentration is drawn, with the greatest accuracy (92.1%) at 21 hours post infusion. However, this was close to the next dose recommending a concentration draw time at 18 hours post infusion to maximize the combination of accuracy and time remaining before the next scheduled dose. This gives the lab time to report the concentration before the next dose is scheduled and achieves an accuracy rate of 87.6%. Conclusions: The use of this nomogram is a valid tool to predict dosing intervals for aminoglycosides in neonates and can aid in saving hospital resources by needing only one concentration measurement to determine dosing interval.

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.

Class of 2011 Abstract / OBJECTIVES: The purpose of this study is to assess three published aminoglycoside dosing protocols (large-dose extended interval), to predict peak and trough concentrations of these protocols and to determine the percentage of patients with peak and trough concentrations within each protocol’s specified ranges. METHODS: This study is a retrospective analysis of clinical data. A database of 515 patients is used to analyze the three different protocols. The variables in this database encompass patients’ age, height, actual body weight (ABW), sex, k, Vd, and dose. From these data, patients' peak and trough concentrations were determined using the three large large, extended interval dosing protocols. RESULTS The results showed Nicolau protocol with the most potential of the three protocols. It had the highest percentages of patients with peak above 15 mg/L and a trough less than 0.5 mg/L. It also had the highest average peak of 19.1 mg/L with 69.9% of patients meeting the protocol’s specified peak range of 13 to 23 mg/L. CONCLUSION: The three examined protocols all showed a percentage of patients within the desired range. Of the three, Nicolau protocol I showed promising results with highest average peak, lowest average trough and high percentage of patients with concentrations within desired ranges. Its percentages above 15 mg/L and less than 0.5 mg/L are greater than protocols II and III. Nicolau dosing protocol may be best in achieving high peak and low trough concentrations.