Abstract AIM Glioblastoma (GB) is the most common malignant primary brain tumor in adults, with a prognosis as poor as 12-15 months with standard treatment. Spermidine/spermine N1-acetyltransferase (SAT1) is a rate limiting enzyme in polyamine metabolism and has been reported to be upregulated in various cancers, including GB. Amantadine is a Health Canada approved drug that is acetylated by SAT1. We established a clinical trial in GB patients to determine if plasma and urine acetyl amantadine (Ac-Am) can be used to measure SAT1 activity and whether levels correlate with their tumor burden. METHODS A clinical trial was established that is currently active and recruiting patients with GB who receive care at CancerCare Manitoba. A total of n=8 participants have been recruited thus far. Participants’ blood and urine were collected two hours after ingesting amantadine (200 mg). Levels of serum and urine Ac-Am were measured using liquid chromatography-tandem mass spectrometry. Acetyl-amantadine levels were correlated with tumour bidimensional diameter and volume measured on MRI. In addition, expression of SAT1 was examined in various cultured GB cells (both cell lines and patient-derived cells) and correlated with Ac-Am. RESULTS Preliminary results indicate that the levels of plasma Ac-Am in study participants positively correlate with their initial tumor burden (r = 0.41). While transient increases in plasma and urine Ac-Am above baseline levels were observed, the clinical significance of these findings is undergoing further analysis. SAT1 was detected in all the GB tumor cells examined. The production of Ac-Am in cultured GB cells varied as a function of SAT1 expression. SIGNIFICANCE A diagnostic biomarker, such as Ac-Am, that could effectively and reliably detect tumor progression and recurrence would be an invaluable adjunct to MRI imaging and could significantly impact the timing of appropriate treatment and reduce patients’ morbidity and mortality.

Spermidine/spermine N1-acetyltransferase 1 (SAT1) responsible for cell polyamine catabolism is overexpressed in glioblastoma multiforme (GB). Its role in tumor survival and promoting resistance towards radiation therapy has made it an interesting target for therapy. In this study, we prepared a lipid nanoparticle-based siRNA delivery system (LNP-siSAT1) to selectively knockdown (KD) SAT1 enzyme in a human glioblastoma cell line. The LNP-siSAT1 containing ionizable DODAP lipid was prepared following a microfluidics mixing method and the resulting nanoparticles had a hydrodynamic size of around 80 nm and a neutral surface charge. The LNP-siSAT1 effectively knocked down the SAT1 expression in U251, LN229, and 42MGBA GB cells, and other brain-relevant endothelial (hCMEC/D3), astrocyte (HA) and macrophage (ANA-1) cells at the mRNA and protein levels. SAT1 KD in U251 cells resulted in a 40% loss in cell viability. Furthermore, SAT1 KD in U251, LN229 and 42MGBA cells sensitized them towards radiation and chemotherapy treatments. In contrast, despite similar SAT1 KD in other brain-relevant cells no significant effect on cytotoxic response, either alone or in combination, was observed. A major roadblock for brain therapeutics is their ability to cross the highly restrictive blood-brain barrier (BBB) presented by the brain microcapillary endothelial cells. Here, we used the BBB circumventing approach to enhance the delivery of LNP-siSAT1 across a BBB cell culture model. A cadherin binding peptide (ADTC5) was used to transiently open the BBB tight junctions to promote paracellular diffusion of LNP-siSAT1. These results suggest LNP-siSAT1 may provide a safe and effective method for reducing SAT1 and sensitizing GB cells to radiation and chemotherapeutic agents.

Aim: The assessment of tumor response to therapy is of critical importance as it permits for a prospective end point evaluation and provides a guide to clinicians for making future treatment decisions. However, current practices in early evaluation of chemotherapy are insufficient. Amantadine is a substrate for SSAT-1. The present pilot study tests the hypothesis that SSAT-1 activity within the tumor, as measured by plasma acetylamantadine concentrations, can be used to monitor patient response to therapy. Results: In cases with evidence of disease response, there was a reduction in the plasma acetylamantadine concentration at 4 h by approximately 32%. There was a mean increase of approximately 34% at the 4 h collection in the nonresponders. Conclusion: Although large-scale studies are required these findings suggest that the amantadine test could allow for determination of the efficacy of therapeutic interventions earlier, providing an effective test to assess response to treatment and for better management of patients.

The objective of this research is to use metabolomic techniques to discover and validate plasma metabolite biomarkers for the diagnosis of early-stage non-small cell lung cancer (NSCLC). The study included plasma samples from 156 patients with biopsy-confirmed NSCLC along with age and gender-matched plasma samples from 60 healthy controls. A fully quantitative targeted mass spectrometry (MS) analysis (targeting 138 metabolites) was performed on all samples. The sample set was split into a discovery set and validation set. Metabolite concentration data, clinical data, and smoking history were used to determine optimal sets of biomarkers and optimal regression models for identifying different stages of NSCLC using the discovery sets. The same biomarkers and regression models were used and assessed on the validation models. Univariate and multivariate statistical analysis identified β-hydroxybutyric acid, LysoPC 20:3, PC ae C40:6, citric acid, and fumaric acid as being significantly different between healthy controls and stage I/II NSCLC. Robust predictive models with areas under the curve (AUC) > 0.9 were developed and validated using these metabolites and other, easily measured clinical data for detecting different stages of NSCLC. This study successfully identified and validated a simple, high-performing, metabolite-based test for detecting early stage (I/II) NSCLC patients in plasma. While promising, further validation on larger and more diverse cohorts is still required.

e14536 Background: In the evolving role of biomarkers, proteinomic signatures related to up-regulation of polyamine synthesis including spermine/spermidine acetyltransferase-1 (SSAT-1) appear to be promising markers of malignancy. Acetylamantadine (AA) excretion, a measure of SSAT-1 up-regulation, has been shown to be a marker for malignant proliferation. In preliminary analyses of ostensibly normal individuals from Canada (Winnipeg) and Bangladesh (Dhaka), a proportion were identified to have evidence of SSAT-1 up-regulation above the expected non-malignant range (outliers). These outliers were assessed and followed for development of identifiable medical conditions. Methods: SSAT-1 up regulation was assessed in 60 ostensibly normal individuals by analysis of urinary excretion of AA after ingestion of amantadine 200 mg x 1 dose. AA was assayed using HPLC-mass spec techniques as previously described. Outliers who consented were followed-up by clinical examinations, routine biochemical and hematological tests and radiographs, where indicated. Results: Total AA average (median) excretion at 6 hrs in the Winnipeg cohort was 579+/-252 vs 1699+/-633 ng in the Dhaka cohort. Average urinary concentration at 6 hrs was 3.75+/-0.75 vs 21+/-20 ng/ml. In outliers consenting to follow-up, 1 case of invasive malignancy was identified, 6 cases of pre-malignant neoplastic change, chronic liver disease in 7 cases and chronic inflammation in 2 cases. In others, no malignant or inflammatory conditions have yet been identified. The range of SSAT-1 activity in individuals living in Bangladesh was significantly higher than in the Canadian cohort. Bangladesh volunteers live in an area of known to have high natural contamination with arsenic, a recognized carcinogen and they are exposed to high levels of air pollution. Conclusions: By this assay, high SSAT-1 activity has been confirmed in patients harbouring malignancy; however, pre-malignant and inflammatory conditions may result in a positive test. Follow-up of individuals with elevated SSAT-1 activity may reveal unrecognized malignancies, pre-malignant neoplasms, liver disease, inflammatory conditions and possibly false-positivity in individuals exposed to carcinogens such as arsenic. In further work, SSAT-1 up-regulation is being correlated with 5 other putative polyamine metabolite markers for accurate diagnosis of lung cancer.

Aim: Spermidine/spermine N1-acetyltransferase (SSAT-1) regulates cell growth, proliferation and death. Amantadine is converted by SSAT-1 to acetylamantadine (AA). In our earlier studies, although SSAT-1 was activated in patients with cancer, a number of ostensibly healthy adult volunteers had higher than expected AA concentration. This study was therefore undertaken to examine the outlier group. Materials & methods: A follow up of urine analysis for AA by liquid chromatography-tandem mass spectrometry as well as clinical assessments and additional blood analyses were conducted. Results: In some of the outlier controls, higher than expected AA concentration was linked to increased serum carcinoembryonic antigen. Clinical and radiographic assessments revealed underlying abnormalities in other cases that could represent premalignant conditions. Hematology tests revealed elevations in white blood cells and platelets, which are markers of inflammation. Conclusion: High urine concentration of AA could be used as a simple and useful test for screening of cancer in high-risk populations.

Aim: Spermidine/spermine N-1-acetyltransferase (SSAT-1) plays a critical role in cell growth, proliferation and death, and is known to be activated in human cancer cells. Amantadine, a US FDA-approved antiviral drug, is a substrate for SSAT-1 and can be used to indirectly measure SSAT-1 activity because of its conversion to acetylamantadine (AA). This study was undertaken to further validate SSAT-1 activity in breast and lung cancer patients. Results: An increase in the urinary concentration of AA in lung and breast cancer patients was observed. The 0-2 h collection time point was determined to be optimal in revealing significant differences in urinary AA concentration between healthy controls and cancer patients. Conclusion: The high urine concentration of AA could be used as a simple and useful test for the detection of breast and lung cancer. [GRAPHICS] Lay abstract: This study describes a novel noninvasive urine test for detecting and screening of breast and lung cancer using a safe and approved drug called amantadine. Higher concentration of the acetylated form of amantadine in the urine are detectable in the urine of both breast and lung cancer patients as compared with healthy adult volunteers. This test is simple and may serve as a useful tool for determining the presence of breast and lung cancer.

Background: Lung cancer is the most common cause of cancer-related deaths worldwide. Early diagnosis is crucial to increase the curability chance of the patients. Low dose CT screening can reduce lung cancer mortality, but it is associated with several limitations. Metabolomics is a promising technique for cancer diagnosis due to its ability to provide chemical phenotyping data. The intent of our study was to explore metabolomic effects and profiles of lung cancer patients to determine if metabolic perturbations in the SSAT-1/polyamine pathway can distinguish between healthy participants and lung cancer patients as a diagnostic and treatment monitoring tool. Patients and Methods: Plasma samples were collected as part of the SSAT1 Amantadine Cancer Study. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify and quantify metabolite concentrations in lung cancer patient and control samples. Standard statistical analyses were performed to determine whether metabolite concentrations could differentiate between healthy subjects and lung cancer patients, as well as risk prediction modeling applied to determine whether metabolic profiles could provide an indication of cancer progression in later stage patients. Results: A panel consisting of 14 metabolites, which included 6 metabolites in the polyamine pathway, was identified that correctly discriminated lung cancer patients from controls with an area under the curve of 0.97 (95% CI: 0.875-1.0). Conclusion: When used in conjunction with the SSAT-1/polyamine pathway, these metabolites may provide the specificity required for diagnosing lung cancer from other cancer types and could be used as a diagnostic and treatment monitoring tool.

Aim: SSAT-1 is an enzyme that plays a critical role in cell growth. Amantadine, a FDA-approved antiviral drug, is a substrate for SSAT-1. The utility of amantadine as an agent to demonstrate elevated SSAT-1 activity linked to cancer was conducted. Results: High levels of SSAT-1 expression were measured in tumor human cell lines, and in breast, prostate and lung tumor tissue. An increase in the urinary levels of acetylated amantadine in cancer patients was observed. Conclusion: Increases in SSAT-1 contents in tumor tissue could be of value in targeting cancers with high SSAT-1 expression for confirmation/quantification. The high levels of acetylated amantadine could be used as a simple and useful screening test for the presence of cancer.

While assessing the ability of mammalian lung tissue to metabolize theophylline, a new metabolite was isolated and characterized. The metabolite was produced by the microsomal fraction of lungs from several species, including rat, rabbit, dog, pig, sheep and human tissue. Metabolite production was blocked by boiling the microsomal tissue. This new metabolite, theophylline-7β-d-ribofuranoside (theonosine), was confirmed by several spectral methods and by comparison to an authentic synthetic compound. Tissue studies from rats, rabbits, dogs, and humans for cofactor involvement demonstrated an absolute requirement for NADP and enhanced metabolite production in the presence of magnesium ion. It remains to be demonstrated whether theonosine may contribute to the known pharmacological effects of theophylline.

Background: Owing to its ability to form spores and toxins, Bacillus anthracis is considered a bioterror agent. Although current therapeutic strategies can be effective, treatment does not prevent sporulation and toxin production. Objectives: To quantify the combined effect of a protein synthesis inhibitor and a bactericidal agent on B. anthracis toxin production, sporulation and cell growth.Methods: Susceptibility and synergy titrations were conducted on B. anthracis Sterne and 03-0191 strains using linezolid and levofloxacin. The effect of antibiotic exposure on cell viability was evaluated using a continuous medium replacement model. In vitro static models were used to study the effect of linezolid and levofloxacin on sporulation and toxin production. Spores were quantified using the heat shock method. Toxin was quantified via commercial ELISA.Results: Synergy titrations indicated that the combination was synergistic or indifferent; however, in all models antagonism was observed. In the spore model, linezolid resulted in the lowest sporulation rates, while combination therapy resulted in the highest. In the toxin model, linezolid prevented toxin production altogether.Conclusions: This study advances our understanding of the effects of combination therapy on B. anthracis infection. Used alone, linezolid therapy abolishes toxin production and reduces sporulation. These results suggest that studies using a step-wise approach using linezolid initially to stop sporulation and toxin production followed by levofloxacin to rapidly kill vegetative B. anthracis can be recommended.

Anita L. Kozyrskyj, BScPhm, MSc; G. Elske Hildes-Ripstein, MD; Sally E. A. Longstaffe, MD;J. Leigh Wincott, MD; Daniel S. Sitar, BScPhm, PhD; Terry P. Klassen, MD, MSc; Michael E. K. Moffatt, MD, MScObjective.— To conduct a meta-analysis of randomized controlled trials of an-tibiotic treatment of acute otitis media in children to determine whether outcomeswere comparable in children treated with antibiotics for less than 7 days or at least7 days or more.Data Sources.— MEDLINE (1966-1997), EMBASE (1974-1997), Current Con-tents, and Science Citation Index searches were conducted to identify randomizedcontrolled trials of the treatment of acute otitis media in children with antibiotics ofdifferent durations.Study Selection.— Studies were included if they met the following criteria: sub-jects aged 4 weeks to 18 years, clinical diagnosis of acute otitis media, no antimi-crobial therapy at time of diagnosis, and randomization to less than 7 days of an-tibiotic treatment vs 7 days or more of antibiotic treatment.Data Extraction.— Trial methodological quality was assessed independently by7 reviewers; outcomes were extracted as the number of treatment failures,relapses, or reinfections.Data Synthesis.— Included trials were grouped by antibiotic used in the shortcourse: (1) 15 short-acting oral antibiotic trials (penicillin V potassium, amoxicillin[-clavulanate],cefaclor,cefixime,cefuroxime,cefpodoximeproxetil,cefprozil),(2)4intramuscularceftriaxonesodiumtrials,and(3)11oralazithromycintrials.Thesum-maryoddsratiofortreatmentoutcomesat8to19daysinchildrentreatedwithshort-acting antibiotics for 5 days vs 8 to 10 days was 1.52 (95% confidence interval [CI],1.17-1.98) but by 20 to 30 days outcomes between treatment groups were com-parable (odds ratio, 1.22; 95% CI, 0.98 to 1.54). The risk difference (2.3%; 95%CI,−0.2% to 4.9%) at 20 to 30 days suggests that 44 children would need to betreated with the long course of short-acting antibiotics to avoid 1 treatment failure.This similarity in later outcomes was observed for up to 3 months following therapy(oddsratio,1.16;95%CI,0.90-1.50).Comparableoutcomeswereshownbetweentreatment with ceftriaxone or azithromycin, and at least 7 days of other antibiotics.Conclusion.— Thismeta-analysissuggeststhat5daysofshort-actingantibioticuse is effective treatment for uncomplicated acute otitis media in children.

Higher doses of cefazolin are required in obese patients for preoperative antibiotic prophylaxis, owing to its low lipophilicity. An ultra high performance liquid chromatography-tandem mass spectrometry method was developed to quantify cefazolin in serum and adipose tissue from 6 obese patients undergoing cesarean delivery, and using stable-isotope labeled cefazolin as an internal standard. The method has a 2μg/g lower limit of quantitation. The concentration in adipose tissue was 3.4±1.6μg/mL, which is less than half of the reported minimum inhibitory concentration of 8μg/mL for cefazolin. Serum cefazolin concentrations were more than 30-fold higher than in adipose tissue.

This issue of the Journal of Clinical Pharmacology, the official publication of the American College of Clinical Pharmacology, represents my final effort as its Editor-in-Chief for the past 5 years. Reflecting on the discipline, Clinical Pharmacology continues to evolve at an accelerating pace. Papers published in the Journal have in the past and continue to reflect advances in the discipline. Concepts published only 5 years ago not unexpectedly are incorporated into today's clinical trial protocols and are often no longer perceived as innovative contributions to the discipline. I am confident that this evolution will continue into the future under the leadership of the incoming Editor-in-Chief, Dr. Joseph Bertino. The Journal has changed considerably over the past 5 years. Review articles are appearing more often, while maintaining the majority of its content as original studies on human pharmacological advances, both pharmacokinetic and pharmacodynamic. We have had the opportunity to publish important supplements related to the growth of pharmacometrics,1 and to revisit the confounding impact of renal impairment on optimization of drug therapy.2 There is an increasing presence of drug studies from the pediatric cohort and of results from patient investigations, for whom new and improved drug treatment strategies continue to be developed. Ultimately, these kinds of papers should impact dramatically on optimization of therapeutics. It is reassuring to see the increasing inclusion of genetic and other biomarker data as parameters that may further address the ultimate goal of individualized drug therapy. Evolution of population pharmacokinetic and pharmacodynamic modeling strategies should allow for improved paradigms for drug treatment of high risk populations, especially where sample access is considerably limited. The increasing success of biological therapies will pose unique challenges to clinical pharmacologists, and provide substantial opportunities for innovation in clinical pharmacokinetic and pharmacodynamic clinical trials. Submissions of manuscripts to the Journal for consideration continue to increase and now exceed 500 yearly, making it much more difficult to select among the many excellent papers for the published content of monthly issues, while reflecting the diversity of the discipline. At this point it is essential to thank my Associate Editors, Drs. Hartmut Derendorf and Michael Reed for their wise counsel, my Editorial Board for its thorough and insightful peer review of the many manuscripts assigned to them, and many guest reviewers for manuscripts where content is reflecting areas of growth and change in the discipline. Of course, all of these efforts were orchestrated via the outstanding efforts of our Managing Editor, Ms. Marjory Spraycar, and Assistant Managing Editor, Ms. Elizabeth Marshall. Our Journal remains a widely read and cited source of knowledge directed at the improvement of patient quality of life when drug therapies are chosen as interventions to minimize consequences of pathological processes. I am grateful to all for having been offered the opportunity to contribute to such a laudable goal.

Copyright: © 2012 Sitar DS. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. It is widely appreciated that the increasing fraction of public and private resources devoted to the maintenance of human health is unsustainable into the future [1,2]. With increasing age comes an increasing probability of acquiring chronic diseases that involve drug therapy as a major component of the management plan. It is increasingly clear that the elderly demonstrate a greater variability in their response to drug administration in comparison to their younger cohorts [3]. The Institute of Medicine Report indicated that drug therapy is associated with a substantial burden of morbidity and mortality that impacts resource utilization [4]. Thus, it is in the interest of society to implement strategies that are likely to minimize the fraction of health care resources devoted to resolving these drugrelated adverse patient events.

Major advances produced by healthcare research have resulted in an increasing number of drugs that may be used to modify disease expression and improve quality of life. These discoveries have been used by clinical pharmacologists as a basis to identify new drug candidates and to develop strategies for their optimal delivery to maximize benefit while simultaneously minimizing adverse events. Unfortunately, many of these studies do not include sufficient older persons in whom most of these drug therapy interventions are likely to apply. This article examines selected physiological, pathological and healthcare interventional changes with age that impact clinical drug studies and the decision to use drugs as therapy in older adults. Clinical examples are provided that illustrate confounders to the accomplishment of an ideal outcome, the improved quality of life that remains for this population.

Background: Intensive sampling of patients for drugs with complex pharmacokinetic profiles is difficult to perform in the clinic or hospitalized patient setting. We seek to address whether sparse sampling can obtain pharmacokinetic parameter values similar to those with traditional modeling from a post hoc analysis of 2 previous clinical trials.Objective: This study investigated whether population-guided, sparse-sampling pharmacokinetic analysis of morphine in 14 healthy volunteers allowed for optimal characterization of concentration-time profiles for a validation population of 5 young male patients receiving morphine.Methods: Data were analyzed using nonparametric adaptive grid (NPAG) population modeling to investigate optimal compartmental structure and the influence of sparse sampling (ie, 9 versus 3 samples per subject) on parameter identification. These results were compared with traditional standard 2-stage (STS) pharmacokinetic modeling. The coefficients of determination (R-2), mean error (ME), and root-mean-square error were used to assess the predictive performance of the various sampling models against a validation population.Results: Seventy-nine percent of the healthy volunteers were male, with a mean age of 36 (17) years and a mean weight of 68 (10) kg. NPAG modeling identified that intravenous morphine was best represented by a 3-compartment pharmacokinetic profile and that sparse sampling with a least 3 blood samples per subject resulted in virtually identical measures of central tendency as the more intensively sampled dataset. A validation cohort of 5 male patients undergoing elective surgery had a mean age of 26 (4) years and a mean weight of 80 (13) kg. Using mean parameter estimates generated from sparse sampling and the 3-compartment model structure, simulated profiles were compared against measured concentrations in this validation cohort. Sparse sampling using NPAG achieved similar values of predictive performance as mean parameter values from the more intensively sampled, with an ME of -1.0 ng/mL and precision of 26.2 ng/mL compared with 0.76 ng/mL and 25.8 ng/mL, respectively. Traditional (STS) modeling techniques resulted in the greatest degree of underprediction within the validation group (ME = 4.43 versus 0.76 ng/mL, STS and NPAG-9, respectively; P < 0.0001).Conclusions: This post hoc analysis suggests that intensive sampling for discerning complex, 3-compartment pharmacokinetic models, such as morphine, may not be necessary. Sparse sampling achieved accurate model structure recognition and parameter identification for predicting concentrations of very complex drug-dosage regimens. (Clin Ther. 2012;34:668-676) (C) 2012 Elsevier HS Journals, Inc. All rights reserved.