Mode of Action,In Vitro Activity,and In Vivo Efficacy of AFN-1252, a Selective Antistaphylococcal FabI Inhibitor

The mechanism of action of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), which is involved in fatty acid biosynthesis, was confirmed by using biochemistry, macromolecular synthesis, genetics, and cocrystallization of an AFN-1252–FabI complex. AFN-1252 demonstrated a low propensity for spontaneous resistance development and a time-dependent reduction of the viability of both methicillin-susceptible and methicillin-resistant S. aureus, achieving a ≥2-log₁₀ reduction in S. aureus counts over 24 h, and was extremely potent against clinical isolates of S. aureus (MIC₉₀, 0.015 μg/ml) and coagulase-negative staphylococci (MIC₉₀, 0.12 μg/ml), regardless of their drug resistance, hospital- or community-associated origin, or other clinical subgroup. AFN-1252 was orally available in mouse pharmacokinetic studies, and a single oral dose of 1 mg/kg AFN-1252 was efficacious in a mouse model of septicemia, providing 100% protection from an otherwise lethal peritoneal infection of S. aureus Smith. A median effective dose of 0.15 mg/kg indicated that AFN-1252 was 12 to 24 times more potent than linezolid in the model. These studies, demonstrating a selective mode of action, potent in vitro activity, and in vivo efficacy, support the continued investigation of AFN-1252 as a targeted therapeutic for staphylococcal infections.     

Discovery of N-(3-(1-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-indol-6-yl) thiophene-2-carboximidamide as a selective inhibitor of human neuronal nitric oxide synthase (nNOS) for the treatment of pain

3,6-Disubstituted indole derivatives were designed, synthesized, and evaluated as inhibitors of human nitric oxide synthase (NOS). Bulky amine containing substitution on the 3-position of the indole ring such as an azabicyclic system showed better selectivity over 5- and 6-membered cyclic amine substitutions. Compound (-)-19 showed the best selectivity for neuronal NOS over endothelial NOS (90-fold) and inducible NOS (309-fold) among the current series. Compounds 16 and (-)-19 were shown to be either inactive or very weak inhibitors of human cytochrome P450 enzymes, indicating a low potential for drug-drug interactions. Compound 16 was shown to reverse thermal hyperalgesia in vivo in the Chung model of neuropathic pain. Compound 16 was also devoid of any significant vasoconstrictive effect in human coronary arteries, associated with the inhibition of human eNOS. These results suggest that 16 may be a useful tool for evaluating the potential role of selective nNOS inhibitors in the treatment of pain such as migraine and CTTH.     

1,2,3,4-tetrahydroquinoline-based selective human neuronal nitric oxide synthase (nNOS) inhibitors: lead optimization studies resulting in the identification of N-(1-(2-(methylamino)ethyl)-1,2,3,4-tetrahydroquinolin-6-yl)thiophene-2-carboximidamide as a preclinical development candidate

Numerous studies have shown that selective nNOS inhibitors could be therapeutic in many neurological disorders. Previously, we reported a series of 1,2,3,4-tetrahydroquinoline-based potent and selective nNOS inhibitors, highlighted by 1 ( J. Med. Chem. 2011 , 54 , 5562 - 5575 ). Despite showing activity in two rodent pain models, 1 suffered from low oral bioavailability (18%) and moderate hERG channel inhibition (IC(50) = 4.7 μM). To optimize the properties of 1, we synthesized a small focused library containing various alkylamino groups on the 1-position of the 1,2,3,4-tetrahydroquinoline scaffold. The compounds were triaged based on their activity in the NOS and hERG manual patch clamp assays and their calculated physicochemical parameters. From these studies, we identified 47 as a potent and selective nNOS inhibitor with improved oral bioavailability (60%) and no hERG channel inhibition (IC(50) > 30 μM). Furthermore, 47 was efficacious in the Chung model of neuropathic pain and has an excellent safety profile, making it a promising preclinical development candidate.     

Intraoperative insertion of Greenfield filters: lessons learned in a personal series of 152 cases

The objective of this study was to define outcomes of 151 patients who underwent insertion of 152 Greenfield filters in the operating room by general and vascular surgery residents with supervision by one attending vascular surgeon. Each patient was taken to the operating room for inferior vena cava (IVC) interruption immediately after a vena cavagram was performed. One patient required a subsequent return to the operating room after developing paradoxical arterial embolism from a large venous thromboembolism which was trapped by and spanned both sides of the first IVC filter. In this case a second suprarenal filter was placed at the time of arterial embolectomy. In each of these 152 cases intraoperative venacavography was performed using a mobile C-arm. Complications such as hemothorax, filter misplacement, and vena cava perforation were identified. Late survival was defined using the Social Security Death Index. Of the 151 patients undergoing intraoperative insertion of Greenfield filters there was one hemothorax from attempts at acquiring venous access via percutaneous puncture of the internal jugular vein. This required transfusion but not thoracotomy, and IVC interruption was achieved. A separate patient had insertion of a Greenfield filter into a gonadal vein which required placement of a second filter into the IVC. There was one IVC perforation from a transfemoral filter insertion which required placement of a second filter above this perforation and laparotomy to retrieve the filter and repair the IVC. In one more patient the IVC filter initially failed to open, and a second filter was placed above the first filter. In this experience the misplacement rate was 0.7 per cent and the serious complication rate was 1.3 per cent. None of the patients was adversely affected per se by transfer to the operating room for Greenfield filter insertion. No patient died from filter insertion, but in two cases serious associated complications contributed to the adverse outcomes in these already terminally ill patients. Overall 30-day mortality rate was 6.6 per cent. Late survival was defined as follows: survival at one year after filter insertion was 75 per cent, at 2 years 63 per cent, at 3 years 60 per cent, at 4 years 57 per cent, and at 5 years 54 per cent. Mean survival after filter placement was 4.96 years. We conclude that Greenfield filters can be inserted in the operating room by general and vascular surgery residents with attending supervision with reasonable safety and with a low rate of filter misplacement. The caval perforation and gonadal vein filter misplacement could both have been avoided by use of an over-the-wire filter deployment system, which at the time of these specific complications was not available. Vena cava filter insertion should remain within the scope of practice of surgeons and can be done with reasonable safety under C-arm guidance in the operating room. Use of over-the-wire systems could have helped reduce the likelihood of all but one of the filter-related complications experienced in this series.     

Design, synthesis, and biological evaluation of 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline-based selective human neuronal nitric oxide synthase (nNOS) inhibitors

Neuronal nitric oxide synthase (nNOS) inhibitors are effective in preclinical models of many neurological disorders. In this study, two related series of compounds, 3,4-dihydroquinolin-2(1H)-one and 1,2,3,4-tetrahydroquinoline, containing a 6-substituted thiophene amidine group were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS). A structure-activity relationship (SAR) study led to the identification of a number of potent and selective nNOS inhibitors. Furthermore, a few representative compounds were shown to possess druglike properties, features that are often difficult to achieve when designing nNOS inhibitors. Compound (S)-35, with excellent potency and selectivity for nNOS, was shown to fully reverse thermal hyperalgesia when given to rats at a dose of 30 mg/kg intraperitonieally (ip) in the L5/L6 spinal nerve ligation model of neuropathic pain (Chung model). In addition, this compound reduced tactile hyperesthesia (allodynia) after oral administration (30 mg/kg) in a rat model of dural inflammation relevant to migraine pain.     

Low-level expression of miR-375 correlates with poor outcome and metastasis while altering the invasive properties of head and neck squamous cell carcinomas

Small, noncoding microRNAs (miRNAs) have been shown to be abnormally expressed in every tumor type examined. We used comparisons of global miRNA expression profiles of head and neck squamous cell carcinoma (HNSCC) samples and adjacent normal tissue to rank those miRNAs that were most significantly altered in our patient population. Rank Consistency Score analysis revealed miR-375 to have the most significantly lowered miRNA levels in tumors relative to matched adjacent nonmalignant tissue from the same patient among 736 miRNAs that were evaluated. This result has been previously observed by other groups; however, we extend this finding with the unique observation that low miR-375 expression levels correlate significantly with cancer survival and distant metastasis. In a study of 123 primary HNSCC patients using multivariable Cox proportional hazard ratios (HR) and 95% confidence intervals (CI), both death from disease (HR: 12.8, 95% CI: 3 to 49) and incidence of distant metastasis (HR: 8.7, 95% CI: 2 to 31) correlated with lower expression levels of miR-375 regardless of the site or stage of the tumor. In addition, we found that oral cavity tumor cell lines (eg, UMSCC1 and UMSCC47) overexpressing miR-375 were significantly less invasive in vitro than their matched empty vector controls. We conclude that miR-375 represents a potential prognostic marker of poor outcome and metastasis in HNSCC and that it may function by suppressing the tumor's invasive properties.     

Career perceptions of young academic oral and maxillofacial surgeons.

PURPOSE: To evaluate the perceptions and motivation of young full-time academic oral and maxillofacial surgeons who chose a career in academia and their experiences as faculty members. MATERIALS AND METHODS: An 11-item questionnaire was sent to the chairman of all nonmilitary American Dental Association-accredited oral and maxillofacial surgery training programs (total of 90 training programs), requesting that this questionnaire be completed by all full-time faculty who completed surgical residency between 1997 and 2003. RESULTS: Forty-eight completed surveys were included in this study. CONCLUSIONS: The primary motivation to pursue a career in academic oral and maxillofacial surgery was the opportunity to perform major maxillofacial surgery, closely followed by a desire to teach dental students and surgical residents. Most respondents noted that they had the opportunity to pursue a faculty practice, and most believed that they worked in a supportive environment. However, several noted that they did not have faculty practice opportunities, and most did not have protected time to carry out research, administrative, or other scholarly activities.     

First-in-class, dual-action, 3,5-disubstituted indole derivatives having human nitric oxide synthase (nNOS) and norepinephrine reuptake inhibitory (NERI) activity for the treatment of neuropathic pain

A family of different 3,5-disubstituted indole derivatives having 6-membered rings were designed, synthesized, and demonstrated inhibition of human nitric oxide synthase (NOS) with norepinephrine reuptake inhibitory activity (NERI). The structure-activity relationship (SAR) within the cyclohexane ring showed the cis-isomers to be more potent for neuronal NOS and selective over endothelial NOS compared to their trans-counterparts. Compounds, such as cis-(+)-37, exhibited dual nNOS and NET inhibition (IC(50) of 0.56 and 1.0 μM, respectively) and excellent selectivity (88-fold and 12-fold) over eNOS and iNOS, respectively. The lead compound (cis-(+)-37) showed lack of any direct vasoconstriction or inhibition of ACh-mediated vasorelaxation in isolated human coronary arteries. Additionally, cis-(+)-37 was effective at reversing both allodynia and thermal hyperalgesia in a standard Chung (spinal nerve ligation) rat neuropathic pain model. Overall, the data suggest that cis-(+)-37 is a promising dual action development candidate having therapeutic potential for the treatment of neuropathic pain.     

Discovery of cis-N-(1-(4-(methylamino)cyclohexyl)indolin-6-yl)thiophene-2-carboximidamide: a 1,6-disubstituted indoline derivative as a highly selective inhibitor of human neuronal nitric oxide synthase (nNOS) without any cardiovascular liabilities

A series of 1,6-disubstituted indoline derivatives were synthesized and evaluated as inhibitors of human nitric oxide synthase (NOS) designed to mitigate the cardiovascular liabilities associated with previously reported tetrahydroquinoline-based selective neuronal NOS inhibitors due to higher lipophilicity ( J. Med. Chem. 2011 , 54 , 5562 - 5575 ). This new series produced similar potency and selectivity among the NOS isoforms and was devoid of any cardiovascular liabilities associated with QT prolongation due to hERG activity or endothelial NOS mediated vasoconstriction effect. The SAR studies led to the identification of cis-45, which was shown to reverse thermal hyperalgesia in vivo in the spinal nerve ligation model of neuropathic pain with excellent safety profile (off-target activities at 80 CNS related receptors/ion channels/transporters). The results presented in this report make cis-45 as an ideal tool for evaluating the potential role of selective nNOS inhibitors in CNS related disorders where excess NO produced by nNOS is thought to play a crucial role.     

The elimination profiles of oxaprozin in equine urine and serum after a 4.8-g dose.

A method for the extraction of oxaprozin from equine urine and serum and its quantitation by high-performance liquid chromatography-ultraviolet detection is presented. Confirmation of oxaprozin in postadministration extracts was accomplished by gas chromatographic- mass spectrometric analysis of methylated extracts or liquid chromatography with tandem mass spectrometry daughter ion mass spectra of underivatized extracts. Daypro, a formulation of oxaprozin, was administered orally at a dose of 4.8 g to four standardbred mares. Urine and serum samples were collected to 120 h postadministration. Base hydrolysis of equine urine before extraction resulted in an increase in the amount of oxaprozin measured, an indication of conjugation by ester formation. The urinary elimination profiles of each horse were significantly different from each other with more than one peak in oxaprozin concentration before the 29-31-h collection time. After this collection time, the differences between the oxaprozin urinary concentrations of each horse follow each other more closely. The peak average urinary concentrations of oxaprozin were 25.1 and 17.0 microg/mL at collection times of 8-10 and 18-22 h, respectively. The latest detection of oxaprozin in urine was at the last collection time of 119-121 h postadministration at a concentration close to the detection limit of approximately 0.1 microg/mL. The serum elimination profiles do not vary between horses as much as the urinary elimination profiles. The peak average serum concentration was 49.0 microg/mL at a collection time of 6 h postadministration. The latest detection was at the last collection time of 120 h. Oxaprozin is metabolized in the horse by hydroxylation. Two major urinary metabolites were isolated and identified as hydroxylated oxaprozin. The two urinary metabolites were isolated from equine postadministration urine and analyzed by mass spectrometry and proton nuclear magnetic resonance spectroscopy, which showed that the hydroxylation had occurred at the para positions of the two aromatic rings.