A high-content AlphaScreen™ identifies E6-specific small molecule inhibitors as potential therapeutics for HPV+ head and neck squamous cell carcinomas

The incidence of human papillomavirus-positive head and neck squamous cell carcinoma (HPV⁺-HNSCC) has increased dramatically over the past decades due to an increase in infection of the oral mucosa by HPV. The etiology of HPV⁺-HNSCC is linked to expression of the HPV oncoprotein, E6, which influences tumor formation, growth and survival. E6 effects this oncogenic phenotype in part through inhibitory protein-protein interactions (PPIs) and accelerated degradation of proteins with tumor suppressor properties, such as p53 and caspase 8. Interfering with the binding between E6 and its cellular partners may therefore represent a reasonable pharmacological intervention in HPV⁺ tumors. In this study, we probed a small-molecule library using AlphaScreen™ technology to discover novel E6 inhibitors. Following a cascade of screens we identified and prioritized one hit compound. Structure activity relationship (SAR) studies of this lead uncovered an analog, 30-hydroxygambogic acid (GA-OH), that displayed improved activity. Further testing of this analog in a panel of HPV⁺ and HPV^– cell lines showed good potency and a large window of selectivity as demonstrated by apoptosis induction and significant inhibition of cell growth, cell survival in HPV⁺ cells. In summary, GA-OH may serve as a starting point for the development of potent E6-specific inhibitors.     

The integrity of the esophageal mucosa. Balance between offensive and defensive mechanisms

Heartburn is the most common and characteristic symptom of gastroesophageal reflux disease. It ultimately results from contact of refluxed gastric acid with nociceptors within the esophageal mucosa and transmission of this peripheral signal to the central nervous system for cognition. Healthy esophageal epithelium provides an effective barrier between refluxed gastric acid and esophageal nociceptors; but this barrier is vulnerable to attack and damage, particularly by acidic gastric contents. How gastric acid is countered by defensive elements within the esophageal mucosa is a major focus of this discussion. When the defense is successful, the subject is asymptomatic and when unsuccessful, the subject experiences heartburn. Those with heartburn commonly fall into one of three endoscopic types: nonerosive reflux disease, erosive esophagitis and Barrett's esophagus. Although what determines endoscopic type remains unknown; it is proposed herein that inflammation plays a key, modulating role.     

In Vitro Activity of Ticarcillin Against Anaerobic Bacteria Compared with That of Carbenicillin and Penicillin

A total of 334 clinical anaerobic isolates were tested in an anaerobic glove box by the agar dilution technique for susceptibility to clinically achievable levels of ticarcillin, carbenicillin, and penicillin. Thirty-two micrograms or less of penicillin per milliliter inhibited 91% of all strains, whereas 100 μg of carbenicillin and ticarcillin per ml inhibited 95 and 98%, respectively. A total of 82% (85 strains) of Bacteroides were inhibited by penicillin, and 93 and 96% were inhibited by carbenicillin and ticarcillin, respectively. Thirteen (24%) of 55 strains of Bacteroides fragilis tested were resistant to 32 μg of penicillin per ml, and 6 (11%) and 3 (5%) were resistant to 100 μg of carbenicillin and ticarcillin per ml, respectively. Within the therapeutic range, ticarcillin was the most effective of the three penicillins tested against B. fragilis subsp. fragilis.     

Characterization of [3H]morphine binding to interleukin-1-activated thymocytes.

We have previously reported that interleukin-1-induced proliferation of thymocytes is accompanied by the appearance of [3H]morphine binding sites on these cells. In the present study, we have characterized these binding sites. They differ from classical opioid receptors in the brain in several ways, including: 1) lack of stereoselectivity; 2) relatively low affinity (Kd = 50 nM) and high capacity (Bmax = 3 pmol/mg of protein); 3) binding is strongly inhibited by Ca++, Mg++, Mn++ and Cl- ions and 4) binding is inhibited by proteinase K or E and by phospholipase A2 but not trypsin treatment of thymocyte membranes. The binding sites, which were found largely on the CD4+ subset of T-cells, also showed a preference for opioid alkaloids over peptides. These [3H]morphine binding sites may mediate a negative feedback effect on interleukin-1-induced proliferation of thymocytes in vivo.     

Multicenter clinical research in adult critical care

OBJECTIVE: To describe the development, organization, and operation of several collaborative groups conducting investigator-initiated multicenter clinical research in adult critical care. DESIGN: To review the process by which investigator-initiated critical care clinical research groups were created using examples from Europe, Australia, the United States, and Canada. Various models of group structure and function are discussed, highlighting complementary approaches to protocol development, multicenter study management, and project funding. DATA SOURCES: Published peer review research and unpublished terms of reference documents on the structure and function of these groups. DATA SYNTHESIS: The overall goal of clinical critical care research groups engaged in multicenter studies is to improve patient outcomes through conducting large, rigorous investigations. Research programs we reviewed included the following: a) multicenter epidemiologic studies and surveys; b) technology evaluations of mechanical ventilation; c) investigations focused on three priority fields (acute lung injury, infection, and acute brain injury); d) a series of randomized trials of treatments for one syndrome (acute respiratory distress syndrome); and e) diverse methodologies addressing several clinical problems. The structure and function of these research groups differ according to their historical development, research culture, and enabling resources. Specific protocols emerge from clinical questions generated by investigators or from collectively prioritized research agendas. Project funding includes government support, peer-review grants, intensive care foundations, industry, local hospital funds, and hybrid models. Infrastructure for study management varies widely. CONCLUSIONS: Several national and international groups have engaged in investigator-initiated multicenter critical care research. The development, organization, and operational methods of these groups illustrate several collaborative models for clinical investigations in the intensive care unit. Common characteristics of these groups are a cohesive spirit, a sense of mission to achieve shared research goals, and acknowledgment that such an organization is much more than the sum of its parts.     

Efficacy, safety, and tolerability of pregabalin treatment for painful diabetic peripheral neuropathy: findings from seven randomized, controlled trials across a range of doses

OBJECTIVE—To evaluate the efficacy, safety, and tolerability of pregabalin across the effective dosing range, to determine differences in the efficacy of three times daily (TID) versus twice daily (BID) dosage schedules, and to use time-to-event analysis to determine the time to onset of a sustained therapeutic effect using data from seven trials of pregabalin in painful diabetic peripheral neuropathy (DPN).RESEARCH DESIGN AND METHODS—Data were pooled across seven double-blind, randomized, placebo-controlled trials using pregabalin to treat painful DPN with dosages of 150, 300, and 600 mg/day administered TID or BID. Only one trial included all three of these dosages, and TID dosing was used in four. All studies shared fundamental selection criteria, and treatment durations ranged from 5 to 13 weeks.RESULTS—Pooled analysis showed that pregabalin significantly reduced pain and pain-related sleep interference associated with DPN (150, 300, and 600 mg/day administered TID vs. placebo, all P ≤ 0.007). Only the 600 mg/day dosage showed efficacy when administered BID (P ≤ 0.001). Pain and sleep interference reductions associated with pregabalin appear to be positively correlated with dosage; the greatest effect was observed in patients treated with 600 mg/day. Kaplan-Meier analysis revealed that the median time to onset of a sustained (≥30% at end point) 1-point improvement was 4 days in patients treated with pregabalin at 600 mg/day, 5 days in patients treated with pregabalin at 300 mg/day, 13 days in patients treated with pregabalin at 150 mg/day, and 60 days in patients receiving placebo. The most common treatment-emergent adverse events were dizziness, somnolence, and peripheral edema.CONCLUSIONS—Treatment with pregabalin across its effective dosing range is associated with significant, dose-related improvement in pain in patients with DPN.The prevalence of diabetic neuropathy is as high as 50% in patients who have had diabetes for 25 years (1), and painful diabetic peripheral neuropathy (DPN) occurs in up to 26% of all people with diabetes (2). Symptoms range from mild dysesthesias to severe unremitting pain that can profoundly impact patients’ lives (3,4).Medications of several different classes are used to treat painful DPN with varying degrees of efficacy, safety, and tolerability. The antiepileptic agents gabapentin and pregabalin have attained widespread use in the treatment of painful DPN. These agents bind to the auxiliary α2-δ subunit of the voltage-sensitive calcium channel, thereby decreasing Ca²⁺ influx at nerve terminals and modulating neurotransmitter release (5).There are seven double-blind, randomized, placebo-controlled trials in painful DPN with pregabalin (6–12), five of which are published in full (7–11). The effective dosing range for treatment of neuropathic pain syndromes is 150 to 600 mg/day, administered either three times daily (TID) or twice daily (BID). Among the seven trials, dosages of 150, 300, and 600 mg/day were used, but only one trial included all three of these dosages. Thus, individually, the seven trials present an incomplete picture of the effective dosing range. In addition, TID dosing was used in the first four trials, whereas the three most recent trials of pregabalin in painful DPN used BID dosing.The objective of the current report is to use the pooled data from these seven trials to evaluate the efficacy, safety, and tolerability of pregabalin across the effective dosing range. We also use these data to determine differences in the efficacy of TID and BID dosing schedules. Finally, we use a time-to-event analysis of the pooled data to determine the time to onset of a sustained therapeutic effect across the range of doses.