Development and evaluation of a thermoreversible ovule formulation of stampidine,a novel nonspermicidal broad-spectrum anti-human immunodeficiency virus microbicide

Stampidine [2',3'-didehydro-2',3'-dideoxythymidine 5'-[p-bromophenyl methoxyalaninyl phosphate], a prodrug of stavudine (STV/d4T) with improved anti-HIV activity, is undergoing development as a novel nonspermicidal microbicide. Here, we report the stability of stampidine as a function of pH, preparation of a novel thermoreversible ovule formulation for mucosal delivery, its dissolution profile in synthetic vaginal fluid, and its mucosal toxicity potential as well as systemic absorption in the rabbit model. Stampidine was most stable under acidic conditions. Stampidine was solubilized in a thermoreversible ovule formulation composed of polyethylene glycol 400, polyethylene glycol fatty acid esters, and polysorbate 80. Does were exposed intravaginally for 14 days to an ovule formulation with and without 0.5%, 1%, or 2% stampidine corresponding to 1 x 107- to 4 x 107-fold higher than its in vitro anti-HIV IC50 value. Vaginal tissues harvested on Day 15 were evaluated for mucosal toxicity and cellular inflammation. Additionally, does were exposed intravaginally to stampidine, and plasma collected at various time points was assayed by analytical HPLC for the prodrug and its bioactive metabolites. Stampidine did not cause mucosal inflammation. The vaginal irritation scores for 0.5-2% stampidine were within the acceptable range for clinical trials. The prodrug and its major metabolites were undetectable in the blood plasma. The marked stability of stampidine at acidic pH, its rapid spreadability, together with its lack of mucosal toxicity or systemic absorption of stampidine via a thermoreversible ovule may provide the foundation for its clinical development as an easy-to-use, safe, and effective broad-spectrum anti-HIV microbicide without contraceptive activity.     

CATCH: a clinical decision rule for the use of computed tomography in children with minor head injury

Background

There is controversy about which children with minor head injury need to undergo computed tomography (CT). We aimed to develop a highly sensitive clinical decision rule for the use of CT in children with minor head injury.

Methods

For this multicentre cohort study, we enrolled consecutive children with blunt head trauma presenting with a score of 13–15 on the Glasgow Coma Scale and loss of consciousness, amnesia, disorientation, persistent vomiting or irritability. For each child, staff in the emergency department completed a standardized assessment form before any CT. The main outcomes were need for neurologic intervention and presence of brain injury as determined by CT. We developed a decision rule by using recursive partitioning to combine variables that were both reliable and strongly associated with the outcome measures and thus to find the best combinations of predictor variables that were highly sensitive for detecting the outcome measures with maximal specificity.

Results

Among the 3866 patients enrolled (mean age 9.2 years), 95 (2.5%) had a score of 13 on the Glasgow Coma Scale, 282 (7.3%) had a score of 14, and 3489 (90.2%) had a score of 15. CT revealed that 159 (4.1%) had a brain injury, and 24 (0.6%) underwent neurologic intervention. We derived a decision rule for CT of the head consisting of four high-risk factors (failure to reach score of 15 on the Glasgow coma scale within two hours, suspicion of open skull fracture, worsening headache and irritability) and three additional medium-risk factors (large, boggy hematoma of the scalp; signs of basal skull fracture; dangerous mechanism of injury). The high-risk factors were 100.0% sensitive (95% CI 86.2%–100.0%) for predicting the need for neurologic intervention and would require that 30.2% of patients undergo CT. The medium-risk factors resulted in 98.1% sensitivity (95% CI 94.6%–99.4%) for the prediction of brain injury by CT and would require that 52.0% of patients undergo CT.

Interpretation

The decision rule developed in this study identifies children at two levels of risk. Once the decision rule has been prospectively validated, it has the potential to standardize and improve the use of CT for children with minor head injury.Each year more than 650 000 children are seen in hospital emergency departments in North America with “minor head injury,” i.e., history of loss of consciousness, amnesia or disorientation in a patient who is conscious and responsive in the emergency department (Glasgow Coma Scale score¹ 13–15). Although most patients with minor head injury can be discharged after a period of observation, a small proportion experience deterioration of their condition and need to undergo neurosurgical intervention for intracranial hematoma.^2–4 The use of computed tomography (CT) in the emergency department is important in the early diagnosis of these intracranial hematomas.Over the past decade the use of CT for minor head injury has become increasingly common, while its diagnostic yield has remained low. In Canadian pediatric emergency departments the use of CT for minor head injury increased from 15% in 1995 to 53% in 2005.^5,6 Despite this increase, a small but important number of pediatric intracranial hematomas are missed in Canadian emergency departments at the first visit.³ Few children with minor head injury have a visible brain injury on CT (4%–7%), and only 0.5% have an intracranial lesion requiring urgent neurosurgical intervention.^5,7 The increased use of CT adds substantially to health care costs and exposes a large number of children each year to the potentially harmful effects of ionizing radiation.^8,9 Currently, there are no widely accepted, evidence-based guidelines on the use of CT for children with minor head injury.A clinical decision rule incorporates three or more variables from the history, physical examination or simple tests^10.11 into a tool that helps clinicians to make diagnostic or therapeutic decisions at the bedside. Members of our group have developed decision rules to allow physicians to be more selective in the use of radiography for children with injuries of the ankle¹² and knee,¹³ as well as for adults with injuries of the ankle,^14–17 knee,^18–20 head^21,22 and cervical spine.^23,24 The aim of this study was to prospectively derive an accurate and reliable clinical decision rule for the use of CT for children with minor head injury.     

Genetic Analysis of DNA Replication in Bacteria: dna B Mutations That Suppress dnaC Mutations and dnaQ Mutations That Suppress dnaE Mutations in SALMONELLA TYPHIMURIUM

We have isolated and characterized extragenic suppressors of mutations in two different target genes that affect DNA replication in Salmonella typhimurium. Both the target and the suppressor genes are functional homologues of known replication genes of E. coli that were identified in intergeneric complementation tests. Our results point to interactions in vivo involving the dnaB and dnaC proteins in one case and the dnaQ and dnaE proteins in the other case. The suppressor mutations, which were isolated as derivatives of lambda-Salmonella in vitro recombinants, were detected by an adaptation of the red plaque complementation assay. This method was applicable even when the locus of suppressor mutations was not chosen in advance.     

Potential Consequences of Virus Infection for Shade-Sun Acclimation in Leaves

Attention is drawn to literature evidence that leaf virus infection may interfere with synthesis of ribulose-1,5-bisphosphate carboxylase oxygenase, and with the activity of photosystem II, two main requirements for shade-sun acclimation. Preliminary experiments which show that virus infections resulting in these interactions can lead to photosynthetic physiology diagnostic of nitrogen limited plants and/or shade ecotypes are presented. Other observations on the diversity of virus infections in sun populations of wild species commonly found in the shade are presented. Together, this evidence leads to the hypothesis that virus infection may well confine individuals of some species to shaded habitats because infection prevents acclimation to bright light, and predisposes them to photoinhibition.