Pattern of Brain Injury Predicts Long-Term Epilepsy Following Neonatal Encephalopathy.

OBJECTIVE: To determine if patterns of hypoxic-ischemic brain injury on magnetic resonance imaging (MRI) in term newborns predict subsequent childhood epilepsy. METHODS: This retrospective cohort study includes term newborns with encephalopathy (n = 181) born between 2004-2012 and admitted to British Columbia Children's Hospital. MRI was performed between 3 and 5 days of age. The predominant patterns of hypoxic-ischemic injury were classified as Normal, Watershed, Basal Nuclei, Total, and Focal-Multifocal. Lesions in hippocampus, motor and occipital cortex were noted. RESULTS: Of 181 newborns, 166 (92%) survived the neonatal period, and 132 (80%) had follow-up with a median duration of 61 months (IQR: 28-95). Twenty-three children (17%) developed epilepsy. A higher proportion with Watershed, Basal Nuclei, or Total patterns developed epilepsy (P < .001). Injury to motor cortex, hippocampus, and occipital lobe (P < .01) were independent risk factors for epilepsy. In the adjusting logistic model, Watershed (odds ratio = 16.0, 95% CI [1.3, 197.2], P = .03) and Basal Nuclei injury (odds ratio = 19.4, 95% CI [1.9, 196.3], P = .01) remained independent risk factors. Therapeutic hypothermia did not alter these associations. Severity of brain injury and recurrent neonatal seizures are other clinical risk factors. SIGNIFICANCE: In term newborns with hypoxic-ischemic encephalopathy, the predominant pattern of Watershed and Basal Nuclei injury are valuable predictors for development of epilepsy in later childhood.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Discovery and Optimization of 5-Amino-1,2,3-triazole-4-carboxamide Series against Trypanosoma cruzi.

Chagas' disease, caused by the protozoan parasite Trypanosoma cruzi, is the most common cause of cardiac-related deaths in endemic regions of Latin America. There is an urgent need for new safer treatments because current standard therapeutic options, benznidazole and nifurtimox, have significant side effects and are only effective in the acute phase of the infection with limited efficacy in the chronic phase. Phenotypic high content screening against the intracellular parasite in infected VERO cells was used to identify a novel hit series of 5-amino-1,2,3-triazole-4-carboxamides (ATC). Optimization of the ATC series gave improvements in potency, aqueous solubility, and metabolic stability, which combined to give significant improvements in oral exposure. Mitigation of a potential Ames and hERG liability ultimately led to two promising compounds, one of which demonstrated significant suppression of parasite burden in a mouse model of Chagas' disease.

Use of Calculated Physicochemical Properties to Enhance Quantitative Response When Using Charged Aerosol Detection.

Universal quantitative detection without the need for analyte reference standards would offer substantial benefits in many areas of analytical science. The quantitative capability of high-performance liquid chromatography (HPLC) with charged aerosol detection (CAD) was investigated for 50 compounds with a wide range of physical and chemical properties. It is widely believed that CAD is a mass detector. Quantification of the 50 compounds using a generic calibrant and mass calibration achieved an average error of 11.4% relative to 1H NMR. Correction factors are proposed that estimate the relative surface area of particles in the detector, taking into account the effects of the density and charge of analytes. Performing these corrections and quantifying with surface area calibration, rather than mass, shows considerably improved linearity and uniformity of detection, reducing the average error relative to 1H NMR to 7.1%. The accuracy of CAD quantification was most significantly improved for highly dense compounds, with traditional mass calibration showing an average error of 34.7% and the newly proposed surface area calibration showing an average error of 5.8%.

Reliability of Early Magnetic Resonance Imaging (MRI) and Necessity of Repeating MRI in Noncooled and Cooled Infants With Neonatal Encephalopathy.

In cooled newborns with encephalopathy, although late magnetic resonance imaging (MRI) scan (10-14 days of age) is reliable in predicting long-term outcome, it is unknown whether early scan (3-6 days of life) is. We compared the predominant pattern and extent of lesion between early and late MRI in 89 term neonates with neonatal encephalopathy. Forty-three neonates (48%) were cooled. The predominant pattern of lesions and the extent of lesion in the watershed region agreed near perfectly in noncooled (kappa = 0.94; k = 0.88) and cooled (k = 0.89; k = 0.87) infants respectively. There was perfect agreement in the extent of lesion in the basal nuclei in noncooled infants (k = 0.83) and excellent agreement in cooled infants (k = 0.67). Changes in extent of lesions on late MRI occurred in 19 of 89 infants, with higher risk in infants with hypoglycemia and moderate-severe lesions in basal nuclei. In most term neonates with neonatal encephalopathy, early MRI (relative to late scan) robustly predicts the predominant pattern and extent of injury.

Detection of a novel intragenic rearrangement in the creatine transporter gene by next generation sequencing.

Deficiency caused by mutations in the creatine transporter gene (SLC6A8/CT1) is an X-linked form of intellectual disability. The presence of highly homologous pseudogenes and high GC content of SLC6A8 genomic sequence complicates the molecular diagnosis of this disorder. To minimize the pseudogene interference, exons 2 to 13 of SLC6A8 were amplified as a single PCR product using gene-specific long-range PCR (LR-PCR) primers. The GC-rich exon 1 and its flanking intronic sequences were amplified separately in a short fragment under GC-rich conditions and a touchdown PCR program. Traditional Sanger sequence analysis of all coding exons of SLC6A8 from a 3-year-old boy with creatine transporter deficiency did not detect deleterious mutations. The long-range PCR product was used as template followed by massively parallel sequencing (MPS) on HiSeq2000. We were able to detect a tandem duplication involving part of exons 11 and 12 in the SLC6A8 gene. The deduced c.1592_1639dup133 mutation was confirmed to be a hemizygous insertion by targeted genomic DNA and cDNA Sanger sequencing. Combination of deep sequencing technology with long-range PCR revealed a novel intragenic duplication in the SLC6A8 gene, providing a definitive molecular diagnosis of creatine transporter deficiency in a male patient.

Evolution of pattern of injury and quantitative MRI on days 1 and 3 in term newborns with hypoxic-ischemic encephalopathy.

BACKGROUND: Brain injury in term neonatal hypoxic-ischemic encephalopathy (HIE) emerges on magnetic resonance imaging (MRI) by day 3. This study aimed to address the relationship of MRI, diffusion tensor imaging (DTI), and MR spectroscopic imaging (MRSI) findings on days 1 and 3 in a prospective cohort of term newborns with HIE. METHODS: A total of 24 term newborns with HIE were prospectively studied with MRI on days 1 and 3; 19 were imaged with DTI and MRSI on days 1 and 3. MRI was assessed using validated scores. The relationship between MRI, DTI, and MRSI values on days 1 and 3 was determined using linear regression for repeated measures. RESULTS: Conventional MRI showed a complex variation of findings from day 1 to 3. In gray matter, mean diffusivity (Dav) and metabolite ratios measured on day 1 were predictive of values on day 3 (all P ≤ 0.04). In white matter, Dav, fractional anisotropy (FA), and N-acetylaspartate (NAA)/choline on days 1 and 3 were strongly related (all P ≤ 0.003). Hypothermia appeared to attenuate the severity and progression of brain injury in the six treated newborns. CONCLUSION: In term newborns with HIE, quantitative MR values on days 1 and 3 are strongly associated, providing an objective measure of injury before qualitative images.

Synthesis and structure-activity relationships of indazole arylsulfonamides as allosteric CC-chemokine receptor 4 (CCR4) antagonists.

A series of indazole arylsulfonamides were synthesized and examined as human CCR4 antagonists. Methoxy- or hydroxyl-containing groups were the more potent indazole C4 substituents. Only small groups were tolerated at C5, C6, or C7, with the C6 analogues being preferred. The most potent N3-substituent was 5-chlorothiophene-2-sulfonamide. N1 meta-substituted benzyl groups possessing an α-amino-3-[(methylamino)acyl]-group were the most potent N1-substituents. Strongly basic amino groups had low oral absorption in vivo. Less basic analogues, such as morpholines, had good oral absorption; however, they also had high clearance. The most potent compound with high absorption in two species was analogue 6 (GSK2239633A), which was selected for further development. Aryl sulfonamide antagonists bind to CCR4 at an intracellular allosteric site denoted site II. X-ray diffraction studies on two indazole sulfonamide fragments suggested the presence of an important intramolecular interaction in the active conformation.

Getting physical in drug discovery II: the impact of chromatographic hydrophobicity measurements and aromaticity.

Here, we review the performance of chromatographic hydrophobicity measurements in a data set of 100,000 GlaxoSmithKline compounds, demonstrating the advantages of the method over octanol-water partitioning and highlighting new insights for drug discovery. The value of chromatographic measurements, versus other hydrophobicity estimates, was supported by improved relationships with solubility, permeation, cytochrome P450s, intrinsic clearance, hERG binding and promiscuity. We also observed marked differentiation of the relative influence of intrinsic and effective hydrophobicity. The summing of hydrophobicity values plus aromatic ring count [logD(pH7.4) (or logP)+#Ar], indicated a wide relevance for simplistic 'property forecast indices' in developability assays, clearly enhanced by chromatographic values; therefore establishing new foundations for enriching property-based drug design.

The role of hypoxia-ischemia in term newborns with arterial stroke.

The role of generalized hypoxia-ischemia in the genesis of perinatal focal arterial stroke remains puzzling. Animal studies have demonstrated that hypoxia-ischemia may alter blood flow through the ductus venosus, thereby increasing the risk for placental emboli entering the cerebral circulation. A retrospective review was performed of clinical records of all term newborns admitted to a tertiary perinatal center between January 1995 and May 2007 with acute arterial stroke on neuroimaging during the first week of life. Newborns were classified into 2 groups on the basis of neuroimaging abnormalities: stroke alone, or stroke and nonfocal hypoxic-ischemic brain injury. A total of 62 newborns had focal or multifocal stroke, 36 with stroke alone and 26 with stroke with nonfocal hypoxia-ischemia. Multiple risk factors for hypoxia-ischemia occurred in most newborns in both groups. These data indicate that hypoxia-ischemia may play a role in the genesis of stroke in the term newborn with or without evidence of nonfocal hypoxic-ischemic brain injury on neuroimaging.

Estimating unbound volume of distribution and tissue binding by in vitro HPLC-based human serum albumin and immobilised artificial membrane-binding measurements.

The in vivo unbound volume of distribution (V(du)) can be used to estimate the free steady-state plasma concentration with a given dose of a drug administered intravenously. We have demonstrated that the calibrated HPLC retention times obtained on biomimetic stationary phases, such as immobilised human serum albumin and phosphatidyl-choline, can be used to estimate compounds' in vivo behaviour. The mechanistic models are based on the assumption that the sum of the albumin and phospholipid binding has the most significant impact on reducing compounds' free concentration both in plasma and in tissues. The model equations were obtained using the literature human volume of distribution and fraction unbound in plasma values of 135 known drug molecules and have been tested on a further 300 in-house compounds. The model can be used to design compounds with low V(du) values and high fraction unbound in tissues which will minimise the required dose to achieve the efficacious free concentration at the target organ (excluding possible active transport processes).

Show me the money. Developing the right compensation structure for your practice.

Keep lines of communication open to avoid common pitfalls associated with financial responsibility.

How your practice can earn $88,000 per physician over the next five years.

Your medical practice could earn as much as $88,000 per physician over the next five years through three new programs provided through the federal stimulus package. By participating in electronic health records, using electronic prescribing, and performing standard quality procedures that your practice most likely already follows, your practice could earn part of $19 billion that has been designated in the American Recovery and Reinvestment Act of 2009 to improve health information technology. You won't want to delay, however, because the funding is front-loaded, with of the funding provided in the first two years.

Getting physical in drug discovery: a contemporary perspective on solubility and hydrophobicity.

Suboptimal physical properties have been identified as a particular shortcoming of compounds in contemporary drug discovery, contributing to high attrition levels. An analysis of the relationship between hydrophobicity (calculated and measured) and approximately 100k measured kinetic solubility values has been undertaken. In line with the General Solubility Equation, estimates of hydrophobicity, particularly ACD clogD(pH7.4), give a useful indication of the likely solubility classification of particular molecules. Taking ACD clogD(pH7.4) values together with the number of aromatic rings in a given molecule provides enhanced prediction. The 'Solubility Forecast Index' (SFI=clogD(pH7.4)+#Ar) is proposed as a simple, yet effective, guide to predicting solubility. Moreover, analysis of measured distribution/partition coefficient values highlighted statistically significant shortcomings in the applicability of octanol/water as a model system for hydrophobicity determination with poorly soluble compounds.

Comparison of computer tomography and magnetic resonance imaging scans on the third day of life in term newborns with neonatal encephalopathy.

OBJECTIVE: Our goal was to compare the patterns of brain injury detected by computed tomography, conventional MRI (T1- and T2-weighted sequences), and diffusion-weighted MRI in a cohort of term newborns with neonatal encephalopathy studied uniformly with all 3 modalities on the third day of life. METHODS: Term newborns (> or =36 weeks' gestation) admitted to our center with neonatal encephalopathy were scanned with computed tomography, MRI, and diffusion-weighted MRI at 72 (+/-12) hours of life (n = 48). Each modality was scored independently of the other with previously validated scoring systems. The predominant pattern of brain injury was classified as: normal, watershed, basal nuclei, total (maximal basal nuclei and watershed), and focal-multifocal (presence of strokes and/or white matter injury alone). RESULTS: The agreement for the predominant pattern of injury was excellent between MRI and diffusion-weighted MRI (77% agreement). The agreement for the pattern of injury was also good for computed tomography and diffusion-weighted MRI (67% agreement). The extent of cortical injury and focal-multifocal lesions, such as strokes and white matter injury, were less apparent on computed tomography than diffusion-weighted MRI. In 19 newborns with a repeat MRI in the second week of life, the predominant pattern seen on the day 3 diffusion-weighted MRI was confirmed. CONCLUSIONS: Diffusion-weighted MRI is the most sensitive technique with which to assess brain injury on day 3 of life in term newborns with neonatal encephalopathy, particularly for cortical injury and focal-multifocal lesions such as stroke and white matter injury. All 3 modalities identify the most serious patterns of brain injury similarly.

White matter injury in term newborns with neonatal encephalopathy.

White matter injury (WMI) is the characteristic pattern of brain injury detected on magnetic resonance imaging in the premature newborn. Focal noncystic WMI is increasingly recognized in populations of term newborns. The aim of this study was to describe the occurrence of focal noncystic WMI in a cohort of 48 term newborns with encephalopathy studied with magnetic resonance imaging at 72 +/- 12 h of life, and to identify clinical risk factors for this pattern of injury. Eleven newborns (23%; 95% CI 11-35) were found to have WMI (four minimal, three moderate, and four severe). In 10 of the 11 newborns, the WMI was associated with restricted diffusion on apparent diffusion coefficient maps. An increasing severity of WMI was associated with lower gestational age at birth (p = 0.05), but not lower birth weight. Newborns with WMI had milder encephalopathy and fewer clinical seizures relative to other newborns in the cohort. Other brain injuries were seen in three of the 11 newborns: basal nuclei predominant pattern of injury in one and cortical strokes in two. These findings suggest that WMI in the term newborn is acquired near birth and that the state of brain maturation is an important determinant of this pattern of brain injury.

Dimeric zanamivir conjugates with various linking groups are potent, long-lasting inhibitors of influenza neuraminidase including H5N1 avian influenza.

The synthesis, antiviral and pharmacokinetic properties of zanamivir (ZMV) dimers 8 and 13 are described. The compounds are highly potent neuraminidase (NA) inhibitors which, along with dimer 3, are being investigated as potential second generation inhaled therapies both for the treatment of influenza and for prophylactic use. They show outstanding activity in a 1 week mouse influenza prophylaxis assay, and compared with ZMV, high concentrations of 8 and 13 are found in rat lung tissue after 1 week. Retention of compounds in rat lung tissue correlated both with molecular weight (excluding 3 and 15) and with a capacity factor K' derived from immobilized artificial membrane (IAM) chromatography (including 3 and 15). Pharmacokinetic parameters for 3, 8 and 13 in rats show the compounds have short to moderate plasma half-lives, low clearances and low volumes of distribution. Dimer 3 shows NA inhibitory activity against N1 viruses including the recent highly pathogenic H5N1 A/Chicken/Vietnam/8/2004. In plaque reduction assays, 3, 8 and 13 show good to outstanding potency against a panel of nine flu A and B virus strains. Consistent with its shorter and more rigid linking group, dimer 8 has been successfully crystallized.

Denitrification potential in relation to lithology in five headwater riparian zones.

The influence of riparian zone lithology on nitrate dynamics is poorly understood. We investigated vertical variations in potential denitrification activity in relation to the lithology and stratigraphy of five headwater riparian zones on glacial till and outwash landscapes in southern Ontario, Canada. Conductive coarse sand and gravel layers occurred in four of the five riparian areas. These layers were thin and did not extend to the field-riparian perimeter in some riparian zones, which limited their role as conduits for ground water flow. We found widespread organic-rich layers at depths ranging from 40 to 300 cm that resulted from natural floodplain processes and the burial of surface soils by rapid valley-bottom sedimentation after European settlement. The organic matter content of these layers varied considerably from 2 to 5% (relic channel deposit) to 5 to 21% (buried soils) and 30 to 62% (buried peat). Denitrification potential (DNP) was measured by the acetylene block method in sediment slurries amended with nitrate. The highest DNP rates were usually found in the top 0- to 15-cm surface soil layer in all riparian zones. However, a steep decline in DNP with depth was often absent and high DNP activity occurred in the deep organic-rich layers. Water table variations in 2000-2002 indicated that ground water only interacted frequently with riparian surface soils between late March and May, whereas subsurface organic layers that sustain considerable DNP were below the water table for most of the year. These results suggest that riparian zones with organic deposits at depth may effectively remove nitrate from ground water even when the water table does not interact with organic-rich surface soil horizons.

Cerebellar vermian atrophy after neonatal hypoxic-ischemic encephalopathy.

BACKGROUND AND PURPOSE: Although pathologic evidence of cerebellar injury due to birth asphyxia is well described, neuroimaging evidence is sparse. The primary purpose of this retrospective study was to evaluate the early and late imaging findings in the cerebellum of patients who had neonatal hypoxic-ischemic encephalopathy with thalamic edema shown by neonatal CT. The secondary aims were to validate thalamic edema shown by neonatal CT as a marker of thalamic injury and to assess the late cerebral cortical abnormalities associated with neonatal thalamic edema. METHODS: Fifty-five neonates with thalamic edema shown by CT performed when patients were 3 days old were identified from a cohort of full-term neonates with hypoxic-ischemic encephalopathy. Twenty-six of the 55 underwent follow-up neuroimaging. All sonograms, CT scans, and MR images of the brains of the 55 neonates were retrospectively reviewed by two pediatric neuroradiologists. The examinations were reviewed for evidence of hemorrhage, edema, atrophy, and CT attenuation or MR signal intensity abnormalities in the cerebellum, basal ganglia, and cerebral cortex. The neonatal autopsy findings in four cases were reviewed separately by a pediatric neuropathologist. RESULTS: Of the 55 neonates with thalamic edema shown by neonatal CT, 28 (51%) had thalamic edema with diffuse cerebral cortical edema, and 27 (49%) had thalamic edema without diffuse cortical edema. The cerebellar vermes appeared normal on all neonatal sonograms, CT scans, and MR images. However, atrophy of the cerebellar vermis was found in 12 (46%) of 26 patients by use of follow-up studies (95% CI, 27-65%). One of the 12 patients also had cerebellar hemispheric atrophy. Cerebellar vermian atrophy was shown at follow-up in eight (67%) of 12 patients who had neonatal thalamic edema with cortical sparing, compared with four (29%) of 14 patients who had thalamic edema with diffuse cortical edema. The difference did not reach statistical significance. The thalami appeared abnormal on follow-up neuroimages in 25 of 26 cases. Different patterns of cortical atrophy were observed on the images of patients who had thalamic edema with cortical sparing compared with those obtained in patients who had thalamic edema with cortical involvement. CONCLUSION: Cerebellar vermian atrophy is a frequent finding on follow-up images of patients in whom neonatal CT showed hypoxic-ischemic encephalopathy with abnormal thalami.

Polyploid cells in the mouse ovary.

Cell ploidy in the ovarian follicle and corpus luteum was investigated by DNA in situ hybridization to a reiterated, chromosome 3 transgene in mice that were hemizygous for the transgene. This approach was first validated by analysis of mouse kidney, pancreas and liver control tissues, which contain different frequencies of polyploid nuclei. Polyploid nuclei (with multiple hybridization signals) were seen in histological sections of both ovarian follicles and corpora lutea. The frequency of polyploid nuclei in follicles showed no consistent relationship with age (between 6 weeks and 10 months) but polyploid nuclei were significantly more abundant in corpora lutea than follicles (6.3% vs. 2.5%). This implies that production of polyploid cells is more closely associated with differentiation of ovarian follicles into corpora lutea than with the age of the female. Polyploidy tended to be more frequent in corpora lutea of mice that had mated even if they did not become pregnant. This study has highlighted the presence of polyploid cells in the mouse ovarian follicle and corpus luteum and has identified mating as a possible trigger for polyploidy in the corpus luteum. Further work is required to determine the physiological role of polyploid ovarian cells in reproduction.