Correlation Between Rod Fracture and Shear Stress: A Novel Parameter.

BACKGROUND: We sought to assess the accuracy of a novel parameter proportional to the rod shear stress (RSS) in identifying patients at risk of rod fracture (RF) after surgery for correction of adult spinal deformity. METHODS: We performed a retrospective medical record review of patients aged ≥18 years treated for adult spinal deformity between 2004 and 2014 with ≥24 months of follow-up. The primary outcome was RFs identified radiographically. Patient weight (w), number of instrumented levels (N), and minimum rod diameter (d) were recorded and used to calculate the RSS parameter (RSS=Nwd2). Receiver operating characteristic curves were produced and the area under the curve (AUC ± 95% confidence interval [CI]) was calculated to compare this parameter's discriminative accuracy to that of its constituent variables. The sensitivity, specificity, and likelihood ratios (LRs) were calculated. RESULTS: A total of 28 RF-positive and 154 RF-negative patients were included. The average age was 59.2 ± 9.6 years, and 93.4% were women. The RSS parameter produced the greatest AUC (0.73 ± 0.11). At an RSS cutoff of 30.1, it achieved a sensitivity of 71.4% and specificity of 71.4% (LR, 2.5; 95% CI, 1.8-3.5). The number of instrumented levels produced the next-greatest AUC (0.65 ± 0.12), with a sensitivity of 78.6% and specificity of 50.0% at a cutoff of 15 (LR, 1.6; 95% CI, 1.2-2.0). CONCLUSIONS: The RSS is calculated using easily obtainable information and shows potential as a tool for predicting patient-specific risk of RF after spinal fusion. The number of instrumented levels also correlates strongly with the occurrence of RFs and is not significantly less accurate than the RSS. A larger sample size and prospective validation would be useful in determining with greater confidence which parameter is superior for predicting RFs after spinal fusion.

Improved 2-pyridyl reductive homocoupling reaction using biorenewable solvent Cyrene™ (dihydrolevoglucosenone).

The synthesis of 5,5'-bis(trifluoromethyl)-2,2'-bipyridine using 2-bromo-5-(trifluoromethyl) pyridine was achieved at 50 °C using palladium acetate, tetrabutylammonium iodide (TBAI), potassium carbonate, and isopropanol in Cyrene™ (dihydrolevoglucosenone), a bio-renewable "green" solvent formed by a two-step process from cellulose. Improvements were achieved with 50% of γ-valerolactone (GVL) in Cyrene™ resulting in a 95% yield and 99% product purity without the use of column chromatography or recrystallization. At 80 °C, the reaction was completed within 1 h. Full conversion with 1 mol% instead of 15 mol% of palladium acetate was observed within 10 h. We showed that the formed 2,2'-bipyridine product significantly accelerated the reaction probably due to the stabilization of the catalytic species. The addition of TBAI was essential for the rapid homocoupling, however, 20 mol% of TBAI was sufficient to reach full conversion of 2-bromo-5-(trifluoromethyl) pyridine within 6 h at 80 °C. Another improvement was observed with the substitution of isopropanol by 1,4-butanediol achieving full conversion within 6 h. 2-Bromopyridines with electron withdrawing substituents in the 6, 5, 4 ring position reacted under these conditions. 2-Bromopyridines with an electron donating substituent reacted slower. Overall, we demonstrated that the 50% GVL in Cyrene™ blend is a superior "green" and less toxic alternative to dimethylformamide for the reductive homocoupling reaction. Using a quantitative scoring for twelve principles of green chemistry (DOZN™), we found significant improvements that were mediated by higher yield (atom economy), shorter heating time and lower reaction temperature (energy efficiency), safer solvent (hazardous chemical synthesis), and safer chemistry (accident prevention).

Design, Synthesis, and Biological Evaluation of Novel Spiro Imidazobenzodiazepines to Identify Improved Inhaled Bronchodilators.

Novel gamma-aminobutyric acid receptor (GABAAR) ligands structurally related to imidazobenzodiazepine MIDD0301 were synthesized using spiro-amino acid N-carboxyanhydrides (NCAs). These compounds demonstrated increased resistance to phase 2 metabolism and avoided the formation of a 6H isomer. Compound design was guided by molecular docking using the available crystal structure of the α1ß3γ2 GABAAR and correlated with in vitro binding data. The carboxylic acid containing GABAAR ligands have high aqueous solubility, low permeability, and low cell toxicity. The inability of GABAAR ligands to cross the blood-brain barrier was confirmed in vivo by the absence of sensorimotor inhibition. Pharmacological activities at lung GABAARs were demonstrated by ex vivo relaxation of guinea pig airway smooth muscle and reduction of methacholine-induced airway hyperresponsiveness (AHR) in conscious mice. We identified bronchodilator 5c with an affinity of 9 nM for GABAARs that was metabolically stable in the presence of human and mouse microsomes.

Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization.

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAAR is a ligand-operated Cl- channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABAARs, affording a seemingly logical site of action, the mechanism(s) by which GABAAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction in vivo and relaxes peripheral airways preconstricted with MCh ex vivo using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAAR-dependent, as it is not inhibited by the GABAAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca2, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca2+ influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca2+ oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca2+ mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP3). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca2+ mobilization in ASM, likely by inhibiting Ca2+ release through IP3Rs (IP3 receptors).

Comparative pharmacodynamic and pharmacokinetic study of MIDD0301 and its (S) enantiomer.

MIDD0301 is being developed as an oral drug to relax airway smooth muscle (ASM) and reduce lung inflammation in asthma. We report a comparative study of MIDD0301 and its S isomer (MIDD0301S), and found that the compounds have equivalent affinity for γ-aminobutyric acid type A receptor (GABAA R) expressed in rat brain, with half maximal inhibitory concentration values of 25.1 and 26.3 nM for the S and R enantiomers, respectively. Both compounds relaxed substance P contracted ASM within 30 min and neither enantiomer revealed affinity to 48 receptors in an off-target screen. Both enantiomers reduced airway hyperresponsiveness (AHR) with nebulized and oral dosing in two mouse models of bronchoconstriction. In A/J mice, which are very sensitive to methacholine-induced bronchoconstriction, we observed reduction of AHR at 10.8 mg/kg MIDD0301 and 15 mg/kg MIDD0301S. Using oral administration, 100 mg/kg/day for 3 days of either enantiomer was sufficient to reduce AHR. In a model of severe airway inflammation induced by interferon-γ and lipopolysaccharide (LPS), we observed reduction of AHR at 7.2 mg/kg for both enantiomers using nebulized administration, and at 100 mg/kg for oral administration. MIDD0301 and MIDD0301S did not undergo Phase I metabolism. Glucuronidation was observed for both compounds, whereas only MIDD0301 formed the corresponding glucoside in the presence of kidney microsomes. Pharmacokinetic analysis identified glucuronides as the major metabolite with concentrations up to 20-fold more than the parent compound. MIDD0301 glucuronide and MIDD0301 taurine bind GABAA Rs, although 10-fold weaker than MIDD0301. In mouse blood, the taurine adduct was only observed for MIDD0301. Overall, both compounds exhibited similar receptor binding and pharmacodynamic properties with subtle differences in metabolism and greater oral availability and blood concentrations of MIDD0301S.

Development of Inhaled GABAA Receptor Modulators to Improve Airway Function in Bronchoconstrictive Disorders.

We report the modification of MIDD0301, an imidazodiazepine GABAA receptor (GABAAR) ligand, using two alkyl substituents. We developed PI310 with a 6-(4-phenylbutoxy)hexyl chain as used in the long-acting ß2-agonist salmeterol and PI320 with a poly(ethylene glycol) chain as used to improve the brain:plasma ratio of naloxegol, a naloxone analogue. Both imidazodiazepines showed affinity toward the GABAAR binding site of clonazepam, with IC50 values of 576 and 242 nM, respectively. Molecular docking analysis, using the available α1ß3γ2 GABAAR structural data, suggests binding of the diazepine core between the α1+/γ2- interface, whereas alkyl substituents are located outside the binding site and thus interact with the protein surface and solvent molecules. The physicochemical properties of these compounds are very different. The solubility of PI310 is low in water. PEGylation of PI320 significantly improves aqueous solubility and cell permeability. Neither compound is toxic in HEK293 cells following exposure at >300 µM for 18 h. Ex vivo studies using guinea pig tracheal rings showed that PI310 was unable to relax the constricted airway smooth muscle. In contrast, PI320 induced muscle relaxation at organ bath concentrations as low as 5 µM, with rapid onset (15 min) at 25 µM. PI320 also reduced airway hyper-responsiveness in vivo in a mouse model of steroid-resistant lung inflammation induced by intratracheal challenge with INFγ and lipopolysaccharide (LPS). At nebulized doses of 7.2 mg/kg, PI320 and albuterol were equally effective in reducing airway hyper-responsiveness. Ten minutes after nebulization, the lung concentration of PI320 was 50-fold that of PI310, indicating superior availability of PI320 when nebulized as an aqueous solution. Overall, PI320 is a promising inhaled drug candidate to quickly relax airway smooth muscle in bronchoconstrictive disorders, such as asthma. Future studies will evaluate the pharmacokinetic/pharmacodynamic properties of PI320 when administered orally.

Identification and Quantification of MIDD0301 Metabolites.

BACKGROUND: MIDD0301 is an oral asthma drug candidate that binds GABAA receptors on airway smooth muscle and immune cells. OBJECTIVE: The objective of this study is to identify and quantify MIDD0301 metabolites in vitro and in vivo and determine the pharmacokinetics of oral, IP, and IV administered MIDD0301. METHODS: In vitro conversion of MIDD0301 was performed using liver and kidney microsomes/S9 fractions followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). A LC-MS/MS method was developed using synthesized standards to quantify MIDD0301 and its metabolites in urine and feces. Blood, lung, and brain were harvested from animals that received MIDD0301 by oral, IP, and IV administration, followed by LCMS/ MS quantification. Imaging mass spectrometry was used to demonstrate the presence of MIDD0301 in the lung after oral administration. RESULTS: MIDD0301 is stable in the presence of liver and kidney microsomes and S9 fractions for at least two hours. MIDD0301 undergoes conversion to the corresponding glucuronide and glucoside in the presence of conjugating cofactors. For IP and IV administration, unconjugated MIDD0301 together with significant amounts of MIDD0301 glucoside and MIDD0301 taurine were found in urine and feces. Less conjugation was observed following oral administration, with MIDD0301 glucuronide being the main metabolite. Pharmacokinetic quantification of MIDD0301 in blood, lung, and brain showed very low levels of MIDD0301 in the brain after oral, IV, or IP administration. The drug half-life in these tissues ranged between 4-6 hours for IP and oral and 1-2 hours for IV administration. Imaging mass spectrometry demonstrated that orally administered MIDD0301 distributes uniformly in the lung parenchyma. CONCLUSION: MIDD0301 undergoes no phase I and moderate phase II metabolism.

Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Prevalence of obesity in korean adolescents and its relationship with the weekly frequency of the physical education classes.

The purpose of this study was to investigate differences in the prevalence of obesity among Korean adolescents and to determine the relationship of obesity prevalence with weekly frequency of physical education (PE) classes. In 2009, 72,399 students from grades 7 to 12 participated in the fifth Korea Youth Risk Behavior Web-based Survey (KYRBWS-V) project. Body mass index (BMI) and the frequency of PE classes attended were assessed by the KYRBWS- V. BMI was computed to classify the participants as underweight, normal weight, overweight, and obese. The association between the frequency of PE classes and BMI were examined using one-way ANOVA and logistic regression analysis. The differences in the weekly frequency of PE classes and the BMI values among both the boys and girls were significant (p < 0.001). A post-hoc test showed that underweight boys and girls attended the PE classes more frequently (p < 0.001), and overweight girls attended these classes less frequently (p < 0.01) than the other groups did; moreover, obese boys and girls, compared to boys and girls in the other groups, attended less number of PE classes per week while at school (p < 0.05). Besides, the odds ratio (95% confidence interval, CI) for normal-weight vs. underweight boys attending 1 PE class, 2 PE classes, and ≥ 3 PE classes per week were 1.168 (1.011-1.349, p = 0.035), 1.621 (1.450-1.812, p < 0.001), and 3.023 (2.704-3.381, p < 0.001), respectively, compared with those for boys who did not attend PE classes. The OR (95% CI) of normal-weight vs. obese boys attending ≥ 3 PE classes attended across normal vs. obese boys was 0.862 (0.762-0.974, p = 0.017), compared with those of boys who did not attend PE classes. The OR (95% CI) for normal-weight vs. underweight girls who attended 2 PE classes and ≥ 3 PE classes per week were 1.235 (1.131-1.349, p < 0.001) and 2.238 (2.048-2.446, p < 0.001), respectively, compared with those of girls who did not attend PE classes. The OR (95% CI) of for normal-weight vs. overweight girls who attended ≥ 3 PE classes per week were 0.886 (0.787- 0.997, p = 0.045) and 0.772 (0.679-0.878, p < 0.001), respectively, compared with those of girls who did not attend PE classes. The OR (95% CI) for normal-weight vs. obese girls who attended 2 PE classes and ≥ 3 PE classes per week were 0.788 (0.675-0.919, p = 0.002) and 0.709 (0.599-0.838, p < 0.001), respectively, compared with those of girls who did not attend the PE class. Increase in the frequency of PE classes should be considered in any attempt for curbing weight-related problems in Korean adolescents. Key pointsIncrease in the frequency of PE classes is a factor that should be considered to improve weight status.

Persistent syringomyelia following pediatric Chiari I decompression: radiological and surgical findings.

OBJECT: The incidence of syringomyelia in patients with Chiari I malformation is reported to be between 20 and 75%. In the series reported herein 10.6% of patients with both clinical entities have continued to have a significant syrinx following their first decompressive procedure. All but one patient had resolution of their syrinx after a second posterior fossa operation. The authors have analyzed this smaller group of patients to look for possible radiological or surgical findings that may aid in predicting which patients are less likely to respond to the initial first decompressive procedure. METHODS: The authors retrospectively reviewed radiological and operative data in eight patients who continued to have syringomyelia following a decompressive procedure. Seven (88%) of these patients had complete resolution of their syrinx following a second operation. At repeated operation, obstruction at the foramen of Magendie was seen in six patients. In one patient in whom the dura was not opened during first operation, the second operation revealed an arachnoid veil that occluded the foramen of Magendie. CONCLUSIONS: No single radiological measurement was found to aid in the prediction of which patients would not respond to the first decompressive procedure. Furthermore, no operative finding was extraordinarily unique to any single patient. All but one patient in whom confirmation of a patent foramen of Magendie was made at repeated operation-that is, lysing of arachnoid veils, stent placement, unilateral tonsillar coagulation-had resolution of their syringomyelia. Surgical reexploration should be considered in cases of persistent syringomyelia.

Posterior cranial fossa volume in patients with rickets: insights into the increased occurrence of Chiari I malformation in metabolic bone disease.

OBJECTIVE: Some have proposed that the calvarial thickening seen in patients with rickets results in an increased rate of Chiari I malformation (CIM) in these patients. The present study measures the posterior fossa volume in children with rickets to verify previous case reports indicting a small posterior fossa as the cause for an increased rate of CIM in children with rickets. METHODS: Patients were chosen by use of a computer database to search for individuals diagnosed with rickets. Nineteen patients were identified with this diagnosis. Seven patients were found from this cohort to have imaging of the head. Axial computed tomographic and magnetic resonance images were analyzed by use of the Cavalieri method to define posterior fossa volumes. These data were then compared with those from age-matched control subjects. RESULTS: Mean volumes of the posterior fossa were significantly reduced in all patients compared with age-matched control subjects (P < 0.0001). CONCLUSION: We have found that the volume of the posterior fossa is significantly smaller in children with rickets versus age-matched control subjects. Furthermore, 29% of our study group had an associated CIM. We may hope that these data will aid in the further understanding of the pathophysiology of CIM in cases of metabolic bone disease.

A colormetric assay for catechol-O-methyltransferase.

A series of catechol diazo dyes were synthesized and tested as substrates for the enzyme catechol-O-methyltransferase (COMT) with the aim of developing a sensitive HPLC assay method using visible wavelength light detection. A method was developed which allowed for the determination of the two regioisomeric methylated products of the COMT catalyzed reaction of 4-[(3,4-dihydroxyphenyl)azo]benzenesulfonate with S-adenosylmethionine (AdoMet). Separation of the assay components was achieved by reverse phase chromatography using an isocratic mobile phase. No pre-preparation of the assay samples was required.

Delayed resolution of Chiari I-associated hydromyelia after posterior fossa decompression: case report and review of the literature.

OBJECTIVE AND IMPORTANCE: The expected time interval for resolution of hydromyelia after Chiari I decompression is lacking in the literature. This case report highlights one instance of delayed resolution of Chiari-induced hydromyelia. CLINICAL PRESENTATION: We report an adolescent girl with a Chiari I malformation and hydromyelia. INTERVENTION: A suboccipital craniectomy and C1 laminectomy with intradural exploration and duraplasty were performed. Serial imaging at 1 and 2 years after posterior cranial fossa decompression with duraplasty demonstrated no change in the size of the hydromyelia. At 3 years after surgery and before reoperation for continued hydromyelia, repeat magnetic resonance imaging demonstrated significant diminution of the fluid cavity. If this hydromyelia did resolve as a result of surgery, the interval for radiological observation clearly needs to be reconsidered. CONCLUSION: Unfortunately, this is an area in which the literature is lacking. With this case as a nidus, studies are now necessary to determine the range of time necessary for Chiari I malformation-related hydromyelia to resolve.

Magnetic resonance imaging evidence of posterior pharynx denervation in pediatric patients with Chiari I malformation and absent gag reflex.

OBJECT: The authors hypothesized that children with preoperative Chiari I malformation and an absent gag reflex may harbor pharyngeal musculature atrophy identifiable on magnetic resonance (MR) imaging. METHODS: Thirty patients with preoperative Chiari I malformation and a functioning gag reflex, five patients with preoperative Chiari I malformation and complete absence of gag reflex, and 50 control individuals underwent radiological measurement of the posterior pharyngeal wall thickness. The thickness of the posterior pharyngeal wall in age-matched controls was significantly thinner (p < 0.0001) than that in patients with Chiari I malformation and no functioning gag reflex. Additionally, in patients with hindbrain herniation and absent gag reflex, the posterior pharyngeal wall thicknesses were comparable to or thinner than those in age-matched controls. A general decrease in the thickness of the posterior pharyngeal wall was found in control individuals who were older compared with patients with Chiari I malformation and a preserved gag reflex in whom the prevertebral soft tissues were found to increase in thickness with age. Analysis of these data showed that in children with a nonfunctioning gag reflex the prevertebral soft tissue, composed primarily of the superior constrictor muscle, is statistically thinner compared with that in age-matched controls and age-matched children with Chiari I malformation and a functioning gag reflex. The authors theorize that the discrepancy between this measurement in controls and patients with hindbrain herniation is due to the thickening of the craniocervical ligaments that is known to occur in this clinical entity. CONCLUSIONS: The finding that prevertebral soft tissue thickens with age in patients with Chiari I malformation and functioning gag reflex alone may aid in the interpretation of soft-tissue injury following cervical spine injuries in this group.

Effect of lameness on milk yield in dairy cows.

OBJECTIVE: To examine the relationship between lameness and milk yield in dairy cows. DESIGN: Cohort study. ANIMALS: 531 dairy cows. PROCEDURE: Cows affected with lameness were classified into 1 of 3 groups on the basis of type of diseases or lesions observed, including interdigital phlegmon (foot rot), papillomatous digital dermatitis (foot warts), or claw lesions. Cows not affected with lameness were classified as healthy. From Dairy Herd Improvement Association records, 305-day mature equivalent milk yield data were collected at the end of lactation or when the cow left the herd. Milk yield was compared between cows affected with lameness and healthy cows. RESULTS: 167 (31%) cows were affected with lameness during lactation. Lame cows had claw lesions (60%), papillomatous digital dermatitis (31%), or interdigital phlegmon (9%). Milk yield in lame cows with interdigital phlegmon (mean, 17,122 lb) was significantly less, compared with healthy cows (19,007 lb). CONCLUSIONS AND CLINICAL RELEVANCE: In this herd, interdigital phlegmon was associated with a 10% decrease in milk production. Lame cows with claw lesions or papillomatous digital dermatitis produced less milk than healthy cows, but the difference was not significant.