Discovery and characterization of LY2784544, a small-molecule tyrosine kinase inhibitor of JAK2V617F.

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P<0.05) Ba/F3-JAK2V617F-GFP tumor burden in the JAK2V617F-induced MPN model (TED50=13.7 mg/kg, twice daily). In contrast, LY2784544 showed no effect on erythroid progenitors, reticulocytes or platelets. These data suggest that LY2784544 has potential for development as a targeted agent against JAK2V617F and may have properties that allow suppression of JAK2V617F-induced MPN pathogenesis while minimizing effects on hematopoietic progenitor cells.

PP023. Unexpected random urinary protein: creatinine ratio results - Insightsfrom clinician-laboratory medicine collaboration.

INTRODUCTION: Proteinuria assessment is important in pregnancy, particularly in determining whether or not a woman has pre-eclampsia. The random protein to creatinine ratio (PrCr) has been recommended as a confirmatory test for dipstick proteinuria in pregnancy, defined as random PrCr ⩾30mg/mmol. However, it has been our clinical impression that women with normal pregnancy outcomes have fluctuating or persistently elevated PrCr values. OBJECTIVES: As the primary goal of proteinuria testing in pregnancy should be to identify women at increased risk of adverse outcomes, we sought to explore our clinical impression that an elevated PrCr is seen not infrequently in pregnancies with normal outcome. METHODS: In this prospective cohort study, consecutive inpatients or outpatients (attending high-risk maternity clinics) were evaluated at a tertiary care facility. Random midstream urine samples were obtained as part of normal clinical care. Urine protein was measured using a pyrocatechol violet molybdate dye-binding method, and urine creatinine by an enzymatic method, both on an automated analyser (Vitros® 5.1 FS or Vitros® 5600, Ortho-Clinical Diagnostics, Rochester, NY) followed by PrCr calculation. Maternal and perinatal outcomes were abstracted from the hospital case notes. RESULTS: 160 women (81.9% outpatients) were screened at one/more antenatal visits providing a total of 233 samples for analysis. Ninety one (39.1%) samples had a random PrCr ⩾30 mg/mmol. This result was more common when urinary creatinine concentration was <3mM [64 (94.1%)] compared with ⩾3mM [27 (16.4%)], even among the 32 (20.0%) women with known normal pregnancy outcome [(13 (92.9%) vs. 0 (0%), respectively] (Panel A). In dilution studies using the same automated analyser, urinary protein (at a concentration of 0.12g/L) was 'detected' in deionised, double-distilled water. Method-specific re-analysis of data from two other published cohorts from our centre revealed substantially less inflation of PrCr values in dilute 24h urine samples tested using a pyrogallol red dye-binding based protein assay. When results were categorized according to urinary creatinine <3mM vs. ⩾3mM, PrCr ⩾30mg/mmol occurred in 12 (66.7%) vs. 99 (55.3%) respectively (p=0.35) in a 24-h urine completeness cohort and 92 (73.6%) vs. 313 (64.9%) respectively (p=0.07) in a cohort of women hospitalised for pre-eclampsia (Panel B). CONCLUSION: Random urinary PrCr results may be inflated in dilute urines because of overestimation of proteinuria in a common pyrocatechol violet dye-based method. This inflation was reduced but not eliminated when the dye used was pyrogallol red. Analytical methods do matter in the assessment of proteinuria in pregnant women. It may be prudent to consider the potential for falsely positive PrCr ⩾30mg/mmol in dilute urine, and to order PrCr testing on first voided (concentrated) urines whenever possible.

PP024. Random urine albumin: Creatinine ratio in high-risk pregnancy - Is it clinically useful?

INTRODUCTION: The albumin:creatinine ratio (ACr) is the newest of available methods of proteinuria assessment in pregnancy. Published cut-offs for detection of ⩾0.3g/d proteinuria vary from 2mg/mmol to 8mg/mmol. Up to 20% of women have an elevated ACr in pregnancy but normal outcome. In addition, it is our impression that the urine albumin component of the ACr is frequently below the detection limit of the assay. OBJECTIVES: To evaluate the frequency with which a measurable ACr can be obtained in a high-risk outpatient maternity population. METHODS: In this prospective cohort study, consecutive inpatients or outpatients (attending primarily morning high-risk maternity clinics) were evaluated at a tertiary care facility. Random midstream urine samples were obtained as part of normal clinical care. In the hospital laboratory, urinary albumin was measured using an immunoturbidimetric method, and urinary creatinine by an enzymatic method, both on an automated analyser (Vitros® 5,1 FS or Vitros® 5600, Ortho-Clinical Diagnostics, Rochester NY). ACr was calculated for samples with measurable urine albumin, and for samples with albumin below the assay range, ACr was calculated using the assay cut-off for albumin of 6.00mg/L. RESULTS: One hundred and sixty women (81.9% outpatients) were screened at one/more antenatal visits, providing a total of 233 urine samples for analysis. 68 (29.2%) urine samples were dilute (i.e., had urinary creatinine <3mM); only 13 (19.1%) of these had measurable urinary albumin for calculation of the ACr. Overall, 117/233 samples (50.2%) had measurable urine albumin that could be used to calculate the ACr. 76 (65.0%) had ACr >2mg/mmol and 34 (29.1%) had ACr >8mg/mmol. For the 116/233 (49.8%) samples with urine albumin below the assay detection limit, ACr was calculated using 6.00mg/L as the value for urine albumin. All of the 55 dilute samples had an ACr >2mg/mmol and 3 (2.6%) had an ACr >8mg/mmol. If dilute samples were excluded, none of the remaining 61 samples had an ACr value >2mg/mmol. CONCLUSION: Among a population of pregnant women attending primarily morning high-risk maternity clinics, urine is often dilute and urine albumin is often below the assay detection limit. This combination may result in uninterpretable ACr values if an ACr cut-off of 2mg/mmol is used as the decision limit for proteinuria ⩾0.3g/d. ACr may be best performed on first voided (concentrated) urine if ACr is used to assess proteinuria in pregnancy.

PP025. Urinary dipstick proteinuria testing - Does automated strip analysis offer an advantage over visual testing?

INTRODUCTION: The visual urinary test strip is widely accepted for screening for proteinuria in pregnancy, given the convenience of the method and its low cost. However, test strips are known to lack sensitivity and specificity. The 2010 NICE (National Institute for Health and Clinical Excellence) guidelines for management of pregnancy hypertension have recommended the use of an automated test strip reader to confirm proteinuria (http://nice.org.uk/CG107). Superior diagnostic test performance of an automated (vs. visual) method has been proposed based on reduced subjectivity. OBJECTIVES: To compare the diagnostic test properties of automated vs. visual read urine dipstick testing for detection of a random protein:creatinine ratio (PrCr) of ⩾30mg/mmol. METHODS: In this prospective cohort study, consecutive inpatients or outpatients (obstetric medicine and high-risk maternity clinics) were evaluated at a tertiary care facility. Random midstream urine samples (obtained as part of normal clinical care) were split into two aliquots. The first underwent a point-of-care testing for proteinuria using both visual (Multistix 10SG, Siemens Healthcare Diagnostics, Inc., Tarrytown NY) and automated (Chemstrip 10A, Roche Diagnostics, Laval QC) test strips, the latter read by an analyser (Urisys 1100®, Roche Diagnostics, Laval QC). The second aliquot was sent to the hospital laboratory for analysis of urinary protein using a pyrocatechol violet molybdate dye-binding method, and urinary creatinine using an enzymatic method, both on an automated analyser (Vitros® 5,1 FS or Vitros® 5600, Ortho-Clinical Diagnostics, Rochester NY); random PrCr ratios were calculated in the laboratory. Following exclusion of dilute samples with urinary creatinine concentration <3mM (given inflation of PrCr values in dilute urine by our method), diagnostic test properties were determined for visual and automated dipstick proteinuria testing (⩾1+) for detection of a random PrCr ⩾30mg/mmol. RESULTS: 160 women (81.9% outpatients) were screened at one/more antenatal visits, providing a total of 233 urine samples for analysis. Both visual and automated read urinary dipstick testing showed low sensitivity (56.0% and 53.9%, respectively). Positive likelihood ratios (LR+) and 95% CI were 15.0 [5.9,37.9] and 24.6 [7.6,79.6], respectively. Negative LR (LR-) were 0.46 [0.29,0.71] and 0.47 [0.31,0.72], respectively. CONCLUSION: Automated dipstick urinalysis is not more sensitive than visual read urinalysis for detection of proteinuria in a primarily outpatient setting in pregnancy. Both have excellent LR+ but only fair to poor LR- as previously recognised for visual dipstick testing. Performance of automated strip analysis testing may vary with the test strips and analyser used.

Validation of a method for the simultaneous determination of four schisandra lignans in the raw herb and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi) by RP-LC with DAD.

A rapid and specific reversed-phase high performance liquid chromatography (RP-LC) method with photodiode array detection (DAD) was developed and validated for the determination of four common schisandra lignans, schisandrin (1), schisandrol B (2), deoxyshisandrin (3) and gamma-schisandrin (4), in raw herb materials and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi). The extraction solvent and extraction method were optimised where it was found that a 4 h Soxhlet extraction using methanol was successful at extracting >99.5% of each of the schisandra lignans analysed from the raw herb material. The sample preparation process for the dried aqueous extract samples involved sonication using methanol for 2 x 30 min. The herb and extract solutions were separated on a Varian Microsorb-MV 100-5 C18 column using a gradient mixture of 0.1% aqueous phosphoric acid and acetonitrile. Subsequent detection and quantitation of the schisandra lignans was performed at 210 nm. The correlation coefficients of the linear regression analysis performed on these calibration curves were >0.9996 for all four schisandra lignans assayed. The detection limits and quantification limits ranged from 0.12 to 0.57 and 0.41 to 1.89 mg g(-1), respectively. The mean recoveries of the various analytes ranged from 92.20 to 107.01%. The method was used to investigate the levels of the four mentioned components in herb samples and dried aqueous extracts. The identities of the chromatographic peaks were confirmed by (+) electrospray ionisation LC-MS/MS.

Dental radiology and oral pathology.

This article represents an overview of normal and pathological findings of the oral structures for the practicing radiologist. Some of the disease processes discussed are of dental etiology, whereas others are manifestations of systemic diseases. Normal tooth development is described followed by an overview of radiolucent, radiopaque, and mixed lucent-opaque lesions. In the radiolucent category, normal anatomical landmarks of the jaws include the mental foramen, manidbular canal, incisive canal and maxillary sinuses. Other lucencies that represent normal structures or variations include developing tooth buds and root apices, healing dental extraction sites, prominent submandibular fossae and nutrient canals. In the radiopaque category, normal anatomical landmarks of the jaws include the genial tubercle, mental ridge, external oblique ridge, walls of the submandibular canal, walls of the maxillary sinus, nasal septum, stylohyoid ligament, and zygomatic process. Other opacities that represent normal structures or variations include dental restoration material, dental endodontic material, retained roots, sialoliths, salivary duct calculi, calcified lymph nodes, recent extraction sites, hypercementosis, and foreign bodies. Pathological entities which present radiographically as lucencies, opacities, and mixed lucent-opaque areas include inflammatory lesions, cysts, fibro-osseous disease, benign neoplasms, malignant neoplasms, and lesions secondary to metabolic disorders.