Comparison of porcine urine and bile as matrices to screen for the residues of two sulfonamides using a semi-automated enzyme immunoassay.

Porcine urine enzyme immunoassays for sulfamethazine and sulfadiazine have previously been employed as screening tests to predict the concentrations of the drugs in the corresponding tissues (kidneys), If a urine was found positive (> 800 ng ml(-1)) the corresponding kidney was then analysed by an enzyme immunoassay and, if found positive, a confirmatory analysis by HPLC was performed. Urine was chosen as the screening matrix since sulfonamides are mainly eliminated through this body fluid, However, after obtaining a number of false positive predictions, an investigation was carried out to assess the possibility of using an alternative body fluid which would act as a superior indicator of the presence of sulfonamides in porcine kidney, An initial study indicated that serum, plasma and bile could all be used as screening matrices. From these, bile was chosen as the preferred sample matrix and an extensive study followed to compare the efficiencies of sulfonamide positive bile and urine at predicting sulphonamide positive kidneys, Bile was found to be 17 times more efficient than urine at predicting a sulfamethazine positive kidney and 11 times more efficient at predicting a sulfadiazine positive kidney, With this enhanced performance of the initial screening test, the need for the costly and time consuming kidney enzyme immunoassay, prior to HPLC analysis, was eliminated

Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor

During the course of our research efforts to develop a potent and selective gamma-secretase inhibitor for the treatment of Alzheimer's disease, we investigated a series of carboxamide-substituted sulfonamides. Optimization based on potency, Notch/amyloid-beta precursor protein selectivity, and brain efficacy after oral dosing led to the discovery of 4 (BMS-708163). Compound 4 is a potent inhibitor of gamma-secretase (A beta 40 IC50 = 0.30 nM), demonstrating a 193-fold selectivity against Notch. Oral administration of 4 significantly reduced A beta 40 levels for sustained periods in brain, plasma, and cerebrospinal fluid in rats and dogs.

Biosensor assay of sulfadiazine and sulfamethazine residues in pork

Biosensor-based immunochemical screening assays for the detection of sulfadiazine (SDZ) and sulfamethazine (SMT) in muscle extract from pigs were developed. Samples were extracted with aqueous buffer and then centrifuged. This simple and straightforward preparation allowed up to 40 samples to be processed and analysed in 1 d. The limits of detection for the assays were found to be 5.6 ng g(-1) for SDZ and 7.4 ng g(-1) for SMT. These figures were well below the European and US legal limits for sulfonamides (100 ng g(-1)). The precision (RSD) between runs was

Multi sulfonamide screening in porcine muscle using a surface plasmon resonance biosensor

A binding protein displaying broad-spectrum cross-reactivity within the sulfonamide group was used in conjunction with a sulfonamide specific sensor chip and a surface plasmon resonance biosensor to develop a rapid broad spectrum screening assay for sulfonamides in porcine muscle. Results for 40 samples were available in just over 5 h after the completion of a simple sample preparation protocol. Twenty sulfonamide compounds were detected. Acetylated metabolites were not recognised by the binding protein. Limit of detection (mean-three times standard deviation value when n = 20) was calculated to be 16.9 ng g(-1) in tissue samples. Intra-assay precision (n = 10) was calculated at 4.3 %CV for a sample spiked at 50 ng g(-1) with sulfamethazine, 3.6 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 7.2 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 3.1 %CV for a sample spiked at 100 ng g-1 with sulfadiazine. Inter-assay precision (n = 3) was calculated at 9.7 %CV for a sample spiked at 50 ng g-1 with sulfamethazine, 3.8 %CV for a sample spiked at 100 ng g(-1) with sulfamethazine, 3.5 %CV for a sample spiked at 50 ng g(-1) with sulfadiazine and 2.8 %CV for a sample spiked at 100 ng g(-1) with sulfadiazine. (C) 2004 Elsevier B.V. All rights reserved.

c-Met inhibition in a HOXA9/Meis1 model of CN-AML

BACKGROUND: Hematopoiesis is a paradigm for developmental processes, hierarchically organized, with stem cells at its origin. Hematopoietic stem cells (HSCs) replenish progenitor and precursor cells of multiple lineages, which normally differentiate into short-lived mature circulating cells. Hematopoiesis has provided insight into the molecular basis of tissue homeostasis and malignancy. Malignant hematopoiesis, in particular acute myeloid leukemia (AML), results from impaired development or differentiation of HSCs and progenitors. Co-overexpression of HOX and TALE genes, particularly the HOXA cluster and MEIS1, is associated with AML. Clinically relevant models of AML are required to advance drug development for an aging patient cohort.

RESULTS: Molecular analysis identified altered gene, microRNA, and protein expression in HOXA9/Meis1 leukemic bone marrow compared to normal controls. A candidate drug screen identified the c-Met inhibitor SU11274 for further analysis. Altered cell cycle status, apoptosis, differentiation, and impaired colony formation were shown for SU11274 in AML cell lines and primary leukemic bone marrow.

CONCLUSIONS: The clonal HOXA9/Meis1 AML model is amenable to drug screening analysis. The data presented indicate that human AML cells respond in a similar manner to the HOXA9/Meis1 cells, indicating pre-clinical relevance of the mouse model.

BRAF mutation testing algorithm for vemurafenib treatment in melanoma: recommendations from an expert panel.

The incidence of melanoma has increased rapidly over the past 30 years, and the disease is now the sixth most common cancer among men and women in the U.K. Many patients are diagnosed with or develop metastatic disease, and survival is substantially reduced in these patients. Mutations in the BRAF gene have been identified as key drivers of melanoma cells and are found in around 50% of cutaneous melanomas. Vemurafenib (Zelboraf(®) ; Roche Molecular Systems Inc., Pleasanton, CA, U.S.A.) is the first licensed inhibitor of mutated BRAF, and offers a new first-line option for patients with unresectable or metastatic melanoma who harbour BRAF mutations. Vemurafenib was developed in conjunction with a companion diagnostic, the cobas(®) 4800 BRAF V600 Mutation Test. The purpose of this paper is to make evidence-based recommendations to facilitate the implementation of BRAF mutation testing and targeted therapy in patients with metastatic melanoma in the U.K. The recommendations are the result of a meeting of an expert panel and have been reviewed by melanoma specialists and representatives of the National Cancer Research Network Clinical Study Group on behalf of the wider melanoma community. This article is intended to be a starting point for practical advice and recommendations, which will no doubt be updated as we gain further experience in personalizing therapy for patients with melanoma.