Metabolic activation of fluoropyrrolidine dipeptidyl peptidase-IV inhibitors by rat liver microsomes.

The current study evaluated the potential for two dipeptidyl peptidase-IV (DPP-IV) inhibitor analogs (1S)-1-(trans-4-([(4-trifluoromethoxyphenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride and (1S)-1-(trans-4-([(2,4-difluorophenyl)sulfonyl]amino)cyclohexyl)-2-[(3S)-3-fluoropyrrolidin-1-yl]-2-oxoethanaminium chloride (MRL-A and MRL-B), containing a fluoropyrrolidine moiety in the structure, to undergo metabolic activation. The irreversible binding of these tritium-labeled compounds to rat liver microsomal protein was time- and NADPH-dependent and was attenuated by the addition of reduced glutathione (GSH) or N-acetylcysteine (NAC) to the incubation, indicating that chemically reactive intermediates were formed and trapped by these nucleophiles. Mass spectrometric analyses and further trapping experiments with semicarbazide indicated that the fluoropyrrolidine ring had undergone sequential oxidation and defluorination events resulting in the formation of GSH or NAC conjugates of the pyrrolidine moiety. The bioactivation of MRL-A was catalyzed primarily by rat recombinant CYP3A1 and CYP3A2. Pretreatment of rats with prototypic CYP3A1 and 3A2 inducers (pregnenolone-16alpha-carbonitrile and dexamethasone) enhanced the extent of bioactivation which, in turn, led to a higher degree of in vitro irreversible binding to microsomal proteins (5- and 9-fold increase, respectively). Herein, we describe studies that demonstrate that the fluoropyrrolidine ring is prone to metabolic activation and that GSH or NAC can trap the reactive intermediates to form adducts that provide insight into the mechanisms of bioactivation.     

Oncologic outcomes between open and robotic-assisted radical cystectomy: a propensity score matched analysis

Purpose

To compare oncologic outcomes between open radical cystectomy (ORC) and robotic-assisted radical cystectomy (RARC) using propensity score (PS) matching of preoperative variables.

Methods

A group of 51 consecutive patients who underwent RARC between 2009 and 2012 were matched by propensity scoring with an equal number of patients who underwent ORC. Patient demographics, clinical staging, pathologic staging, pathologic grading, histology, positive margin status, lymph node yield, duration of hospital stay, and overall survival were examined.

Results

PS-matched ORC and RARC cohorts demonstrated no significant differences with respect to preoperative variables, pathologic stage, grade, histology, metastasis at preoperative staging, and postoperative positive margin status. There were statistically significant differences in nodal status (66.7 % N0 for ORC vs. 80.4 % N0 for RARC, p = 0.039) and median lymph node yield (6 for ORC vs. 18 for RARC, p < 0.0001). No positive soft tissue margins were observed in the RARC group compared to 5.9 % in the ORC group (p = 0.332). There were no significant differences in mean duration of hospital stay or mean overall survival between ORC and RARC.

Conclusion

ORC and RARC represent effective surgical approaches for the treatment of bladder cancer. Histopathologic outcomes for RARC compare favorably to ORC with respect to soft tissue margin rates and lymph node yield. These data suggest that RARC is an acceptable surgical approach for treatment of bladder cancer that can achieve outcomes that are equal or superior to those of ORC.     

Discovery and optimization of triazolopyridazines as potent and selective inhibitors of the c-Met kinase

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.     

Assessment of the implementation of HIV-rapid test kits at different levels of health institutions in Ethiopia

OBJECTIVE: Evaluation and monitoring of Human Immunodeficiency Virus (HIV) testing reagents at the point of service is helpful to prevent the occurrence of problems related to testing and interpretation. To evaluate the implementation of HIV rapid test kits at the point of services in voluntarily counseling and testing (VCT) and diagnostic centers in Ethiopia. METHODS: The assessment was the third phase of evaluation of HIV rapid test kits in Ethiopia followed from phase-I and phase-II. Known proficiency testing panels, well-structured questionnaire (addressing type of tests, human resource and problems related to tests), onsite supervision and retesting of samples collected from sites were used to evaluate the performances of reagents and laboratories. RESULTS: Forty-four health institutions were included. Thirty-six (90.0%) health institutions had trained human resource on HIV testing. In 27 (61.4%) three types of HIV rapid test kits (Determine, Capillus and Unigold) were available. Serial-algorithm was used in all the laboratories. In 31 (70.4%) of them external quality control specimens were not used. Twenty two (50.0%) of the laboratories reported frequent shortage of reagents. All (100%) were able to identify negative specimens distributed. Positive proficiency panel samples were identified in 37 (94.8%) of the 39 laboratories. There was 98.3% agreement at a screening level between the sites and the central laboratory. Rate of discrepancy between screening and confirmatory assays was found to be 3.0% and 2.1% at the sites and at central laboratory, respectively. CONCLUSION: The test kits showed a good performance at the point of services in the field sites. However, continuous assessment of HIV test kits at the point of service and training of professionals on newly arrived techniques are recommended to have effective testing performance with acceptable sensitive and specific testing algorithm. Effective quality assurance program should be in place to support programs such as VCT, prevention of mother-to-child-transmission and antiretroviral therapy.     

Network compensation of cyclic GMP-dependent protein kinase II knockout in the hippocampus by Ca2+-permeable AMPA receptors

Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca²⁺ signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca²⁺-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca²⁺-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.Some gene deletions yield no phenotypic changes because of functional compensation by closely related or duplicate genes (1). However, such duplicate gene activity may not be the main compensatory mechanism in mouse (2), although this possibility is still controversial (3). A second mechanism of compensation is provided by alternative metabolic pathways or regulatory networks (4). Although such compensatory mechanisms have been extensively studied, especially in yeast and nematode (1), the roles of metabolic and network compensatory pathways are not well understood in mouse.Long-term potentiation (LTP) and long-term depression (LTD) are long-lasting forms of synaptic plasticity that are thought to be the cellular basis for learning and memory and proper formation of neural circuits during development (5). NMDA receptor (NMDAR)-mediated synaptic plasticity is a generally agreed postsynaptic mechanism in the hippocampus (5). In particular, synaptic Ca²⁺ influx through NMDARs is critical for LTP and LTD through control of various protein kinases and phosphatases (6). LTP is in part dependent upon the activation of protein kinases, which phosphorylate target proteins (6). Several kinases are activated during the induction of LTP, including cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinases (cGKs) (6). In contrast, LTD results from activation of phosphatases that dephosphorylate target proteins (6), and calcineurin, a Ca²⁺/calmodulin-dependent protein phosphatase, is important for LTD expression (7). AMPA receptors (AMPARs) are postsynaptic glutamate receptors that mediate rapid excitatory transmission in the central nervous system (8). During LTP, activated kinases phosphorylate AMPARs, leading to synaptic trafficking of the receptors to increase synapse activity (5). For LTD, activation of postsynaptic phosphatases induces internalization of AMPARs from the synaptic membrane, thereby reducing synaptic strength (5). Therefore, both protein kinases and phosphatases control synaptic trafficking of AMPARs, underlying LTP and LTD.AMPARs are tetrameric ligand-gated ion channels that consist of a combinatorial assembly of four subunits (GluA1–4) (9). Studies of GluA1 knockout (KO) mice show that GluA1 is critical for LTP in the CA1 region of the hippocampus (10). GluA1 homomers, like all GluA2-lacking/GluA1-containing receptors, are sensitive to polyamine block and are Ca²⁺-permeable, whereas GluA2-containing AMPARs are Ca²⁺-impermeable (9). Moreover, GluA1 is the major subunit that is trafficked from recycling endosomes to the synaptic membrane in response to neuronal activity (11). Phosphorylation of GluA1 within its intracellular carboxyl-terminal domain (CTD) can regulate AMPAR membrane trafficking (12). Several CTD phosphorylations regulate trafficking (6). In particular, PKA and cGKII both phosphorylate serine 845 of GluA1, increasing the level of extrasynaptic receptors (13, 14). Therefore, activation of PKA and cGKII during LTP induction increases GluA1 phosphorylation, which enhances AMPAR activity at synapses. On the other hand, calcineurin dephosphorylates serine 845 of GluA1, which enables GluA1-containing AMPARs to be endocytosed from the plasma membrane during LTD (15, 16). This removes synaptic AMPARs, leading to reduction of receptor function during LTD. Taken together, the activity-dependent trafficking of synaptic GluA1 is regulated by the status of phosphorylation in the CTD, which provides a critical mechanism underlying LTP and LTD.Several studies have shown that acute inhibition of cGKII impairs hippocampal LTP (13, 17, 18). However, cGKII KO animals show apparently normal LTP in the hippocampus (19), suggesting that a form of functional compensation takes place in the KO hippocampus. Here, we show that cGKII KO reduces Ca²⁺ signals by decreasing cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors (IP3Rs), which in turn lowers calcineurin activity in hippocampal neurons, which stabilizes phosphorylation of GluA1 in homomeric, Ca²⁺-permeable AMPARs (CPARs). This elevates CPARs at the synapse as a previously unidentified compensatory mechanism for hippocampal LTP in cGKII-deficient animals that is alternative to the form of LTP expressed in WT.     

Thyroid neoplasms in Tikur Anbessa Hospital, Addis Ababa: a retrospective review with emphasis on cancer

Reportedly, thyroid cancer is a rare disease in most parts of the world. In Ethiopia, the prevalence of the disease is not known. This study aims to present the experience on thyroid cancer of a main central referral hospital. During the period 1993-02, 137 cases of thyroid neoplasms were operated on in Tikur Anbessa Teaching Hospital, Addis Ababa. Of these, 114 of 130 patients whose records were retrieved were retrospectively reviewed Neoplastic disease of the thyroid was found to be more common in younger age groups (mean age, 37 years) and in females (F: M ratio, 1.7: 1.0). Features of advanced malignant disease were not rare (21 cases). Of the 75 thyroid cancers, papillary carcinoma was the most common histologic type (72%). The extent of surgical treatment included partial, subtotal, near total and total thyroidectomy. In 15 cases, the lesions were found to be unresectable. The long-term outcome of treatment was difficult to evaluate, as the follow-up time was short. We believe that this paper will give base-line information on the frequency of thyroid cancer (7.5%) for a more comprehensive study in the future.     

Identification of quinone imine containing glutathione conjugates of diclofenac in rat bile

High-resolution accurate MS with an LTQ-Orbitrap was used to identify quinone imine metabolites derived from the 5-hydroxy (5-OH) and 4 prime-hydroxy (4'-OH) glutathione conjugates of diclofenac in rat bile. The initial quinone imine metabolites formed by oxidation of diclofenac have been postulated to be reactive intermediates potentially involved in diclofenac-mediated hepatotoxicity; while these metabolites could be formed using in vitro systems, they have never been detected in vivo. This report describes the identification of secondary quinone imine metabolites derived from 5-OH and 4'-OH diclofenac glutathione conjugates in rat bile. To verify the proposed structures, the diclofenac quinone imine GSH conjugate standards were prepared synthetically and enzymatically. The novel metabolite peaks displayed the identical retention times, accurate mass MS/MS spectra, and the fragmentation patterns as the corresponding authentic standards. The formation of these secondary quinone metabolites occurs only under conditions where bile salt homeostasis was experimentally altered. Standard practice in biliary excretion experiments using bile duct-cannulated rats includes infusion of taurocholic acid and/or other bile acids to replace those lost due to continuous collection of bile; for this experiment, the rats received no replacement bile acid infusion. High-resolution accurate mass spectrometry data and comparison with chemically and enzymatically prepared quinone imines of diclofenac glutathione conjugates support the identification of these metabolites. A mechanism for the formation of these reactive quinone imine containing glutathione conjugates of diclofenac is proposed.     

The discovery and optimization of a novel class of potent, selective, and orally bioavailable anaplastic lymphoma kinase (ALK) inhibitors with potential utility for the treatment of cancer

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.