Pan AMPK Activation Protects Tubules in Rat Ischemic Acute Kidney Injury.

Acute Kidney Injury (AKI) is characterized by an abrupt decline in kidney function and has been associated with excess risks of death, kidney disease progression, and cardiovascular events. The kidney has a high energetic demand with mitochondrial health being essential to renal function and damaged mitochondria has been reported across AKI subtypes. 5' adenosine monophosphate-activated protein kinase (AMPK) activation preserves cellular energetics through improvement of mitochondrial function and biogenesis when ATP levels are low such as under ischemia-induced AKI. We developed a selective potent small molecule pan AMPK activator, compound 1, and tested its ability to increase AMPK activity and preserve kidney function during ischemia/reperfusion injury in rats. A single administration of 1 caused sustained activation of AMPK for at least 24 hours, protected against acute tubular necrosis, and reduced clinical markers of tubular injury such as NephroCheck and Fractional Excretion of Sodium (FENa). Reduction in plasma creatinine and increased Glomerular Filtration Rate (GFR) indicated preservation of kidney function. Surprisingly, we observed a strong diuretic effect of AMPK activation associated with natriuresis both with and without AKI. Our findings demonstrate that activation of AMPK leads to protection of tubular function under hypoxic/ischemic conditions which holds promise as a potential novel therapeutic approach for AKI. Significance Statement No approved pharmacological therapies currently exist for acute kidney injury. We developed Compound 1 which dose-dependently activated AMPK in the kidney and protected kidney function and tubules after ischemic renal injury in the rat. This was accompanied by natriuresis in injured as well as uninjured rats.

ArcKR expression modifies synaptic plasticity following epileptic activity: Differential effects with in vitro and in vivo seizure-induction protocols.

OBJECTIVES: Pathological forms of neural activity, such as epileptic seizures, modify the expression pattern of multiple proteins, leading to persistent changes in brain function. One such protein is activity-regulated cytoskeleton-associated protein (Arc), which is critically involved in protein-synthesis-dependent synaptic plasticity underlying learning and memory. In the present study, we have investigated how the expression of ArcKR, a form of Arc in which the ubiquitination sites have been mutated, resulting in slowed Arc degradation, modifies group I metabotropic glutamate receptor-mediated long-term depression (G1-mGluR-LTD) following seizures. METHODS: We used a knock-in mice line that express ArcKR and two hyperexcitation models: an in vitro model, where hippocampal slices were exposed to zero Mg2+, 6 mM K+; and an in vivo model, where kainic acid was injected unilaterally into the hippocampus. In both models, field excitatory postsynaptic potentials (fEPSPs) were recorded from the CA1 region of hippocampal slices in response to Schaffer collateral stimulation and G1-mGluR-LTD was induced chemically with the group 1 mGluR agonist DHPG. RESULTS: In the in vitro model, ArcKR expression enhanced the effects of seizure activity and increased the magnitude of G1-mGluR LTD, an effect that could be blocked with the mGluR5 antagonist MTEP. In the in vivo model, fEPSPs were significantly smaller in slices from ArcKR mice and were less contaminated by population spikes. In this model, the amount of G1-mGluR-LTD was significantly less in epileptic slices from ArcKR mice as compared to wildtype (WT) mice. SIGNIFICANCE: We have shown that expression of ArcKR, a form of Arc in which degradation is reduced, significantly modulates the magnitude of G1-mGluR-LTD following epileptic seizures. However, the effect of ArcKR on LTD depends on the epileptic model used, with enhancement of LTD in an in vitro model and a reduction in the kainate mouse model.

Arc expression regulates long-term potentiation magnitude and metaplasticity in area CA1 of the hippocampus in ArcKR mice.

Expression of the immediate early gene Arc/Arg3.1 (Arc), a key mediator of synaptic plasticity, is enhanced by neural activity and then reduced by proteasome-dependent degradation. We have previously shown that the disruption of Arc degradation, in an Arc knock-in mouse (ArcKR), where the predominant Arc ubiquitination sites were mutated, reduced the threshold to induce, and also enhanced, the strength of Group I metabotropic glutamate receptor-mediated long-term depression (DHPG-LTD). Here, we have investigated if ArcKR expression changes long-term potentiation (LTP) in CA1 area of the hippocampus. As previously reported, there was no change in basal synaptic transmission at Schaffer collateral/commissural-CA1 (SC-CA1) synapses in ArcKR versus wild-type (WT) mice. There was, however, a significant increase in the amplitude of synaptically induced (with low frequency paired-pulse stimulation) LTD in ArcKR mice. Theta burst stimulation (TBS)-evoked LTP at SC-CA1 synapses was significantly reduced in ArcKR versus WT mice (after 2 h). Group 1 mGluR priming of LTP was abolished in ArcKR mice, which could also potentially contribute to a depression of LTP. Although high frequency stimulation (HFS)-induced LTP was not significantly different in ArcKR compared with WT mice (after 1 h), there was a phenotype in environmentally enriched mice, with the ratio of LTP to short-term potentiation (STP) significantly reduced in ArcKR mice. These findings support the hypothesis that Arc ubiquitination supports the induction and expression of LTP, likely via limiting Arc-dependent removal of AMPA receptors at synapses.

CO2-Sensitive Connexin Hemichannels in Neurons and Glia: Three Different Modes of Signalling?

Connexins can assemble into either gap junctions (between two cells) or hemichannels (from one cell to the extracellular space) and mediate cell-to-cell signalling. A subset of connexins (Cx26, Cx30, Cx32) are directly sensitive to CO2 and fluctuations in the level within a physiological range affect their open probability, and thus, change cell conductance. These connexins are primarily found on astrocytes or oligodendrocytes, where increased CO2 leads to ATP release, which acts on P2X and P2Y receptors of neighbouring neurons and changes excitability. CO2-sensitive hemichannels are also found on developing cortical neurons, where they play a role in producing spontaneous neuronal activity. It is plausible that the transient opening of hemichannels allows cation influx, leading to depolarisation. Recently, we have shown that dopaminergic neurons in the substantia nigra and GABAergic neurons in the VTA also express Cx26 hemichannels. An increase in the level of CO2 results in hemichannel opening, increasing whole-cell conductance, and decreasing neuronal excitability. We found that the expression of Cx26 in the dopaminergic neurons in the substantia nigra at P7-10 is transferred to glial cells by P17-21, displaying a shift from being inhibitory (to neuronal activity) in young mice, to potentially excitatory (via ATP release). Thus, Cx26 hemichannels could have three modes of signalling (release of ATP, excitatory flickering open and shut and inhibitory shunting) depending on where they are expressed (neurons or glia) and the stage of development.

The mechanistic link between Arc/Arg3.1 expression and AMPA receptor endocytosis.

The activity-regulated cytoskeleton associated protein (Arc/Arg3.1) plays a key role in determining synaptic strength through facilitation of AMPA receptor (AMPAR) endocytosis. Although there is considerable data on the mechanism by which Arc induction controls synaptic plasticity and learning behaviours, several key mechanistic questions remain. Here we review data on the link between Arc expression and the clathrin-mediated endocytic pathway which internalises AMPARs and discuss the significance of Arc binding to the clathrin adaptor protein 2 (AP-2) and to endophilin/dynamin. We consider which AMPAR subunits are selected for Arc-mediated internalisation, implications for synaptic function and consider Arc as a therapeutic target.

Transforming Growth Factor Beta 1 Drives a Switch in Connexin Mediated Cell-to-Cell Communication in Tubular Cells of the Diabetic Kidney.

BACKGROUND/AIMS: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFß1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron. METHODS: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFß1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFß1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-ß1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-ß1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression. RESULTS: Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFß1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFß1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFß1 and to 0.42±0.2nS after 7days TGFß1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFß1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06µM in control to 1.99±0.47µM after 48hr incubation with TGFß1 (10ng/ml; P<0.05, n=3). TGF-ß1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-ß1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3). CONCLUSION: These data suggest that chronic exposure to glucose-evoked TGFß1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy.

Rapid prediction of stem cell mobilization using volume and conductivity data from automated hematology analyzers.

BACKGROUND: Rapid analytics to predict circulating hematopoietic stem cells are valuable for optimal management of mobilization, particularly for the use of newer and costly mobilization agents such as plerixafor. STUDY DESIGN AND METHODS: We used stepwise, linear multiple regression modeling applied to cell population data collected by routine hematology analyzers (Beckman Coulter DxH 800) on patients undergoing autologous stem cell collection (n = 131). Beta coefficients were used to derive a formula for a stem cell index (SCI). We then tested the correlation of SCI with stem cell counts and performance of the SCI as a predictor of poor mobilization with external validation in a separate cohort (n = 183). RESULTS: The SCI correlated strongly with CD34 counts by flow cytometry (r = 0.8372 in the development cohort, r = 0.8332 in the external validation cohort) and compares favorably with other rapid stem cell enumerating technologies. In the external validation cohort, the SCI performed well as a predictor (receiver operating characteristic area under the curve, 0.9336) of poor mobilization (CD34 count < 10), with a sensitivity of 72% and a specificity of 93%. When prevalence of poor mobilization was 33%, this resulted in a positive predictive value of 83% and a negative predictive value of 87%. The SCI also showed promise in tracking responses to plerixafor administration. CONCLUSION: The findings demonstrate the utility of the cell population data collected by hematology analyzers to provide rapid data beyond standard complete blood counts, particularly for stem cell count prediction, requiring no additional reagents, specimen, or instrumentation.

Discovery and optimization of a novel series of pyrazolyltetrahydropyran N-type calcium channel (Cav 2.2) blockers for the treatment of pain.

A novel series of pyrazolyltetrahydropyran N-type calcium channel blockers are described. Structural modifications of the series led to potent compounds in both a cell-based fluorescent calcium influx assay and a patch clamp electrophysiology assay. Representative compounds from the series were bioavailable and showed efficacy in the rat CFA and CCI models of inflammatory and neuropathic pain.

Discovery of N-arylpyrroles as agonists of GPR120 for the treatment of type II diabetes.

The discovery of a novel series of N-arylpyrroles as agonists of GPR120 (FFAR4) is discussed. One lead compound is a potent GPR120 agonist, has good selectivity for related receptor GPR40 (FFAR1), has acceptable PK properties, and is active in 2 models of Type 2 Diabetes in mice.

Modeling microelectrode biosensors: free-flow calibration can substantially underestimate tissue concentrations.

Microelectrode amperometric biosensors are widely used to measure concentrations of analytes in solution and tissue including acetylcholine, adenosine, glucose, and glutamate. A great deal of experimental and modeling effort has been directed at quantifying the response of the biosensors themselves; however, the influence that the macroscopic tissue environment has on biosensor response has not been subjected to the same level of scrutiny. Here we identify an important issue in the way microelectrode biosensors are calibrated that is likely to have led to underestimations of analyte tissue concentrations. Concentration in tissue is typically determined by comparing the biosensor signal to that measured in free-flow calibration conditions. In a free-flow environment the concentration of the analyte at the outer surface of the biosensor can be considered constant. However, in tissue the analyte reaches the biosensor surface by diffusion through the extracellular space. Because the enzymes in the biosensor break down the analyte, a density gradient is set up resulting in a significantly lower concentration of analyte near the biosensor surface. This effect is compounded by the diminished volume fraction (porosity) and reduction in the diffusion coefficient due to obstructions (tortuosity) in tissue. We demonstrate this effect through modeling and experimentally verify our predictions in diffusive environments.NEW & NOTEWORTHY Microelectrode biosensors are typically calibrated in a free-flow environment where the concentrations at the biosensor surface are constant. However, when in tissue, the analyte reaches the biosensor via diffusion and so analyte breakdown by the biosensor results in a concentration gradient and consequently a lower concentration around the biosensor. This effect means that naive free-flow calibration will underestimate tissue concentration. We develop mathematical models to better quantify the discrepancy between the calibration and tissue environment and experimentally verify our key predictions.

Safety and Efficacy of Isolated Limb Infusion Chemotherapy for Advanced Locoregional Melanoma in Elderly Patients: An Australian Multicenter Study.

BACKGROUND: Isolated limb infusion (ILI) offers a minimally invasive treatment option for locally advanced extremity melanoma. OBJECTIVE: The aim of the current study was to evaluate the safety and efficacy of ILI in elderly patients in an Australian multicenter setting. METHODS: The results of 316 first ILI procedures, performed between 1992 and 2008 in five Australian institutions, were identified and analyzed, with the main focus on elderly patients (≥75 years of age). All institutions used the same protocol: melphalan was circulated in the isolated limb for 20-30 min (±actinomycin D), and toxicity, responses, and survival were recorded. RESULTS: Characteristics of patients aged ≥75 years (n = 148) were similar to those aged <75 years (n = 168), except that older patients had more melanoma deposits (median 4 vs. 5; p = 0.035) and lower limb volumes (5.4 vs. 6.5 L; p = 0.001). Median drug circulation times were lower in the older group (21 vs. 24 min; p = 0.04), and older patients experienced less limb toxicity (grade III/IV in 22 and 37% of patients, respectively; p = 0.003). A complete response (CR) was seen in 27% of patients aged ≥75 years and in 38% of patients aged <75 years (p = 0.06), while overall response rates were 72 and 77%, respectively (p = 0.30). No difference in survival was seen (p = 0.69). CONCLUSIONS: The ILI technique proved safe and effective in elderly patients. When present, toxicity was localized, and lower compared with younger patients, possibly due to shorter drug circulation times. CR rates were higher in younger patients, although not significantly, while overall response and survival were equal. Optimization of perioperative factors in elderly patients may allow response rates to be raised further, while maintaining low toxicity.

Design, synthesis and SAR of a novel series of heterocyclic phenylpropanoic acids as GPR120 agonists.

A novel series of 5-membered heterocycle-containing phenylpropanoic acid derivatives was discovered as potent GPR120 agonists with low clearance, high oral bioavailability and in vivo antidiabetic activity in rodents.

Intracellular soluble α-synuclein oligomers reduce pyramidal cell excitability.

KEY POINTS: The presynaptic protein α-synuclein forms aggregates during Parkinson's disease. Accumulating evidence suggests that the small soluble oligomers of α-synuclein are more toxic than the larger aggregates appearing later in the disease. The link between oligomer toxicity and structure still remains unclear. In the present study, we have produced two structurally-defined oligomers that have a similar morphology but differ in secondary structure. These oligomers were introduced into neocortical pyramidal cells during whole-cell recording and, using a combination of experimentation and modelling, electrophysiological parameters were extracted. Both oligomeric species had similar effects on neuronal properties reducing input resistance, time constant and increasing capacitance. The net effect was a marked reduction in neuronal excitability that could impact on network activity. ABSTRACT: The presynaptic protein α-synuclein (αSyn) aggregates during Parkinson's disease (PD) to form large proteinaceous amyloid plaques, the spread of which throughout the brain clinically defines the severity of the disease. During early stages of aggregation, αSyn forms soluble annular oligomers that show greater toxicity than much larger fibrils. These oligomers produce toxicity via a number of possible mechanisms, including the production of pore-forming complexes that permeabilize membranes. In the present study, two well-defined species of soluble αSyn oligomers were produced by different protocols: by polymerization of monomer and by sonication of fibrils. The two oligomeric species produced were morphologically similar, with both having an annular structure and consisting of approximately the same number of monomer subunits, although they differed in their secondary structure. Oligomeric and monomeric αSyn were injected directly into the soma of pyramidal neurons in mouse neocortical brain slices during whole-cell patch clamp recording. Using a combined experimental and modelling approach, neuronal parameters were extracted to measure, for the first time in the neocortex, specific changes in neuronal electrophysiology. Both species of oligomer had similar effects: (i) a significant reduction in input resistance and the membrane time constant and (ii) an increase in the current required to trigger an action potential with a resultant reduction in the firing rate. Differences in oligomer secondary structure appeared to produce only subtle differences in the activity of the oligomers. Monomeric αSyn had no effect on neuronal parameters, even at high concentrations. The oligomer-induced fall in neuronal excitability has the potential to impact both network activity and cognitive processing.

Australian Multicenter Study of Isolated Limb Infusion for Melanoma.

PURPOSE: Isolated limb infusion (ILI) offers a less invasive alternative to isolated limb perfusion (ILP) for the treatment of locally advanced extremity melanoma. In Australia, ILI has essentially completely replaced ILP. The aim of this study was to collect and evaluate the results of ILI in an Australian multicenter setting. PATIENTS AND METHODS: The results of 316 first ILI procedures, performed between 1992 and 2008 in five Australian institutions, were collectively analyzed, with all five institutions using the same protocol. Melphalan was circulated in the isolated limb for 20-30 min (±actinomycin D). Response was determined using the World Health Organization criteria, and limb toxicity was assessed using the Wieberdink scale. RESULTS: The median patient age was 74 years (range 28-100) and 59 % of patients were female. Overall response rate was 75 % (complete response [CR] 33 %; partial response 42 %). Stable disease was seen in 18 % of patients and progressive disease in 7 %. Wieberdink grade III or higher was seen in 30 % of the cases. No toxicity-related amputations occurred, and median survival was 44 months. In patients with a CR, median survival was 80 months (p = 0.014). On multivariate analysis, Breslow thickness, lower-limb ILI, and a procedure performed at the Melanoma Institute Australia remained significant predictors for response, although not for survival. CONCLUSIONS: This Australian multicenter study of ILI is the largest reported to date. ILI is a useful technique that can be safely and effectively performed across tertiary referral centers for the successful management of advanced extremity melanoma. Increased optimization of perioperative factors might allow response rates to be raised further, while maintaining acceptable toxicity.

Experimentally Verified Parameter Sets for Modelling Heterogeneous Neocortical Pyramidal-Cell Populations.

Models of neocortical networks are increasingly including the diversity of excitatory and inhibitory neuronal classes. Significant variability in cellular properties are also seen within a nominal neuronal class and this heterogeneity can be expected to influence the population response and information processing in networks. Recent studies have examined the population and network effects of variability in a particular neuronal parameter with some plausibly chosen distribution. However, the empirical variability and covariance seen across multiple parameters are rarely included, partly due to the lack of data on parameter correlations in forms convenient for model construction. To addess this we quantify the heterogeneity within and between the neocortical pyramidal-cell classes in layers 2/3, 4, and the slender-tufted and thick-tufted pyramidal cells of layer 5 using a combination of intracellular recordings, single-neuron modelling and statistical analyses. From the response to both square-pulse and naturalistic fluctuating stimuli, we examined the class-dependent variance and covariance of electrophysiological parameters and identify the role of the h current in generating parameter correlations. A byproduct of the dynamic I-V method we employed is the straightforward extraction of reduced neuron models from experiment. Empirically these models took the refractory exponential integrate-and-fire form and provide an accurate fit to the perisomatic voltage responses of the diverse pyramidal-cell populations when the class-dependent statistics of the model parameters were respected. By quantifying the parameter statistics we obtained an algorithm which generates populations of model neurons, for each of the four pyramidal-cell classes, that adhere to experimentally observed marginal distributions and parameter correlations. As well as providing this tool, which we hope will be of use for exploring the effects of heterogeneity in neocortical networks, we also provide the code for the dynamic I-V method and make the full electrophysiological data set available.

Localized adenosine signaling provides fine-tuned negative feedback over a wide dynamic range of neocortical network activities.

Although the patterns of activity produced by neocortical networks are now better understood, how these states are activated, sustained, and terminated still remains unclear. Negative feedback by the endogenous neuromodulator adenosine may potentially play an important role, as it can be released by activity and there is dense A1 receptor expression in the neocortex. Using electrophysiology, biosensors, and modeling, we have investigated the properties of adenosine signaling during physiological and pathological network activity in rat neocortical slices. Both low- and high-rate network activities were reduced by A1 receptor activation and enhanced by block of A1 receptors, consistent with activity-dependent adenosine release. Since the A1 receptors were neither saturated nor completely unoccupied during either low- or high-rate activity, adenosine signaling provides a negative-feedback mechanism with a wide dynamic range. Modeling and biosensor experiments show that during high-rate activity increases in extracellular adenosine concentration are highly localized and are uncorrelated over short distances that are certainly<500 µm. Modeling also predicts that the slow rise of the purine waveform cannot be from diffusion from distal release sites but more likely results from uptake and metabolism. The inability to directly measure adenosine release during low-rate activity, although it is present, is probably a consequence of small localized increases in adenosine concentration that are rapidly diminished by diffusion and active removal mechanisms. Saturation of such removal mechanisms when higher concentrations of adenosine are released results in the accumulation of inosine, explaining the strong purine signal during high-rate activity.

Resolubilization of precipitated intact membrane proteins with cold formic acid for analysis by mass spectrometry.

Protein precipitation in organic solvent is an effective strategy to deplete sodium dodecyl sulfate (SDS) ahead of MS analysis. Here we evaluate the recovery of membrane and water-soluble proteins through precipitation with chloroform/methanol/water or with acetone (80%). With each solvent system, membrane protein recovery was greater than 90%, which was generally higher than that of cytosolic proteins. With few exceptions, residual supernatant proteins detected by MS were also detected in the precipitation pellet, having higher MS signal intensity in the pellet fraction. Following precipitation, we present a novel strategy for the quantitative resolubilization of proteins in an MS-compatible solvent system. The pellet is incubated at -20 °C in 80% formic acid/water and then diluted 10-fold with water. Membrane protein recovery matches that of sonication of the pellet in 1% SDS. The resolubilized proteins are stable at room temperature, with no observed formylation as is typical of proteins suspended in formic acid at room temperature. The protocol is applied to the molecular weight determination of membrane proteins from a GELFrEE-fractionated sample of Escherichia coli proteins.

Australian multi-center experience outside of the Sydney Melanoma Unit of isolated limb infusion chemotherapy for melanoma.

INTRODUCTION: Isolated limb infusion (ILI) is a minimally invasive alternative to isolated limb perfusion (ILP) for delivering high-dose regional chemotherapy to treat locally advanced limb melanoma. The current study aimed to evaluate the applicability of ILI in four Australian tertiary referral centers outside of its originating institution, the Sydney Melanoma Unit (SMU; currently known as the Melanoma Institute Australia). METHODS: Data of 131 patients, treated between 1992 and 2008 were collectively analyzed. The ILI procedures were based on the Sydney Melanoma Unit protocol using melphalan. Response was determined using the WHO criteria and toxicity was assessed using the Wieberdink scale. RESULTS: The median patient age was 74 years (range 28-100). Fifty-six percent were female. Overall response (OR) rate to ILI was 63% (CR 27%; PR 36%). Wieberdink toxicity grade III or higher was seen in 13%. No toxicity-related amputations occurred. Median follow-up was 24 months; median survival was 58 months. In patients with a complete response (CR), median survival was 101 months; in patients with a partial response (PR) this was 41 months (P = 0.026). On univariate analysis a younger age, lower-limb procedures and a lower Breslow thickness of the primary melanoma were associated with a favorable response. On multivariate analysis Breslow thickness and lower-limb ILI remained significant predictors for response. CONCLUSION: In this, to date, largest multi-center study of ILI for melanoma the results are comparable to other reports and demonstrate that ILI can be widely implemented and safely applied across tertiary referral centers.

Discovery and SAR of novel tetrahydropyrrolo[3,4-c]pyrazoles as inhibitors of the N-type calcium channel.

A novel series of substituted tetrahydropyrrolo[3,4-c]pyrazoles were investigated as blockers of the N-type calcium channel (Cav2.2 channels), a chronic pain target.

Discovery and SAR of a novel series of 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles as N-type calcium channel inhibitors.

A novel series of substituted 2,4,5,6-tetrahydrocyclopenta[c]pyrazoles were investigated as N-type calcium channel blockers (Cav2.2 channels), a chronic pain target. One compound was active in vivo in the rat CFA pain model.