Targeting B-Raf inhibitor resistant melanoma with novel cell penetrating peptide disrupters of PDE8A - C-Raf.

Background: Recent advances in the treatment of melanoma that involve immunotherapy and B-Raf inhibition have revolutionised cancer care for this disease. However, an un-met clinical need remains in B-Raf inhibitor resistant patients where first-generation B-Raf inhibitors provide only short-term disease control. In these cases, B-Raf inhibition leads to paradoxical activation of the C-Raf - MEK - ERK signalling pathway, followed by metastasis.

Prevention of vasospasm after subarachnoid hemorrhage in dogs by continuous intravenous infusion of PD156707.

This randomized, blinded study tested the prophylactic effect of PD156707, a nonpeptide competitive antagonist of endothelin A receptors, against vasospasm after subarachnoid hemorrhage in dogs. Twenty-two dogs were allocated on day 0 to undergo cerebral angiography followed by injection of arterial blood (0.5 ml/kg) into the cisterna magna.

Sodium channel modulators prevent oxygen and glucose deprivation injury and glutamate release in rat neocortical cultures.

Neocortical cultures were deprived of oxygen and glucose to model ischemic neuronal injury. We used a graded series of periods of oxygen and glucose deprivation, providing graded insults. Cell death was measured by release of lactate dehydrogenase (LDH).

An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.

The effect of hypothermia on induction of heat shock protein (HSP)-72 in ischemic brain.

Intra-ischemic hypothermia has been demonstrated to be protective against ischemic neuronal injury. The present study examined the effect of moderate hypothermia on the expression of heat shock protein (HSP)-72 following transient forebrain ischemia in gerbils by immunohistochemistry. Global forebrain ischemia with concurrent moderate hypothermia (30 degrees C) was induced in gerbils by 10-minute bilateral carotid artery occlusion followed by recirculation periods of 1 hour (h), 6h, 24h, and 48h.

A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death.

Calcium/calmodulin-dependent protein kinase-II (CamK-II) is a major neuronal protein which plays a significant role in the cellular process of long-term potentiation (LTP), and vesicular release of neurotransmitters. Here, we show that KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4- phenylpiperazine, a specific cell-permeable inhibitor of CamK-II substantially protected neurons from (1) acute NMDA toxicity and (2) hypoxia/hypoglycemia-induced neuronal injury in fetal rat cortical cultures. KN-62 did not directly inhibit glutamate, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), glycine, or [piperidyl-3,4-(N)]-(N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine) (TCP) binding to rat brain membranes.

(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid attenuates N-methyl-D-aspartate-induced neuronal cell death in cortical cultures via a reduction in delayed Ca2+ accumulation.

The effects of (1S,3R)-ACPD, a selective metabotropic glutamate receptor agonist, on NMDA-induced 45Ca2+ accumulation and delayed neuronal cell death were determined using primary cerebrocortical cultures. Exposure to (1S,3R)-ACPD alone, although causing small increases in 45Ca2+ accumulation, was not neurotoxic. The presence of (1S,3R)-ACPD during exposure to NMDA attenuated the resulting sustained accumulation of 45Ca2+ and delayed neuronal cell death.

Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2.

Effect of varying levels of glucose on oxygen deprivation-induced delayed neuronal cell death in primary cerebrocortical cultures.

Calcium accumulation and neuronal injury of rat cortical cell cultures in vitro were examined following oxygen deprivation under conditions of normal and low glucose. 45Ca2+ uptake and lactate dehydrogenase (LDH) release, measured at 12 and 24 h after oxygen deprivation, were significantly elevated in cultures exposed to combined oxygen deprivation and low glucose (1.7 or 0.

Exploration of N-phosphonoalkyl-, N-phosphonoalkenyl-, and N-(phosphonoalkyl)phenyl-spaced alpha-amino acids as competitive N-methyl-D-aspartic acid antagonists.

A series of N-substituted alpha-amino acids containing terminal phosphonic acid groups has been synthesized as potential N-methyl-D-aspartate (NMDA) receptor antagonists. NMDA receptor affinity was determined by displacement of a known ligand ([3H]CPP) from crude rat brain synaptic membranes; an antagonist action was demonstrated by the inhibition of glutamate-induced accumulation of [45Ca2+] in cultured rat cortical neurons. Receptor affinity was significantly correlated with antagonist activity (Figure 1).

Comparison of vascular perfusion in ischemia-sensitive and ischemia-resistant regions of gerbil brain by an automated laser cytometric device: a preliminary study.

Reduced blood supply in the hippocampus has been considered to be a factor contributory to this region's selective susceptibility to ischemia. To determine if there are differences in vascularization of vulnerable and resistant regions of the brain, a quantitative morphometric assessment of vascular filling was performed in the gerbil brain by using a relatively new and highly sensitive automated laser cytometric device, ACAS (Adherent Cell Analysis and Sorting). Evan's blue, an intravital vascular tracer, was injected intracardiac in 5 Mongolian gerbils to identify perfused blood vessels in the cortex and CA1 region of hippocampus.

Treatment with conotoxin, an 'N-type' calcium channel blocker, in neuronal hypoxic-ischemic injury.

Therapeutic efficacy of calcium channel blockers in stroke remains controversial, but previously used agents bind almost exclusively to L-type calcium channels. The newly-discovered N-type calcium channel is specific to neurons, and therapy involving blockade of this site has not been previously attempted. We assessed the neuroprotective effect of omega-conotoxin GVIA (CgTx), a blocker of N-type calcium channels, using both in vitro hypoxic injury to rat cortical neurons and an in vivo model of reversible spinal cord ischemia in the rabbit.

Comparison of phenytoin with noncompetitive N-methyl-D-aspartate antagonists in a model of focal brain ischemia in rat.

Recent in vitro and in vivo experiments have suggested that excitatory amino acid antagonists, particularly those active at the N-methyl-D-aspartate receptor subtype, are effective in ameliorating ischemic injury due to their antiexcitotoxic activity. However, these drugs are also potent and effective in vivo anticonvulsants. The present experiments compared the noncompetitive N-methyl-D-aspartate antagonists phencyclidine and MK-801 with the anticonvulsant phenytoin in a model of focal brain ischemia.

Hypoxic neuronal injury in tissue culture is associated with delayed calcium accumulation.

Calcium accumulation and neuronal injury were studied after hypoxia in cerebrocortical cell cultures in vitro. Neuronal injury was associated with a delayed calcium accumulation, which was greatest 5-7 hours after hypoxic exposure. Antiexcitotoxic treatments with tetrodotoxin and magnesium chloride or the selective N-methyl-D-aspartate antagonist (+/-)-4-(3-phosphonopropyl)-2-piperazinecarboxylic acid prevented hypoxic calcium accumulation and neuronal injury even when added 3 hours after hypoxia, during reoxygenation.

Cold injury, blood-brain barrier changes, and leukotriene synthesis: inhibition by phenidone.

Transcranial cold injury in rats and guinea pigs induced cerebral extravasation of albumin labeled with Evans blue dye or 125I, respective indicators of the area and amount of blood-brain barrier (BBB) disruption. Radioimmunoassay of brain extracts showed that cold injury induced leukotriene (LT)C4 in rat and guinea pig brains 15 min after injury. In guinea pigs, the LT synthesis inhibitor phenidone (30 mg/kg, i.

Exploration of phenyl-spaced 2-amino-(5-9)-phosphonoalkanoic acids as competitive N-methyl-D-aspartic acid antagonists.

To investigate the preferred spatial relationship of the distal phosphonic acid to the alpha-amino acid group of the established competitive N-methyl-D-aspartic acid (NMDA) antagonists APH (1) and APV (2), we have prepared a series of ortho-, meta-, and para-substituted (phosphonoalkyl)phenylglycine and -phenylalanine derivatives. With use of a [3H]CPP receptor binding assay, significant binding activity was observed to be critically dependent on both the position of substitution and length of alkyl spacing groups. Two compounds, 4-(phosphonomethyl)-phenylglycine (6, PD 129635) and 3-(phosphonomethyl)phenylalanine (15, PD 130527), displayed receptor-binding affinity comparable to that of APH.

Ketamine prevents ischemic neuronal injury.

The dissociative anesthetic ketamine hydrochloride antagonizes the excitotoxic action of excitatory amino acids in the central nervous system. Proposals that the excitatory amino acid neurotransmitters may become excitotoxic and contribute to the pathophysiology of ischemic brain injury prompted us to examine ketamine in a model of global cerebral ischemia in gerbils. Pretreatment with anesthetic doses of ketamine ameliorated in a dose-dependent manner both behavioral and histopathological assessments of ischemic neuronal injury.

Selective necrosis and total necrosis in focal cerebral ischemia. Neuropathologic observations on experimental middle cerebral artery occlusion in the macaque monkey.

Temporary (15 minutes to 24 hours) or permanent focal cerebral ischemia was induced in 87 awake monkeys (Macaca mulatta and Macaca fasicularis) by transorbital snare ligation of the middle cerebral artery (MCA) and neuropathological evaluation was carried out two weeks later. The size, location and histology of lesions varied within each time-period of MCA occlusion. However, most animals that underwent long-term ischemia (eight hours to permanent) had a single, confluent infarct involving deep and sometimes cortical structures.

Variability and reversibility of focal cerebral ischemia in unanesthetized monkeys.

To assess reversibility of focal cerebral ischemia, we performed a neurologic and pathologic study of 27 monkeys subjected to temporary middle cerebral artery occlusion. An implanted snare ligature occluded the artery in awake monkeys for 30 minutes, 4 hours, 8 hours, 16 hours, 24 hours, or permanently. Serial neurologic observations were made for 2 weeks, and systematic neuropathologic examination estimated extent of infarction.

Thresholds of focal cerebral ischemia in awake monkeys.

An awake-primate model has been developed which permits reversible middle cerebral artery (MCA) occlusion during physiological monitoring. This method eliminates the ischemia-modifying effects of anesthesia, and permits correlation of neurological function with cerebral blood flow (CBF) and neuropathology. The model was used to assess the brain's tolerance to focal cerebral ischemia.