Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells.

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.

MK-8825: a potent and selective CGRP receptor antagonist with good oral activity in rats.

Rational modification of the clinically tested CGRP receptor antagonist MK-3207 (3) afforded an analogue with increased unbound fraction in rat plasma and enhanced aqueous solubility, 2-[(8R)-8-(3,5-difluorophenyl)-8-methyl-10-oxo-6,9-diazaspiro[4.5]dec-9-yl]-N-[(6S)-2'-oxo-1',2',5,7-tetrahydrospiro[cyclopenta[b]pyridine-6,3'-pyrrolo[2,3-b]pyridin]-3-yl]acetamide (MK-8825) (6). Compound 6 maintained similar affinity to 3 at the human and rat CGRP receptors but possessed significantly improved in vivo potency in a rat pharmacodynamic model. The overall profile of 6 indicates it should find utility as a rat tool to investigate effects of CGRP receptor blockade in vivo.

Development of orally bioavailable and CNS penetrant biphenylaminocyclopropane carboxamide bradykinin B1 receptor antagonists.

A series of biphenylaminocyclopropane carboxamide based bradykinin B1 receptor antagonists has been developed that possesses good pharmacokinetic properties and is CNS penetrant. Discovery that the replacement of the trifluoropropionamide in the lead structure with polyhaloacetamides, particularly a trifluoroacetamide, significantly reduced P-glycoprotein mediated efflux for the series proved essential. One of these novel bradykinin B1 antagonists (13b) also exhibited suitable pharmacokinetic properties and efficient ex vivo receptor occupancy for further development as a novel approach for the treatment of pain and inflammation.

A protective effect after clearance of orthotopic rat hepatocellular carcinoma by nanosecond pulsed electric fields.

Strategies for treating liver cancer using radiation, chemotherapy combinations and tyrosine kinase inhibitors targeting specific mutations have provided longer survival times, yet multiple treatments are often needed and recurrences with new malignant phenotypes are not uncommon. New and innovative treatments are undoubtedly needed to successfully treat liver cancer. Over the last decade, nanosecond pulsed electric fields (nsPEFs) have shown promise in pre-clinical studies; however, these have been limited to treatment of skin cancers or xenographs in mice. In the present report, an orthotopic hepatocellular carcinoma (HCC) model is established in rats using N1-S1 HCC cells. Data demonstrate a response rate of 80-90% when 1000 pulses are delivered with 100ns durations, electric field strengths of 50kV/cm and repetition rates of 1Hz. N1-S1 tumours treated with nsPEFs expressed significant number of cells with active caspase-3 and caspase-9, but not caspase-8, indicating an intrinsic apoptosis mechanism(s) as well as caspase-independent mechanisms. Most remarkably, rats with successfully ablated tumours failed to re-grow tumours when challenged with a second injection of N1-S1 cells when implanted in the same or different liver lobe that harboured the original tumour. Given this protective effect, infiltration of immune cells and the presence of granzyme B expressing cells within days of treatment suggest the possibility of an anti-tumour adaptive immune response. In conclusion, NsPEFs not only eliminate N1-S1 HCC tumours, but also may induce an immuno-protective effect that defends animals against recurrences of the same cancer.

Induction of Cell Death Mechanisms and Apoptosis by Nanosecond Pulsed Electric Fields (nsPEFs).

Pulse power technology using nanosecond pulsed electric fields (nsPEFs) offers a new stimulus to modulate cell functions or induce cell death for cancer cell ablation. New data and a literature review demonstrate fundamental and basic cellular mechanisms when nsPEFs interact with cellular targets. NsPEFs supra-electroporate cells creating large numbers of nanopores in all cell membranes. While nsPEFs have multiple cellular targets, these studies show that nsPEF-induced dissipation of ΔΨm closely parallels deterioration in cell viability. Increases in intracellular Ca2+ alone were not sufficient for cell death; however, cell death depended of the presence of Ca2+. When both events occur, cell death ensues. Further, direct evidence supports the hypothesis that pulse rise-fall times or high frequency components of nsPEFs are important for decreasing ΔΨm and cell viability. Evidence indicates in Jurkat cells that cytochrome c release from mitochondria is caspase-independent indicating an absence of extrinsic apoptosis and that cell death can be caspase-dependent and -independent. The Ca2+ dependence of nsPEF-induced dissipation of ΔΨm suggests that nanoporation of inner mitochondria membranes is less likely and effects on a Ca2+-dependent protein(s) or the membrane in which it is embedded are more likely a target for nsPEF-induced cell death. The mitochondria permeability transition pore (mPTP) complex is a likely candidate. Data demonstrate that nsPEFs can bypass cancer mutations that evade apoptosis through mechanisms at either the DISC or the apoptosome.

Transient features in nanosecond pulsed electric fields differentially modulate mitochondria and viability.

It is hypothesized that high frequency components of nanosecond pulsed electric fields (nsPEFs), determined by transient pulse features, are important for maximizing electric field interactions with intracellular structures. For monopolar square wave pulses, these transient features are determined by the rapid rise and fall of the pulsed electric fields. To determine effects on mitochondria membranes and plasma membranes, N1-S1 hepatocellular carcinoma cells were exposed to single 600 ns pulses with varying electric fields (0-80 kV/cm) and short (15 ns) or long (150 ns) rise and fall times. Plasma membrane effects were evaluated using Fluo-4 to determine calcium influx, the only measurable source of increases in intracellular calcium. Mitochondria membrane effects were evaluated using tetramethylrhodamine ethyl ester (TMRE) to determine mitochondria membrane potentials (ΔΨm). Single pulses with short rise and fall times caused electric field-dependent increases in calcium influx, dissipation of ΔΨm and cell death. Pulses with long rise and fall times exhibited electric field-dependent increases in calcium influx, but diminished effects on dissipation of ΔΨm and viability. Results indicate that high frequency components have significant differential impact on mitochondria membranes, which determines cell death, but lesser variances on plasma membranes, which allows calcium influxes, a primary determinant for dissipation of ΔΨm and cell death.

Synthesis, structure-activity relationship, and pharmacological profile of analogs of the ASIC-3 inhibitor A-317567.

The synthesis, structure-activity relationship (SAR), and pharmacological evaluation of analogs of the acid-sensing ion channel (ASIC) inhibitor A-317567 are reported. It was found that the compound with an acetylenic linkage was the most potent ASIC-3 channel blocker. This compound reversed mechanical hypersensitivity in the rat iodoacetate model of osteoarthritis pain, although sedation was noted. Sedation was also observed in ASIC-3 knockout mice, questioning whether sedation and antinociception are mediated via a non-ASIC-3 specific mechanism.

Antinociceptive effects of the non-selective cannabinoid receptor agonist CP 55,940 are absent in CB1(-/-) and not CB2(-/-) mice in models of acute and persistent pain.

Previous studies have suggested a role for both CB1 and CB2 cannabinoid receptors in modulation of nociception. To further examine the role of CB1 and CB2 receptors in antinociception, we evaluated the efficacy of the non-selective cannabinoid receptor agonist, CP 55,940, in models of acute, inflammatory, and neuropathic pain in control mice, CB1 receptor knockout mice, and CB2 receptor knockout mice. In control C57BL/6 mice, administration of CP 55,940 (0.03-0.3 mg/kg, i.p.) reversed complete Freund's adjuvant-induced tactile allodynia, reversed tactile allodynia in the spinal nerve ligation model and inhibited the noxious heat-evoked tail withdrawal response. In addition to its antinociceptive effects, CP 55,940 produced an impairment of motor coordination in the rotarod test. The antinociceptive effects produced by CP 55,940 and associated motor deficits were found to be completely abolished in CB1 receptor knockout mice. In contrast, the antinociceptive effects of CP 55,940 in all pain models were fully retained in CB2 receptor knockout mice, along with the associated motor deficits. The results suggest that the antinociceptive effects of CP 55,940 in models of acute and persistent pain, along with the associated motor deficits, are mediated by CB1 receptors, and likely not CB2 receptors.