Jennifer S. McKay

AZD6244 (ARRY-142886), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, and potential for combination in preclinical models

Constitutive activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in human cancers is often associated with mutational activation of BRAF or RAS. MAPK/ERK kinase 1/2 kinases lie downstream of RAS and BRAF and are the only acknowledged activators of ERK1/2, making them attractive targets for therapeutic intervention. AZD6244 (ARRY-142886) is a potent, selective, and ATP-uncompetitive inhibitor of MAPK/ERK kinase 1/2. In vitro cell viability inhibition screening of a tumor cell line panel found that lines harboring BRAF or RAS mutations were more likely to be sensitive to AZD6244. The in vivo mechanisms by which AZD6244 inhibits tumor growth were investigated. Chronic dosing with 25 mg/kg AZD6244 bd resulted in suppression of growth of Colo-205, Calu-6, and SW-620 xenografts, whereas an acute dose resulted in significant inhibition of ERK1/2 phosphorylation. Increased cleaved caspase-3, a marker of apoptosis, was detected in Colo-205 and Calu-6 but not in SW-620 tumors where a significant decrease in cell proliferation was detected. Chronic dosing of AZD6244 induced a morphologic change in SW-620 tumors to a more differentiated phenotype. The potential of AZD6244 in combination with cytotoxic drugs was evaluated in mice bearing SW-620 xenografts. Treatment with tolerated doses of AZD6244 and either irinotecan or docetaxel resulted in significantly enhanced antitumor efficacy relative to that of either agent alone. These results indicate that AZD6244 has potential to inhibit proliferation and induce apoptosis and differentiation, but the response varies between different xenografts. Moreover, enhanced antitumor efficacy can be obtained by combining AZD6244 with the cytotoxic drugs irinotecan or docetaxel. [Mol Cancer Ther 2007;6(8):2209–19]

Glial‐mediated neuroprotection: Evidence for the protective role of the NO‐cGMP pathway via neuron–glial communication in the peripheral nervous system

The NO‐cGMP pathway has emerged as a neuroprotective signaling system involved in communication between neurons and glia. We have previously shown that axotomy or nerve growth factor (NGF)‐deprivation of dorsal root ganglion (DRG) neurons leads to increased production of NO and at the same time an increase in cGMP production in their satellite glia cells. Blockade of NO or its receptor, the cGMP synthesizing enzyme soluble guanylate cyclase (sGC), results in apoptosis of neurons and glia. We now show that co‐culture of neonatal DRG neurons with either Schwann cells pre‐treated with an NO donor or a membrane‐permeant cGMP analogue; or neurons maintained in the medium from Schwann cell cultures treated in the same way, prevents neuronal apoptosis. Both NO donor and cGMP treatment of Schwann cells results in synthesis of NGF and NT3. Furthermore, if the Schwann cells are previously infected with adenoviral vectors expressing a dominant negative sGC mutant transgene, treatment of these Schwann cells with an NO donor now fails to prevent neuronal apoptosis. Schwann cells treated in this way also fail to express neither cGMP nor neurotrophins. These findings suggest NO‐sGC‐cGMP‐mediated NGF and NT3 synthesis by Schwann cells protect neurons.

Evaluation of a convenient method of assessing rodent visual function in safety pharmacology studies: effects of sodium iodate on visual acuity and retinal morphology in albino and pigmented rats and mice.

INTRODUCTION We have evaluated the ability of a semi-automated, optomotor reflex method to assess drug-induced visual dysfunction, in albino and pigmented rats and mice. METHODS Male Han Wistar (HW) and Long Evans (LE) rats and mice (CD-1 and C57BL/6) were tested in a chamber formed by 4 computer monitors displaying a rotating vertical grating, to elicit head-tracking movements. The highest visible grating frequency was taken as the threshold of visual acuity, in cycles per degree (c/d). Animals received an intravenous infusion of either sodium iodate (50mg/kg) or 0.9% w/v NaCl (aq). They were tested 2h later, then re-tested daily for a further 3 days. The time course of the effect was assessed in HW rats over a 6-week period, including electron microscopy, and immunohistochemical analysis of markers of injury and repair in the retina. RESULTS Baseline visual acuities for HW and LE rats were 0.355 ± 0.007 and 0.530 ± 0.004 c/d, respectively, and 0.296 ± 0.003 c/d and 0.370 ± 0.001 c/d for CD-1 and C57BL/6 mice, respectively (n=10 for each). In HW rats there was a dramatic loss of visual acuity 2h after administration of sodium iodate (0.021 ± 0.021 c/d; P<0.001). Less dramatic decreases in visual acuity were seen in LE rats and in the two mouse strains. In HW rats, visual acuity was restored after 4 weeks. This paralleled the histopathological recovery of the peripheral retina, whereas the central retina did not recover. DISCUSSION The method proved to be very convenient, and the stability of visual acuity in vehicle control rats over a 6-week period also demonstrated its suitability for inclusion in long-term toxicity studies. Both albino and pigmented mice and rats are suitable for assessment of retinotoxicity using this method, but albino rats are the most sensitive to sodium iodate.

NO‐cGMP mediated galanin expression in NGF‐deprived or axotomized sensory neurons

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5′ proximal promoter fragment (− 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF‐deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above‐mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF‐deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF‐deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant‐negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO‐cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up‐regulation of galanin, and for DRG neuron survival and regeneration after axotomy.

Regulation of activity-dependent neuroprotective protein (ADNP) by the NO-cGMP pathway in the hippocampus during kainic acid-induced seizure.

Activity-dependent neuroprotective protein (ADNP) is widely distributed in the cytoplasm of neurons and astrocytes of the hippocampus. Kainic acid (KA)-induced seizures increases neuronal nitric oxide synthase (nNOS) in neurons and inducible NOS (iNOS) in glia cells which coincides with a reduction in ADNP in the hippocampus. Inhibitors of NOS or soluble guanylyl cyclase (sGC) activity reduce ADNP under basal conditions in the absence of seizures. Treating animals with these inhibitors prior to KA-induced seizure, in particular, L-NAME (N(G)-nitro-l-arginine methyl ester), advances the onset of the first seizure but reverses the loss of ADNP by 3 days after the first seizure. This suggests that the NO-cGMP pathway has a role in regulating ADNP under both basal physiological conditions and in the pathophysiological changes produced during epileptogenesis.

Nitric Oxide-NGF Mediated PPTA/SP, ADNP, and VIP Expression in the Peripheral Nervous System

Nerve growth factor (NGF)-deprivation or axotomy of dorsal root ganglion (DRG) neurons causes stress, which they cope by triggering various mechanisms. Among several molecular changes, in the present study, we demonstrate preprotachykinin-A–substance P (PPTA–SP) and activity-dependent neuroprotective protein–vasoactive intestinal peptide (ADNP–VIP) expression pattern using DRG neurons–Schwann cells coculture and axotomy model. In the presence of NGF, DRG cultures showed high levels of PPTA and ADNP mRNA expression, which were significantly suppressed in the absence of NGF and/or nitric oxide synthase (NOS) inhibition by NG-nitro-l-arginine methyl ester (l-NAME), suggesting that both NGF and nitric oxide (NO) can regulate PPTA and ADNP expression. However, treating coculture with NO donor, diethylenetriamine nitric oxide (DETA–NO) did not increase PPTA and ADNP expression in the presence or absence of NGF, although there was a marginal increase in ADNP expression in the absence of NGF. NGF-deprivation increases endogenous NO; thus, DETA–NO had no further effect on PPTA and ADNP expression. Alternatively, NGF produced from NO-stimulated Schwann cells influence gene expression. In addition, interestingly, DETA–NO treatment of Schwann cells alone suppresses both PPTA and ADNP, suggesting differential response of DRG neurons–Schwann cells coculture to DETA–NO. SP and ADNP immunostaining of axotomized DRGs revealed significant reduction in SP and ADNP compared to intact DRG, which was partially recovered in neuronal NOS blocker, 7-nitroindazole (7-NI)-treated DRGs, particularly intense ADNP staining in satellite glia. As ADNP is VIP-responsive gene, we further explored VIP expression in DRGs. Axotomy increased VIP in DRG neurons, but 7-NI treatment caused intense VIP staining in satellite glia. These observations suggest a complex interaction of NO–NGF with PPTA/SP and ADNP–VIP in neuron–glial communication when neurons are stressed.

An antibody panel for immunohistochemical analysis of the retina in Davidson's-fixed, paraffin-embedded eyes of rats.

Unlike most other tissues, the optimal fixative for preserving eye morphology is considered to be Davidsons fixative or modified Davidsons rather than formalin. However, the methodology for antibodies to be used in tissues fixed this way is not normally outlined in current antibody datasheets. Additionally, where eyes have been stored in Davidsons fixative, the efficacy of retrospective analysis of eye morphology by immunohistochemistry is largely unknown. The aim of this study was to compare a panel of six antibodies in both Davidsons-fixed and formalin-fixed pigmented and non-pigmented rat eyes, in order to provide optimal methods for future retinal immunohistochemical evaluation with image analysis. The antibodies evaluated were raised against rhodopsin, synaptophysin, glutamine synthetase, glial fibrillary acidic protein (GFAP), cleaved caspase-3 and phospho-histone H3 (PH3). Overall, the staining quality of these antibodies was found to be optimal in Davidsons compared to formalin-fixed tissues after a time period of up to 4 days in fixative. The methods outlined thus provide a platform for future detailed analysis of retinal pathology in Davidsons-fixed eyes.

A commentary on the process of peer review and pathology data locking

JENNIFER S. MCKAY, ERIO BARALE-THOMAS, BRAD BOLON, CATHERINE GEORGE, JERRY HARDISTY, SUNAO MANABE, FREDERIC SCHORSCH, MUNEHIRO TERANISHI, AND KLAUS WEBER AstraZeneca, Macclesfield, SK10 4TG, United Kingdom Johnson and Johnson PRD, 2340 Beerse, Belgium GEMpath Inc., Longmont, CO 80503-2339, USA Ipsen, 91966 Les Ulis Cedex, France Experimental Pathology Laboratories, Research Triangle Park NC 22709, USA Daiichi Sankyo Co., Ltd., Japan Bayer CropScience, 69009 Lyon, France Harlan Laboratories, 4452 Itingen, Switzerland

NO-cGMP mediated neuropeptides regulation in peripheral nerve injury

NO-CGMP MEDIATED NEUROPEPTIDES REGULATION IN PERIPHERAL NERVE INJURY T. Thippeswamy , A.S. Cosgrave , D.A. Arora , J.S. McKay , J.P. Quinn ; a Department of Veterinary Preclinical Science, Veterinary Faculty, University of Liverpool, Liverpool, L69 7ZJ, UK; b AstraZeneca, Alderley Park, Macclesfield, SK10 4TG, UK; c Department of Physiology and Department of Human Anatomy and Cell Biology, School of Biomedical Sciences, Medical School, University of Liverpool, Liverpool L69 3BX, UK