Preclinical toxicity of AZD7969: Effects of GSK3ß inhibition in adult stem cells.

AZD7969 is a potent inhibitor of glycogen synthase kinase 3 (GSK3ß), which is a multifunctional serine/threonine kinase that negatively regulates the Wnt/ß-catenin signaling pathway. Treatment of rats and dogs with AZD7969 for periods of up to 4 weeks resulted in a number of changes, the most significant of which was a dose-dependent, and treatment-related, increase in proliferation in a number of tissues that was thought to arise from derepression of Wnt/ß-catenin signaling in the stem cell compartment. Phenotypically, this resulted in hyperplasia that either maintained normal tissue architecture in the gastrointestinal tract, liver, kidney, and adrenals or effaced normal tissue architecture within the bones, incisor teeth, and femorotibial joint. In addition to these changes, we noted a treatment-related increase in iron loading in the liver and proximal small intestines. This off-target effect was robust, potent, and occurred in both dogs and rats suggesting that AZD7969 might be a useful tool compound to study iron storage disorders in the laboratory.

Effect of the p38 kinase inhibitor, SB 203580, on allergic airway inflammation in the rat.

Tumour necrosis factor-alpha (TNF-alpha) and interleukin 1beta (IL-1beta) have been implicated in the pathogenesis of asthma. The p38 kinase inhibitor, SB 203580 inhibits TNF-alpha and IL-1beta production in vitro and in vivo. In this study the effect of SB 203580 on allergen-induced airway TNF-alpha production and inflammatory cell recruitment was investigated in sensitized Brown Norway rats. The allergen-induced increase in bronchoalveolar lavage (BAL) TNF-alpha was inhibited by SB 203580 at every dose tested (10 - 100 mg kg(-1), p.o.). In contrast, neither ovalbumin-induced eosinophilia or neutrophilia were inhibited by SB 203580 (10 - 100 mg kg(-1), p.o.). In conclusion, SB 203580 inhibits BAL TNF-alpha production by 95% without inhibiting either antigen-induced airway eosinophilia or neutrophilia. This data suggests that either the residual TNF-alpha is sufficient to drive allergen-induced inflammatory cell recruitment into the lung or that TNF-alpha is not involved in allergen-induced inflammatory cell recruitment.

Cerebroprotective effect of the nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl) imidazole and 7-nitro indazole, after transient focal cerebral ischemia in the rat.

The novel neuronal nitric oxide synthase inhibitors, 1-(2-trifluoromethylphenyl)imidazole (TRIM) and 7-nitro indazole (7-NI), were used to investigate the role of nitric oxide in a model of transient focal cerebral ischemia in vivo. In halothane-anesthetized rats, the middle cerebral artery (MCA) was occluded for 2 hours using an intravascular thread and then reperfused for 22 hours before histologic evaluation. TRIM (10, 20, or 50 mg/kg), 7-NI (60 mg/kg), TRIM (50 mg/kg) plus L-arginine (300 mg/kg), or L-arginine (300 mg/kg) alone was administered intraperitoneally, either at 5 or 90 minutes after MCA occlusion. Immediate administration (5 minutes after MCA occlusion) of TRIM produced a dose-related reduction in lesion size, which was reversed with L-arginine coadministration. Similarly, delayed administration of TRIM (90 minutes after MCA occlusion, 50 mg/kg) decreased total lesion volume by 48.4% +/- 13.0% in comparison to a reduction of 39.3% +/- 10.9% when TRIM (50 mg/kg) was administered immediately (5 minutes) after occlusion. 7-NI (60 mg/kg) reduced the total lesion volume by 38.5% +/- 13.7% when administered immediately (5 minutes) after MCA occlusion, but had no effect when administration was delayed (90 minutes). Neither TRIM (50 mg/kg) nor 7-NI (60 mg/kg), administered 5 minutes after MCA occlusion, had any significant effect on mean arterial blood pressure throughout the ischemic period or for up to 10 minutes after reperfusion. These results indicate that immediate or delayed administration of the selective neuronal NOS inhibitor TRIM reduces the lesion volume after transient MCA occlusion. In contrast, only immediate administration of 7-NI reduces lesion volume.

The involvement of calcitonin gene-related peptide (CGRP) and substance P in feline pial artery diameter responses evoked by capsaicin.

The effects of capsaicin and selective neuropeptide antagonists on pial artery diameter were measured using an on-line image analyser in anaesthetised cats, in order to monitor the effects of mediators released in response to activation of trigeminal nerves. Perivascular injection of CGRP (10(-8) M) and the neurokinin-1 (NK1) receptor agonist substance P methyl ester, SPOMe (10(-6) M) produced an increase in pial artery diameter. The vasodilatory action of these agonists was significantly and selectively inhibited using the CGRP receptor antagonist, CGRP8-37 (10(-6) M), and the NK1 receptor antagonist, CP99994 (10(-6) M) respectively. Capsaicin (10(-8)-10(-5) M) produced a biphasic response upon perivascular injection that was concentration dependent. At 10(-6) M capsaicin an initial transient vasoconstriction was observed followed by a longer-lasting vasodilatation. The vasodilator component was significantly reduced by CGRP8-37 (10(-6) M) or CP99994 (10(-6) M). These results show that chemical (capsaicin) activation of trigeminal nerves leads to vasodilatation of feline arteries in situ. This vasodilatation is mediated via the activation of CGRP and NK1 receptors probably via the efferent release of CGRP and a substance P-like peptide.

The modulation of the increase in rat facial skin blood flow observed after trigeminal ganglion stimulation.

Electrical stimulation of the trigeminal ganglion causes an increase in facial skin blood flow in the anaesthetised rat, as measured by laser Doppler flowmetry. We investigated the modulation of this neurogenic vasodilator response using selective receptor agonists for putative prejunctional inhibitory receptors, as well as other pharmacological agents to further characterise this response. [D-Ala2,Me-Phe4,Gly5-ol]enkephalin (DAGO, a mu-opioid receptor agonist) inhibited the vasodilator response in a dose-related (0.058-5.8 mumol/kg i.v.) and naloxone-sensitive manner. A similar inhibitory response was observed with the local anaesthetic lignocaine (2% w/v, s.c. 20 microliters). In contrast, the histamine H3-receptor agonist alpha-methylhistamine (15 or 35 mumol/kg, i.v.) and the 5-HT1D receptor agonists sumatriptan (0.24 or 2.4 mumol/kg, i.v.) and CP 122,288 (0.0003-3 mumol/kg, i.v.) had no effect on these responses. Similarly, atropine (1.5 mumol/kg, i.v.) and indomethacin (28 mumol/kg, i.v.) did not alter the vasodilatation observed in this model. In conclusion, only mu-opioid receptor activation and local anaesthetic had any inhibitory action on the neurogenic vasodilatation observed in this model.

Trigeminal ganglion stimulation increases facial skin blood flow in the rat: a major role for calcitonin gene-related peptide.

Activation of the trigeminovascular system leads to neurogenic inflammation within the dura mater and cerebral vasodilatation. These processes have been implicated in the pathogenesis of migraine headache. Neurogenic vasodilator responses to trigeminal ganglion stimulation were investigated in rat facial skin, an area innervated by the trigeminal nerve. Microvascular blood flow changes in the facial skin were measured in anaesthetised rats, using laser Doppler flowmetry. Electrical stimulation of the trigeminal ganglion caused an ipsilateral increase in facial skin blood flow which was found to be frequency dependent (0.5-10 Hz). The role of several neuropeptides in these blood flow responses was studied using selective receptor antagonists. The calcitonin gene-related peptide antagonist, CGRP8-37 (400 nmol.kg-1, i.v.) had no effect on resting levels of facial skin blood flow, but markedly inhibited responses induced by trigeminal ganglion stimulation (5 Hz, 10 V, 1 ms for 30 s). However, neither the neurokinin-1 (NK1) receptor antagonist, RP67580 (0.23 or 2.3 mumol.kg-1, i.v.) nor the vasoactive intestinal peptide (VIP) antagonist, [p-Cl-D-Phe6,Leu17]-VIP (15 or 30 nmol.kg-1, i.v.) had any effect on these responses. These results suggest that CGRP is the major neuropeptide involved in the vasodilator response to trigeminal ganglion stimulation in rat facial skin. Clarification of the mechanisms involved in this neurogenic vasodilator response may aid the development of drugs that target the trigeminovascular system during migraine headache.

Effect of a calcitonin gene-related peptide antagonist (CGRP8-37) on skin vasodilatation and oedema induced by stimulation of the rat saphenous nerve.

1. The effect of the calcitonin gene-related peptide antagonist (CGRP8-37, 400 nmol kg-1, i.v.) on the increased blood flow induced by calcitonin gene related peptide (CGRP), vasodilator prostaglandins, and topical capsaicin was measured with a laser Doppler blood flow meter in rat abdominal skin. 2. The saphenous nerve was electrically stimulated and the effect of CGRP8-37 (400 nmol kg-1, i.v.) on the increased blood flow (measured by laser Doppler flowmetry) and oedema formation (measured by the extravascular accumulation of [125I]-albumin) was investigated in the rat hind paw. 3. CGRP8-37 (400 nmol kg-1, i.v.) had no effect on basal cutaneous blood flow at uninjected sites and sites injected with Tyrode buffer, but acted selectively to inhibit the increased blood flow induced by intradermal CGRP (10 pmol/site, P < 0.05), but not that induced by prostaglandin E2 (PGE2, 300 pmol/site) or carba-prostacyclin (cPGI2, 100 pmol/site). 4. Capsaicin (0.1-33 mM), applied topically, acted in a dose-related manner to increase blood flow. CGRP8-37 (400 nmol kg-1, i.v.) almost totally inhibited blood flow induced by capsaicin (10 mM; P < 0.05) but did not significantly inhibit blood flow induced by a higher dose of capsaicin (33 mM). 5. The increased blood flow induced by short stimulation of the saphenous nerve (10 V, 1 ms, 2 Hz for 30 s) was inhibited by 76%, 5 min after i.v. CGRP8-37 (400 nmol kg-1, i.v., P < 0.05). 6. A longer (5 min) electrical stimulation of the saphenous nerve caused oedema formation, in addition to increased blood flow. The oedema formation was significantly inhibited by CGRP8-37 (400 nmol kg-1,i.v., P<0.05).7. The results suggest that the potent microvascular vasodilator neuropeptide, CGRP, is responsible for the increased blood flow observed after short stimulation of the saphenous nerve and that endogenous CGRP contributes in a pro-inflammatory manner to neurogenic oedema formation in the rat hind paw.