Frank Cloutier

Kinin receptors in pain and inflammation

Kinins are among the most potent autacoids involved in inflammatory, vascular and pain processes. These short-lived peptides, including bradykinin, kallidin and T-kinin, are generated during tissue injury and noxious stimulation. However, emerging evidence also suggests that kinins are stored in neuronal elements of the central nervous system (CNS) where they are thought to play a role as neuromediators in various cerebral functions, particularly in the control of nociceptive information. Kinins exert their biological effects through the activation of two transmembrane G-protein-coupled receptors, denoted bradykinin B(1) and B(2). Whereas the B(2) receptor is constitutive and activated by the parent molecules, the B(1) receptor is generally underexpressed in normal tissues and is activated by kinins deprived of the C-terminal Arg (des-Arg(9)-kinins). The induction and increased expression of B(1) receptor occur following tissue injury or after treatment with bacterial endotoxins or cytokines such as interleukin-1 beta and tumor necrosis factor-alpha. This review summarizes the most recent data from various animal models which convey support for a role of B(2) receptors in the acute phase of the inflammatory and pain response, and for a role of B(1) receptors in the chronic phase of the response. The B(1) receptor may exert a strategic role in inflammatory diseases with an immune component (diabetes, asthma, rheumatoid arthritis and multiple sclerosis). New information is provided regarding the role of sensory mechanisms subserving spinal hyperalgesia and intrapleural neutrophil migration that occur upon B(1) receptor activation in streptozotocin-treated rats, a model of insulin-dependent diabetes mellitus in which the B(1) receptor seems to be rapidly overexpressed. Although it is widely accepted that the blockade of kinin receptors with specific antagonists could be of benefit in the treatment of somatic and visceral inflammation and pain, recent molecular and functional evidence suggests that the activation of B(1) receptors with an agonist may afford a novel therapeutic approach in the CNS inflammatory demyelinating disorder encountered in multiple sclerosis by reducing immune cell infiltration (T-lymphocytes) into the brain. Hence, the B(1) receptor may exert either a protective or detrimental effect depending on the inflammatory disease. This dual function of the B(1) receptor deserves to be investigated further.

Nuclear Retention of MBP mRNAs in the Quaking Viable Mice

Quaking viable (qk(v)) mice fail to properly compact myelin in their central nervous systems. Although the defect in the qk(v) mice involves a mutation affecting the expression of the alternatively spliced qk gene products, their roles in myelination are unknown. We show that the QKI RNA binding proteins regulate the nuclear export of MBP mRNAs. Disruption of the QKI nucleocytoplasmic equilibrium in oligodendrocytes results in nuclear and perikaryal retention of the MBP mRNAs and lack of export to cytoplasmic processes, as it occurs in qk(v) mice. MBP mRNA export defect leads to a reduction in the MBP levels and their improper cellular targeting to the periphery. Our findings suggest that QKI participates in myelination by regulating the mRNA export of key protein components.

Pharmacological characterization of the cardiovascular responses elicited by kinin B1 and B2 receptor agonists in the spinal cord of streptozotocin-diabetic rats

Kinin receptor agonists and antagonists at the B1 and B2 receptors were injected intrathecally (i.t., at T‐9 spinal cord level) to conscious unrestrained rats and their effects on mean arterial pressure (MAP) and heart rate (HR) were compared in streptozotocin (STZ)‐diabetic rats (65 mg kg−1 STZ, i.p. 3 weeks earlier) and aged‐matched control rats. The B1 receptor agonist, des‐Arg9‐Bradykinin (BK) (3.2–32.5 nmol), evoked dose‐dependent increases in MAP and tachycardia during the first 10 min post‐injection in STZ‐diabetic rats only. The cardiovascular response to 6.5 nmol des‐Arg9‐BK was reversibly blocked by the prior i.t. injection of antagonists for the B1 receptor ([des‐Arg10]‐Hoe 140, 650 pmol or [Leu8]‐des‐Arg9‐BK, 65 nmol) and B2 receptor (Hoe 140, 81 pmol or FR173657, 81 pmol) or by indomethacin (5 mg kg−1, i.a.). The i.t. injection of BK (8.1–810 pmol) induced dose‐dependent increases in MAP which were accompanied either by tachycardiac (STZ‐diabetic rats) or bradycardiac (control rats) responses. The pressor response to BK was significantly greater in STZ‐diabetic rats. The cardiovascular response to 81 pmol BK was reversibly blocked by 81 pmol Hoe 140 or 81 pmol FR173657 but not by B1 receptor antagonists nor by indomethacin in STZ‐diabetic rats. The data suggest that the activation of kinin B1 receptor in the spinal cord of STZ‐diabetic rats leads to cardiovascular changes through a prostaglandin mediated mechanism. Thus, this study affords an accessible model for studying the expression, the pharmacology and physiopathology of the B1 receptor in the central nervous system.

Pharmacologic and autoradiographic evidence for an up‐regulation of kinin B2 receptors in the spinal cord of spontaneously hypertensive rats

The effects of intrathecally (i.t.) injected kinin B1 and B2 receptor agonists and antagonists were measured on mean arterial pressure (MAP) and heart rate (HR) of conscious unrestrained spontaneously hypertensive rats (SHR of 16 weeks old) and age‐matched normotensive Wistar Kyoto (WKY). Quantitative in vitro autoradiographic studies were also performed on the thoracic spinal cord of both strains with specific radioligands for B2 receptors, [125I]‐HPP‐Hoe 140, and B1 receptors, [125I]‐HPP‐[des‐Arg10]‐Hoe140. Bradykinin (BK) (0.81 – 810 pmol) increased MAP dose‐dependently with increases or decreases of HR. The pressor response to BK was significantly greater in SHR. The cardiovascular response to 8.1 pmol BK was reversibly blocked by 81 pmol Hoe 140 (B2 antagonist) but not by 81 – 810 pmol [des‐Arg10]‐Hoe 140 (B1 antagonist) in both strains. The B1 receptor agonist, des‐Arg9‐BK (8100 pmol) produced either no effects or increased MAP with variable effects on HR. These responses were similar in both strains and were reversibly blocked by 81 pmol Hoe 140. Inhibition with 8100 pmol [des‐Arg10]‐Hoe 140 was not specific to B1 agonist‐mediated responses. [125I]‐HPP‐Hoe 140 specific binding sites were predominantly located to superficial laminae of the dorsal horn and were significantly higher in SHR. Low levels of [125I]‐HPP‐[des‐Arg10]‐HOE 140 specific binding sites were found in all laminae of both strains. It is concluded that the hypersensitivity of the cardiovascular response to BK is due to an increased number of B2 receptors in the spinal cord of SHR and that B1 receptors are unlikely involved in spinal cardiovascular regulation in SHR.

Antiangiogenic activity of aganirsen in nonhuman primate and rodent models of retinal neovascular disease after topical administration.

PURPOSE Aganirsen, an antisense oligonucleotide inhibiting insulin receptor substrate (IRS)-1 expression, has been shown to promote the regression of pathologic corneal neovascularization in patients. In this study, the authors aimed to demonstrate the antiangiogenic activity of aganirsen in animal models of retinal neovascularization. METHODS Eyedrops of aganirsen were applied daily in nonhuman primates after laser-induced choroidal neovascularization (CNV; model of wet age-related macular degeneration [AMD]) and in newborn rats after oxygen-induced retinopathy (OIR; model of ischemic retinopathy). Retinal aganirsen concentrations were assessed in rabbits and monkeys after topical delivery (21.5, 43, or 86 μg). Clinical significance was further evaluated by determination of IRS-1 expression in monkey and human retinal biopsy specimens. RESULTS Topical corneal application of aganirsen attenuated neovascular lesion development dose dependently in African green monkeys. The incidence of high-grade CNV lesions (grade IV) decreased from 20.5% in vehicle-treated animals to 1.7% (P < 0.05) at the 86-μg dose. Topical aganirsen inhibited retinal neovascularization after OIR in rats (P < 0.05); furthermore, a single intravitreal injection of aganirsen reduced OIR as effectively as ranibizumab, and their effects were additive. Significantly, topical applications of aganirsen did not interfere with physiological retinal vessel development in newborn rats. Retinal delivery after topical administration was confirmed, and retinal expression of IRS-1 was demonstrated to be elevated in patients with subretinal neovascularization and AMD. CONCLUSIONS Topical application of aganirsen offers a safe and effective therapy for both choroidal and retinal neovascularization without preventing its normal vascularization. Together, these findings support the clinical testing of aganirsen for human retinal neovascular diseases.

Expression of kinin B1 receptors in the spinal cord of streptozotocin-diabetic rat.

Previous studies have reported cardiovascular and nociceptive responses after intrathecal injection of kinin B1 receptor (B1R) agonists in the model of streptozotocin (STZ)-diabetic rat (diabetic). The aim of this study was to measure the early up-regulation of B1R binding sites and mRNA in the thoracic spinal cord of diabetic and control rats. Data show significant increases of specific B1R binding sites in the dorsal horn of diabetic rats 2 days (+315%), 7 days (+303%) and 21 days (+181%) after STZ treatment. Levels of mRNA were significantly increased (+68%) at 2 and 7 days but not at 21 days. These data bring the first molecular evidence for an early up-regulation of B1R in the spinal cord of diabetic rat.

Correlation between brain bradykinin receptor binding sites and cardiovascular function in young and adult spontaneously hypertensive rats

Intracerebroventricular (i.c.v.) effects of bradykinin (BK) B1 and B2 receptor agonists and antagonists were assessed on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR, aged of 8 and 16 weeks) and age‐matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiographic studies were also performed on the brain of both strains with specific radioligands for B2 receptors [125I]HPP‐Hoe 140 and B1 receptors [125I]HPP‐des‐Arg10 and Hoe140. MAP increased linearly with doses of BK (81–8100 pmol) and the amplitudes were significantly greater in SHR, particularly at 16 weeks. While BK evoked a negative linear trend on HR (bradycardia) in WKY, a positive one (tachycardia) was observed in adult SHR. In both strains, BK‐induced pressor response was blocked by equimolar doses of B2 receptor antagonist, D‐Arg‐[Hyp3, Thi5, D‐Tic7, Oic8]‐BK (Hoe 140), but not by B1 receptor antagonist, AcLys[D‐βNal7, Ile8]des‐Arg9‐BK (R‐715). B1 receptor agonists (Sar‐[D‐Phe8]‐des‐Arg9‐BK, des‐Arg9‐BK, des‐Arg10‐Kallidin) and antagonist (R‐715 alone or with Hoe 140) had no or marginal effect on MAP and HR at doses up to 8100 pmol in SHR and WKY. Higher densities of specific [125I]HPP‐Hoe 140 labelling were found in discrete brain areas of SHR, especially in regions associated with cardiovascular function. Low levels of [125I]HPP‐[des‐Arg10]‐Hoe140 binding sites were seen in WKY and SHR, yet densities were significantly greater in midbrain and cortical regions of SHR aged of 16 weeks. Contrary to SHR, ageing caused a downregulation of B2 and B1 receptor binding sites in specific brain nuclei in WKY. It is concluded that the hypersensitivity of the pressor response to i.c.v. BK in SHR occurs during both the early and established phases of hypertension in parallel with the enhancement of B2 receptor binding sites in various cardiovascular brain centres. In contrast, brain B1 receptors do not seem to participate in the central pressor effects of kinins nor in the maintenance of hypertension in SHR.

Upregulation of tachykinin NK-1 and NK-3 receptor binding sites in the spinal cord of spontaneously hypertensive rat: impact on the autonomic control of blood pressure

1 Effects of intrathecally (i.t.) injected tachykinin NK‐1 and ‐3 receptor agonists and antagonists were measured on mean arterial blood pressure (MAP) and heart rate (HR) in awake unrestrained spontaneously hypertensive rats (SHR,15‐week‐old) and age‐matched Wistar Kyoto rats (WKY). Quantitative in vitro autoradiography was also performed on the lower thoracic spinal cord of both strains and Wistar rats using specific radioligands for NK‐1 receptor ([125I]HPP[Arg3,Sar9,Met(O2)11]SP (3–11)) and NK‐3 receptor ([125I]HPP‐Asp‐Asp‐Phe‐N‐MePhe‐Gly‐Leu‐Met‐NH2). 2 The NK‐1 agonist [Sar9,Met(O2)11]SP (650 and 6500 pmol) decreased MAP and increased HR in WKY. The fall in MAP was blunted in SHR and substituted by increases in MAP (65–6500 pmol) and more sustained tachycardia. The NK‐3 agonist senktide (6.5–65 pmol) evoked marked increases in MAP and HR (SHR>>>WKY), yet this response was rapidly desensitized. Cardiovascular effects of [Sar9,Met(O2)11]SP (650 pmol) and senktide (6.5 pmol) were selectively blocked by the prior i.t. injection of LY303870 (NK‐1 antagonist, 65 nmol) and SB235375 (NK‐3 antagonist, 6.5 nmol), respectively. Antagonists had no direct effect on MAP and HR in both strains. 3 Densities of NK‐1 and ‐3 receptor binding sites were significantly increased in all laminae of the spinal cord in SHR when compared to control WKY and Wistar rats. The dissociation constant was however not affected in SHR for both NK‐1 (Kd=2.5 nM) and NK‐3 (Kd=5 nM) receptors. 4 Data highlight an upregulation of NK‐1 and ‐3 receptor binding sites in the thoracic spinal cord of SHR that may contribute to the hypersensitivity of the pressor response to agonists and to the greater sympathetic activity seen in this model of arterial hypertension.

Compensatory mechanisms to maintain blood pressure in paraplegic rats: Implication of central tachykinin NK-1 and NK-3 receptors?

People with high level spinal cord injury (SCI) suffer from both hypotension and spontaneous hypertension due to loss of supraspinal control of spinal sympathetic outflow. Few reports have addressed whether any changes occur in central regulation of blood pressure (BP) and heart rat (HR) at the supraspinal level. Central tachykinin NK-1 and NK-3 receptors are located in many cardiovascular areas in the brain and are known to modulate BP and HR. This study examined the intracerebroventricular (i.c.v.) effects of the selective NK-1 receptor agonist [Sar(9), Met(O(2))(11)]SP (65pmol, n=6) and NK-3 receptor agonist senktide (650pmol, n=6) on mean arterial pressure (MAP) and HR before and after complete spinal cord transection at thoracic level 4 (T4). [Sar(9), Met(O(2))(11)]SP evoked increases in MAP and HR which were still present 4days after the T4 SCI. Further analysis using the beta(1)-adrenoceptor antagonist atenolol (10mgkg(-1)) revealed an increased contribution of HR in the MAP increase after SCI. For senktide, 2 and 5weeks after T4 SCI, the rise in MAP induced by senktide was significantly increased in magnitude and was similar to a normal response at 8weeks. These effects were accompanied by a bradycardia, which was still present and amplified at 8weeks. Our results reveal a transient potentiation of the senktide-mediated MAP effect and a greater contribution of the HR in MAP increase by [Sar(9), Met(O(2))(11)]SP in T4 transected rats. Although the significance of these changes remains to be established. This suggest a reorganization of supraspinal mechanisms regulating BP and HR after a high level SCI. Central NK-1 and NK-3 receptors might therefore contribute to the maintenance of MAP following high thoracic SCI.

Specific targeting of the IL-23 receptor, using a novel small peptide noncompetitive antagonist, decreases the inflammatory response

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rβ1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rβ1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rβ-IL-12Rβ2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.