Optogenetic therapy: high spatiotemporal resolution and pattern discrimination compatible with vision restoration in non-human primates.

Vision restoration is an ideal medical application for optogenetics, because the eye provides direct optical access to the retina for stimulation. Optogenetic therapy could be used for diseases involving photoreceptor degeneration, such as retinitis pigmentosa or age-related macular degeneration. We describe here the selection, in non-human primates, of a specific optogenetic construct currently tested in a clinical trial. We used the microbial opsin ChrimsonR, and showed that the AAV2.7m8 vector had a higher transfection efficiency than AAV2 in retinal ganglion cells (RGCs) and that ChrimsonR fused to tdTomato (ChR-tdT) was expressed more efficiently than ChrimsonR. Light at 600 nm activated RGCs transfected with AAV2.7m8 ChR-tdT, from an irradiance of 1015 photons.cm-2.s-1. Vector doses of 5 × 1010 and 5 × 1011 vg/eye transfected up to 7000 RGCs/mm2 in the perifovea, with no significant immune reaction. We recorded RGC responses from a stimulus duration of 1 ms upwards. When using the recorded activity to decode stimulus information, we obtained an estimated visual acuity of 20/249, above the level of legal blindness (20/400). These results lay the groundwork for the ongoing clinical trial with the AAV2.7m8 - ChR-tdT vector for vision restoration in patients with retinitis pigmentosa.

Autoradiographic analysis of mouse brain kinin B1 and B2 receptors after closed head trauma and ability of Anatibant mesylate to cross the blood-brain barrier.

The potent non-peptide B2 receptor (R) antagonist, Anatibant mesylate (Ms) (LF 16-0687 Ms), reduces brain edema and improves neurological function recovery in various focal and diffuse models of traumatic brain injury in rodents. In the present study, alteration of kinin B1 and B2R after closed head trauma (CHT) and in vivo binding properties of Anatibant Ms (3 mg/kg, s.c.) injected 30 min after CHT were studied in mice by autoradiography using the radioligands [125I]HPP-Hoe 140 (B2R), and [125I]HPP-des-Arg10-Hoe 140 (B1R). Whereas B1R is barely detected in most brain regions, B2R is extensively distributed, displaying the highest densities in the hindbrain. CHT was associated with a slight increase of B1R and a decrease of B2R (10-50%) in several brain regions. Anatibant Ms (Ki = 22 pM) displaced the B2R radioligand from its binding sites in several areas of the forebrain, basal ganglia and hindbrain. Displacement was achieved in 1 h and persisted at 4 h post-injection. The inhibition did not exceed 50% of the total specific binding in non-injured mice. After CHT, the displacement by Anatibant Ms was higher and almost complete in the cortex, caudate putamen, thalamus, hippocampus, medial geniculate nucleus, ventral tegmental area, and raphe. Evans blue extravasation in brain tissue at 4 h after CHT was abolished by Anatibant Ms. It appeared that Anatibant Ms penetrated into the brain in sufficient amounts, particularly after disruption of the blood-brain barrier, to account for its B2R-mediated neuro- and vascular protective effects. The diminished binding of B2R after CHT may reflect the occupancy or internalization of B2R following the endogenous production of bradykinin (BK).

Therapeutic window of bradykinin B2 receptor inhibition after focal cerebral ischemia in rats.

Following cerebral ischemia bradykinin/kinin B(2) receptors mediate inflammatory responses resulting in edema formation and secondary brain damage. However, the therapeutic window for B(2) receptor inhibition determining its potential clinical use has not been investigated so far. The aim of the current study was therefore to investigate the effect of delayed B(2) receptor inhibition on morphological and functional outcome following experimental stroke. Rats were subjected to 90 min of middle cerebral artery occlusion (MCAo) by an intraluminal filament. Animals received 0.9% NaCl or 1.0mg/kg/day Anatibant (LF 16-0687 Ms), a selective bradykinin B(2) receptor antagonist, for 3 days beginning at different time points after MCAo: 1, 2.5, 4.5, or 6.5h (n=10 per group). Neurological recovery was examined daily, infarct volume on day 7 after MCAo. Animal physiology was not influenced by B(2) receptor inhibition. Significant improvement of functional outcome was observed when treatment was delayed up to 4.5h after ischemia (p<0.05 versus vehicle). Inhibition of B(2) receptors during ischemia, i.e. when the inhibitor was given 1h after MCAo, reduced infarct volume in the basal ganglia and in the cortex by 49% (p<0.05) and 26% (p<0.05), respectively. Inhibition of B(2) receptors at later time points (2.5, 4.5, or 6.5 after MCAo) reduced penumbral damage, i.e. cortical infarction, by 19-26% (p<0.05). In conclusion, the current study shows that the therapeutic window of B(2) receptor inhibition extends for up to 6.5h after MCAo. Our data therefore suggest that inhibition of kinin B(2) receptors represents a treatment strategy for ischemic stroke which may warrant clinical validation.

Release of bradykinin and expression of kinin B2 receptors in the brain: role for cell death and brain edema formation after focal cerebral ischemia in mice.

Pharmacological studies using bradykinin B2 receptor antagonists suggest that bradykinin, an early mediator of inflammation and the main metabolite of the kallikrein-kinin system, is involved in secondary brain damage after cerebral ischemia. However, the time-course of bradykinin production and kinin receptor expression as well as the conclusive role of bradykinin B2 receptors for brain damage after experimental stroke have not been elucidated so far. C57/Bl6 mice were subjected to 45 mins of middle cerebral artery occlusion (MCAO) and 2, 4, 8, 24, and 48 h later brains were removed for the analysis of tissue bradykinin concentration and kinin B2 receptor mRNA and protein expression. Brain edema, infarct volume, functional outcome, and long-term survival were assessed in WT and B2-/- mice 24 h or 7 days after MCAO. Tissue bradykinin was maximally increased 12 h after ischemia (three-fold), while kinin B2 receptor mRNA upregulation peaked 24 to 48 h after MCAO (10- to 12-fold versus naïve brain tissue). Immunohistochemistry revealed that kinin B2 receptors were constitutively and widely expressed in mouse brain, were upregulated 2 h after ischemia in cells showing signs of ischemic damage, and remained upregulated in the penumbra up to 24 h after ischemia. B2-/- mice had improved motor function (P<0.05), smaller infarct volumes (-38%; P<0.01), developed less brain edema (-87%; P<0.05), and survived longer (P<0.01) as compared with wild-type controls. The current results show that bradykinin is produced in the brain, kinin B2 receptors are upregulated on dying cells, and B2 receptors are involved in cell death and brain edema formation after experimental stroke.

LF 16-0687 Ms, a bradykinin B2 receptor antagonist, reduces ischemic brain injury in a murine model of transient focal cerebral ischemia.

1. Bradykinin promotes neuronal damage and brain edema through the activation of the B(2) receptor. The neuroprotective effect of LF 16-0687 Ms, a B(2) receptor antagonist, has been described when given prior to induction of transient focal cerebral ischemia in rat, but there are no data regarding the consequence of a treatment when given after injury. Therefore, in a murine model of transient middle cerebral artery occlusion (MCAO), we evaluated the effect of LF 16-0687 Ms given prior to and/or after the onset of ischemia on neurological deficit, infarct volume and inflammatory responses including cerebral edema, blood-brain barrier (BBB) disruption and neutrophil accumulation. 2. LF 16-0687 Ms (1, 2 and 4 mg kg(-1)) administered 0.5 h before and, 1.25 and 6 h after MCAO, decreased the infarct volume by a maximum of 33% and significantly improved the neurological recovery. 3. When given at 0.25 and 6.25 h after MCAO, LF 16-0687 Ms (1.5, 3 and 6 mg kg(-1)) decreased the infarct volume by a maximum of 25% and improved the neurological score. 4. Post-treatment with LF 16-0687 Ms (1.5 mg kg(-1)) significantly decreased brain edema (-28%), BBB disruption (-60%) and neutrophil accumulation (-65%) induced by ischemia. Physiological parameters were not modified by LF 16-0687 Ms. 5. These data emphasize the role of bradykinin B(2) receptor in the development of infarct lesion, neurological deficit and inflammatory responses resulting from transient focal cerebral ischemia. Therefore, B(2) receptor antagonist might represent a new therapeutic approach in the pharmacological treatment of stroke.

Characterization of the molecular interactions of interleukin-8 (CXCL8), growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) with the human CXCR2.

Neutrophil recruitment to inflammatory sites is mediated by two related receptors: CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2). Both receptors share two ligands, interleukin-8 (CXCL8) and GCP-2 (CXCL6), whereas several chemokines, including growth related oncogen alpha (CXCL1) and a non-peptide antagonist (SB 225002) are specific for CXCR2. The objective of this study was to map the different amino acids involved in the binding and activation/inhibition of human CXCR2. This was performed by exchanging non-conserved amino acids of CXCR2 with their counterparts in CXCR1. The mutants generated showed that: (a) for CXCL8 binding, the N-terminus of CXCR1 and the second extra-cellular loop of CXCR2 are determinant, the N-terminus of CXCR2 is not sufficient and the transmembrane domain seven is probably involved; (b) for CXCL1, the N-terminus of CXCR2 is necessary but not sufficient for binding. The activation study indicated that amino acids critical for activation are not necessarily involved in binding process. Finally, the mechanism of binding of a non-peptide antagonist on CXCR2 was investigated: it occurred through epitopes (a) which were disseminated within the receptor, (b) which differed according to the use of CXCL8 or CXCL1 as a competitor and (c) which did not necessarily overlap with agonist binding sites. We also showed that inhibition of binding and inhibition of activation involved different amino acids.

Cloning and characterization of guinea pig interleukin-8 receptor.

CXC-chemokine receptors 1 and 2 and their ligands (CXCL1, 2, 3, 5, 6, 7, and 8) induce the selective recruitment of neutrophils during inflammation. Such receptors have not been characterized yet in guinea pig, an animal inflammation model of interest. We report the identification, cloning, and characterization of a CXCL8 receptor in guinea pig. Human CXCL8 produced in vivo neutrophilia, chemotaxis and intracellular calcium release of guinea pig neutrophils. The expression of this receptor at their neutrophil surface was investigated. The cDNA encoding a functional CXCL8 receptor was cloned from guinea pig neutrophils and sequenced. It was synthesized using RT-PCR, with oligonucleotide primers derived from well conserved regions of published CXCL8 receptors. This sequence presented an open reading frame coding for 352 amino acids and shares, at the amino acid level, 70 and 69% identity with human and rabbit CXCR2, respectively. The receptor was mainly expressed in neutrophils but it was also present in kidney, lung, spleen and, to a less extent, in heart. Cloned receptor transfected cells showed that this receptor displayed high affinity for human CXCL8, slightly lower than the affinity observed with guinea pig neutrophils. CXC chemokines from both rabbit and human were shown to induce inositol phosphate accumulation in these transfected cells. Receptor binding and activation characteristics together with sequence homology suggested that we identified a guinea pig equivalent of the human CXCR2 receptor.

LF 16-0687 Ms, a bradykinin B2 receptor antagonist, reduces brain edema and improves long-term neurological function recovery after closed head trauma in rats.

Bradykinin is an endogenous inflammatory agent that enhances vascular permeability and produces tissue edema. We investigated whether LF 16-0687 Ms, a potent nonpeptide antagonist of bradykinin type-2 (B(2)) receptor, was able to reduce brain swelling and to improve the recovery of neurological function following closed head trauma (CHT) in rats. In dose-effect studies, LF 16-0687 Ms doses of 0.75-4.5 mg/kg given 1 h after trauma significantly reduced the development of edema in the injured hemisphere by a maximum of 70%. It had no effect on the brain water content of sham-operated rats. LF 16-0687 Ms also significantly improved neurological recovery evaluated by a Neurological Severity Score (NSS) based on motor, reflex, and behavioral tests. In time-window studies LF 16-0687 Ms (2.25 mg/kg) was given 1, 2, 4, and 10 h after CHT. The extent of edema was significantly reduced when LF 16-0687 Ms was given 1 h (-45%), 2 h (-52%), and 4 h (-63%) but not 10 h (-24%) after CHT. Given at any time-point, LF 16-0687 Ms significantly improved the recovery of the NSS at 24 h. In duration of treatment studies, rats tended to recover normal neurological function over 14 days after CHT. However, time to recovery was longer in severely than in moderately injured animals, unless they were treated with LF 16-0687 Ms. This study provides further evidence that blockade of bradykinin B(2) receptors represents a potential effective approach to the treatment of focal cerebral contusions.

LF 16-0687 Ms, a new bradykinin B2 receptor antagonist, decreases ex vivo brain tissue prostaglandin E2 synthesis after closed head trauma in rats.

OBJECTIVE: Bradykinin (B) contributes to secondary brain injury. This injury is mediated in part by prostaglandin (PG). Antagonism of B(2) receptors improves neurological status after brain injury, but the effect of B(2) antagonism on brain tissue PG is unknown. This study examined the effect of LF 16-0687 Ms, a new B(2) receptor antagonist, on brain tissue PGE(2) after closed head trauma (CHT). METHODS: Rats were anesthetized and received sham+saline, sham+LF 16-0687 Ms, CHT+saline, or CHT+LF 16-0687 Ms. Brain tissue samples were obtained at 24 h for determination of PGE(2) (after 2 h of ex vivo incubation) and water content. Neurological severity score (NSS) was assessed at 1 and 24 h. RESULTS: In the group receiving CHT+LF 16-0687 Ms, brain tissue PGE(2) (77.7+/-65.9 pg/mg tissue, mean+/-SD) was less than in the group receiving CHT+saline (368.1+/-186.2 pg/mg tissue) and not different than sham+saline (78.7+/-30.7 pg/mg tissue). LF 16-0687 Ms also improved NSS and decreased brain water content by 51%. CONCLUSION: We conclude that the beneficial effect of LF 16-0687 Ms on outcome after CHT is accompanied by blockade of PGE(2) increase in injured brain tissue.

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists.

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.

Targeting the kallikrein-kinin system as a new therapeutic approach to diabetic retinopathy.

Diabetic retinopathy (DR) is a major microvascular complication of diabetes mellitus that can lead to visual impairment and blindness. There is no approved pharmacological treatment for DR; however, laser therapy, steroids and anti-VEGF agents appear to provide some benefit. Hyperglycemia, advanced glycation end products, growth factors, and elevated levels of circulating and vitreous cytokines and chemokines can all trigger an inflammatory response of the retinal vasculature. Features of DR can include diabetic macular edema, microhemorrhage, loss of capillaries, development of avascular areas and the vitreo-retinal proliferation of neovessels. The kallikrein-kinin system (KKS) has long been recognized as a key player of inflammatory processes in various organs. Intravitreally administered recombinant plasma kallikrein has been demonstrated to produce retinal vascular leakage and hemorrhage, while both kinin B1 and B2 receptor agonists have induced retinal edema. Furthermore, kallikrein inhibitors and peptide-based B1 receptor antagonists could reduce or block retinal vascular permeability in diabetic rats. In a diabetic rat model, FOV-2304 (Fovea Pharmaceuticals SA), a non-peptide selective B1 receptor antagonist, consistently blocked retinal vascular permeability, inhibited leukocyte adhesion and abolished the retinal mRNA expression of several inflammatory mediators. Although additional studies are required to investigate the role of the KKS in early capillary loss and late-stage neovascularization processes, the blockade of the KKS is a promising therapeutic strategy for DR.

Bradykinin B2 receptor antagonism with LF 18-1505T reduces brain edema and improves neurological outcome after closed head trauma in rats.

BACKGROUND: We evaluated the effect of LF 18-1505T, a novel nonpeptide bradykinin type-2 receptor antagonist, on brain edema and neurologic severity score (NSS) after closed head trauma (CHT). METHODS: There were 132 rats anesthetized and assigned for sham or CHT; infusion of saline or LF 18-1505T (0.3, 1, 3, 10, or 30 microg x kg x min); and determination of neurologic outcome (brain water content and NSS) or physiologic variables (blood pressure, glucose concentration, etc.). RESULTS: Post-CHT brain water content was less with LF 18-1505T doses of 3 and 10 microg x kg x min (80.1 +/- 3.8 through 81.6 +/- 2.6%, mean +/- SD) than in the untreated group (84.6 +/- 1.9%, p < 0.01). Post-CHT NSS improved with doses of 3, 10, and 30 microg x kg x min (median, 7; range, 0-12 through median, 10; range, 8-18) as compared with that in the untreated group (median, 17; range, 14-23; p < 0.05). LF 18-1505T with or without CHT did not significantly alter physiologic variables. CONCLUSIONS: LF 18-1505T decreased brain edema and improved neurologic status after CTH in rats without significantly altering physiologic values.

LF 16-0687 Ms, a new bradykinin B2 receptor antagonist, improves neurologic outcome but not brain tissue prostaglandin E2 release in a rat model of closed head trauma combined with ethanol intoxication.

BACKGROUND: LF 16-0687 Ms previously was reported to improve Neurological Severity Score (NSS) and decrease cerebral edema and prostaglandin E(2) (PGE(2)) release after closed head trauma (CHT) in rats. Here, we examined whether these beneficial effects of LF 16-0687 Ms are altered when CHT is accompanied by acute ethanol administration. METHODS: Six groups of rats (n = 8 per group) were examined during combination of the following experimental conditions: CHT versus sham operation, LF 16-0687 Ms 3 mg/kg subcutaneously versus saline, and ethanol 2 g/kg versus saline. RESULTS: After CHT, brain water content decreased and NSS improved with ethanol + LF 16-0687 Ms as compared with values after saline or ethanol. PGE(2) release decreased with ethanol (147 +/- 59 pg/mg tissue) but not with ethanol + LF 16-0687 Ms (286 +/- 194 pg/mg tissue). CONCLUSION: Ethanol does not affect the improvement of NSS and the decrease of cerebral edema seen with LF 16-0687 Ms after CHT, but does reverse the ability of LF 16-0687 Ms to minimize the increase of PGE(2) release. In intoxicated patients, bradykinin antagonist therapy may improve post-CHT outcome without altering PGE(2) release.