Exploring the potential for subtype-selective muscarinic agonists in glaucoma

Pilocarpine has been used to lower intraocular pressure (IOP) in glaucoma patients for more than 100 years. Since the identification of five muscarinic receptor subtypes, there has been an interest in separating the IOP-lowering effects from the ocular side effects of pupil constriction and lens accommodation. However, all these actions seem to be mediated by the M3 receptor. A novel muscarinic receptor agonist, AGN 199170, that has no activity on the M3 subtype was compared to pilocarpine in a monkey glaucoma model. This compound lowered IOP suggesting that muscarinic agonists targeted at muscarinic receptors other than the M3 subtype may be able to selectively lower IOP.     

Pathways of the early propagation of virulent and avirulent rabies strains from the eye to the brain.

Penetration of the central nervous system of the adult rat by the CVS strain of rabies virus and its two avirulent derivatives Av01 and Av02 has been studied by inoculation of the virus into the anterior chamber of the eye. The primary sites of penetration of CVS were (i) the intraocular parasympathetic oculomotor fibers, (ii) the retinopetal fibers of pretectal origin, and (iii) the intraocular fibers of the ophthalmic nerve. The mutant strains, however, lost the capacity to invade the two former groups of fibers, although their penetration into the trigeminal system was not impaired. Neither strain CVS nor the mutants infected primarily the intraocular adrenergic terminals and the optic nerve. Mutant strains, but not CVS, were able to infect the lens. These results indicate that the cholinergic receptor may not be the only receptor for rabies virus and that rabies virus is conveyed in the nervous system by retrograde axoplasmic flow. Strain CVS spread throughout the brain and propagated eventually back to the retina. The mutants penetrated the brain as well, but the infection was slow, involved different cerebral structures, and cleared up completely in 3 weeks, probably because of an efficient immune response.     

The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.     

Influence of the Hypothalamic Arcuate Nucleus on Intraocular Pressure and the Role of Opioid Peptides

Background

An opioid peptide neuron/humoral feedback regulation might be involved in changes of intraocular pressure (IOP). The aims of this study are to investigate the effects of arcuate nucleus (ARC) and opioid peptides on intraocular pressure (IOP).

Methods

Fifty-four healthy purebred New Zealand white rabbits (108eyes) were randomly divided into 4 groups, including control group, electrical stimulation group, [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) group, and [D-Pen 2, D-Pen5]- enkephalin (DPDPE) group. Bilateral IOP was measured after unilateral electrical stimulation of the ARC or unilateral microinjection into the ARC of the selective μ-opioid receptor agonist DAMGO or the selective δ opioid receptor agonist DPDPE, both alone and after pre-administration of either the non-selective opioid receptor antagonist naloxone or saline.

Results

Both electrical stimulation in ARC and micro-injection either <mu> or <delta> opioid receptor agonists, DAMGO or DPDPE, respectively, caused a significant bilateral reduction in IOP (P<0.05) which was more pronounced in the ipsilateral than in the contralateral eye. Pretreatment with naloxone prevented some, but not all IOP reductions.

Conclusion

The ARC takes part in the negative regulation of IOP, an action that may involve opioid neurons.     

Endothelin B receptors contribute to retinal ganglion cell loss in a rat model of glaucoma

Glaucoma is an optic neuropathy, commonly associated with elevated intraocular pressure (IOP) characterized by optic nerve degeneration, cupping of the optic disc, and loss of retinal ganglion cells which could lead to loss of vision. Endothelin-1 (ET-1) is a 21-amino acid vasoactive peptide that plays a key role in the pathogenesis of glaucoma; however, the receptors mediating these effects have not been defined. In the current study, endothelin B (ET(B)) receptor expression was assessed in vivo, in the Morrison's ocular hypertension model of glaucoma in rats. Elevation of IOP in Brown Norway rats produced increased expression of ET(B) receptors in the retina, mainly in retinal ganglion cells (RGCs), nerve fiber layer (NFL), and also in the inner plexiform layer (IPL) and inner nuclear layer (INL). To determine the role of ET(B) receptors in neurodegeneration, Wistar-Kyoto wild type (WT) and ET(B) receptor-deficient (KO) rats were subjected to retrograde labeling with Fluoro-Gold (FG), following which IOP was elevated in one eye while the contralateral eye served as control. IOP elevation for 4 weeks in WT rats caused an appreciable loss of RGCs, which was significantly attenuated in KO rats. In addition, degenerative changes in the optic nerve were greatly reduced in KO rats compared to those in WT rats. Taken together, elevated intraocular pressure mediated increase in ET(B) receptor expression and its activation may contribute to a decrease in RGC survival as seen in glaucoma. These findings raise the possibility of using endothelin receptor antagonists as neuroprotective agents for the treatment of glaucoma.     

A possible new syndrome with growth-hormone secreting pituitary adenoma, colonic polyposis, lipomatosis, lentigines and renal carcinoma in association with familial testicular germ cell malignancy: A case report

Introduction

Cytofluorographic and molecular techniques are effective adjuncts in diagnosing intraocular lymphoma. Primary intraocular lymphoma is an uncommon entity predominantly of B cell origin and rarely with a T cell phenotype. The aim of the present paper is to report a case of a CD8-positive, TCR-α/β-negative intraocular T cell lymphoma and review the literature.

Case presentation

T cell neoplasia was detected based on flow cytometric demonstration of an abnormal T cell population and polymerase chain reactions for immunoglobulin and T-cell receptor rearrangements demonstrating evidence of monoclonality. Flow cytometry revealed a T cell population aberrantly expressing T-cell lineage markers. This T cell population expressed CD2, bright CD3, CD8, bright CD7, CD38, CD69, and variable CD25. T-cell receptor γ gene rearrangement studies demonstrated evidence of T-cell gene rearrangement confirming that the T cells were monoclonal.

Conclusion

We herein report the rare case of a TCR α/β-negative CD8+ intraocular T-cell lymphoma suggestive of gamma/delta origin diagnosed by flow cytometry and polymerase chain reaction.     

Effects of alpha 1 and alpha 2 activation of adrenergic receptors on aqueous humor dynamics

Effects of phenylephrine (alpha 1-adrenergic agonist), prazosin (alpha 1-adrenergic antagonist), clonidine (alpha 2-adrenergic agonist), and yohimbine (alpha 2-adrenergic antagonist) on aqueous humor (AH) dynamics were studied with a cat eye model. Phenylephrine (130 microgram/ml) inhibited AH outflow (67% at 90 min. period) more than AH formation (26% at the same period) indicating the intraocular pressure (IOP) might be raised by the administration of phenylephrine. Prazosin (0.1 microgram/ml) produced effects opposite to those of phenylephrine (55% reduction of AH formation and 25% reduction of AH outflow at 3 hr. period) suggesting the alpha 1-adrenergic receptor is responsible for increases rather than decreases of IOP. Both clonidine (10 microgram/ml) and yohimbine (0.1-1.0 microgram/ml) inhibited AH formation (60% inhibition) more than AH outflow (no inhibition for clonidine and 40% inhibition for yohimbine) to lower IOP. The conventional theory of receptor antagonism does not seem to function at alpha 2-receptor sites.     

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 μM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20–30%, respectively. In the phenol animal model, 1A (100 μM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH³ and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.     

S1P₂ receptor regulation of sphingosine-1-phosphate effects on conventional outflow physiology

Elevated intraocular pressure is the main risk factor in primary open-angle glaucoma, involving an increased resistance to aqueous humor outflow in the juxtacanalicular region of the conventional outflow pathway which includes the trabecular meshwork (TM) and the inner wall of Schlemm's canal (SC). Previously, sphingosine-1-phosphate (S1P) was shown to decrease outflow facility in porcine and human eyes, thus increasing outflow resistance and intraocular pressure. Owing to S1P's known effect of increasing barrier function in endothelial cells and the robust expression of the S1P? receptor on the inner wall of SC, we hypothesized that S1P? receptor activation promotes junction formation and decreases outflow facility. The effects of subtype-specific S1P receptor compounds were tested in human and porcine whole-eye perfusions and human primary cultures of SC and TM cells to determine the receptor responsible for S1P effects on outflow resistance. The S1P?-specific agonist SEW2871 failed to both mimic S1P effects in paired human eye perfusions, as well as increase myosin light chain (MLC) phosphorylation in cell culture, a prominent outcome in S1P-treated SC and TM cells. In contrast, the S1P? antagonist JTE-013, but not the S1P? or S1P?,? antagonists, blocked the S1P-promoted increase in MLC phosphorylation. Moreover, JTE-013 prevented S1P-induced decrease in outflow facility in perfused human eyes (P < 0.05, n = 6 pairs). Similarly, porcine eyes perfused with JTE-013 + S1P did not differ from eyes with JTE-013 alone (P = 0.53, n = 3). These results demonstrate that S1P? , and not S1P? or S1P?, receptor activation increases conventional outflow resistance and is a potential target to regulate intraocular pressure.     

The P2X<Subscript>7</Subscript> receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

Retinal ganglion cells process the visual signal and transmit it along their axons in the optic nerve to the brain. Molecular, immunohistochemical, and functional analyses indicate that the majority of retinal ganglion cells express the ionotropic P2X(7) receptor. Stimulation of the receptor can lead to a rise in intracellular calcium and cell death, although death does not involve the opening of a large diameter pore. Adenosine acting at A(3) receptors can attenuate the rise in calcium and death accompanying P2X(7) receptor activation, suggesting that dephosphorylation of ATP into adenosine is neuroprotective and that the balance of extracellular purines can influence neuronal survival. Increased intraocular pressure can lead to release of excessive extracellular ATP in the retina and damage ganglion cells by acting on P2X(7) receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X(7) receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X(7) receptor makes to neurons elsewhere.     

Distinct cardioprotective effects of adenosine mediated by differential coupling of receptor subtypes to phospholipases C and D.

Adenosine released during cardiac ischemia exerts a marked protective effect in the heart that is mediated by the A(1) and A(3) subtypes of adenosine receptors. The signaling pathways activated by these adenosine receptors have now been characterized in a chick embryo ventricular myocyte culture model of cardioprotection against ischemia. Selective A(1) and A(3) receptor agonists were shown to activate phospholipases C and D, respectively, to achieve their distinct cardioprotective effects. The specificity of the A(3) receptor-phospholipase D interaction was also demonstrated in chick embryo atrial myocytes (which do not express endogenous A(3) receptors) that had been transfected with a vector encoding the human A(3) receptor. Activation of both endogenous A(1) and A(3) receptors in ventricular myocytes resulted in a protective response greater than that induced by stimulation of either receptor alone. Agonists that activate both adenosine A(1) and A(3) receptors may thus prove beneficial for the treatment of myocardial ischemia.     

Tolerance is dependent on complement C3 fragment iC3b binding to antigen-presenting cells

Systemic tolerance can be induced by the introduction of antigen into an immune-privileged site. Here we investigated the role of complement in the induction of tolerance after intraocular injection. We found that the development of antigen-specific tolerance is dependent on a complement activation product. The ligation of the complement C3 activation product iC3b to complement receptor type 3 (the iC3b receptor) on antigen-presenting cells resulted in the sequential production of transforming growth factor-beta2 and interleukin-10, which is essential for the induction of tolerance. These observations may extend to the development of both neonatal tolerance and other forms of acquired tolerance.     

Dynamic changes in reactive oxygen species and antioxidant levels in retinas in experimental glaucoma

We investigated the balance of free radicals in retinas at various time points after chronically elevating intraocular pressures in rats. The left eyes of female Wistar rats were divided into five intraocular pressure elevation (IOPE) subgroups after cauterization of three episcleral veins (1 day, 3 days, 1 week, 3 weeks, and 5 weeks) and time-related sham controls. Chemiluminescence levels were examined in isolated retinas. The nitroblue tetrazolium (NBT) incubation method was also performed to confirm the superoxide expression. The activities of antioxidant enzymes and lipid peroxides in isolated retinas were detected by spectrophotometry. In the IOPE group, the intraocular pressure after cauterization was around 22-30 mm Hg; chemiluminescence levels of isolated retinas were significantly elevated on day 3 and week 1; there were more NBT-positive retinal ganglion cells on day 1 and day 3; lipid peroxidation also increased significantly from day 1 and came back to the baseline on week 5; the activities of superoxide dismutases and catalase rose on week 1. Intraocular pressure elevation was shown to change the free radical balance. We suggest that free radicals and their oxidative stresses may play a role in the early stage of glaucoma in causing retinal ganglion cell death in our rat model.     

A novel pharmacological approach to treating cardiac ischemia. Binary conjugates of A1 and A3 adenosine receptor agonists

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A(1) or A(3) receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerted a cardioprotective effect that was significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A(1) and A(3) receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A(1) and A(3) receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A(1) or A(3) receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A(1) and A(3) receptors (K(i) values of 0.7-3.5 nm) versus A(2A) receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A(1) and A(3) receptors, or engineered atrial cells, in which either human A(3) receptors alone or both human A(1) and A(3) receptors were expressed. The binary agonist MRS 1741 coactivated A(1) and A(3) receptors simultaneously, with full cardioprotection (EC(50) approximately 0.1 nm) dependent on expression of both receptors. Thus, co-activation of both adenosine A(1) and A(3) receptors by the binary A(1)/A(3) agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.     

Synthesis of alkyne derivatives of a novel triazolopyrazine as A(2A) adenosine receptor antagonists

A novel [1,2,4]triazolo[1,5-a]pyrazine core was synthesized and coupled with terminal acetylenes. The structure-activity relationship of the alkynes from this novel template was studied for their in vitro and in vivo adenosine A(2A) receptor antagonism. Selected compounds from this series were shown to have potent in vitro and in vivo activities against adenosine A(2A) receptor. Compound 12, in particular, was found to be orally active at 3mg/kg in both a mouse catalepsy model and a 6-hydroxydopamine-lesioned rat model.     

Intraocular pressure in cats is lowered by drops of hornet venom

1. Nine cats were given an intravenous injection of the Oriental hornet (Vespa orientalis, Vespinae; Hymenoptera) venom sac extract (VSE) and seven cats had the same VSE administered as eye drops. 2. When injected intravenously, the hornet VSE decreased the intraocular pressure in both eyes sharply during the first 20 min and with a slower rate later on until the end of the 3 hr experiment. The intraocular pressure dropped to zero in some cases. 3. VSE eye drops decreased the intraocular pressure only in the treated eye, while in the second eye (left as a control) the intraocular pressure remained the same throughout the experiment. 4. The decrease in the intraocular pressure was sharp during the first 20 min and slowed down afterwards until the end of the experiment. 5. The intraocular pressure did not reduce to zero. 6. This study shows that the active components of the hornet venom which caused a decrease in the intraocular pressure can cross the cornea and exert a hypotensive effect in the eye.     

Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.     

MicroRNA Profiling in Intraocular Medulloepitheliomas

Purpose

To study the differential expression of microRNA (miRNA) profiles between intraocular medulloepithelioma (ME) and normal control tissue (CT).

Material and Methods

Total RNA was extracted from formalin fixed paraffin embedded (FFPE) intraocular ME (n=7) and from age matched ciliary body controls (n=8). The clinical history and phenotype was recorded. MiRNA profiles were determined using the Affymetrix GeneChip miRNA Arrays analyzed using expression console 1.3 software. Validation of significantly dysregulated miRNA was confimed by quantitaive real-time PCR. The web-based DNA Intelligent Analysis (DIANA)-miRPath v2.0 was used to perform enrichment analysis of differentially expressed (DE) miRNA gene targets in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway.

Results

The pathologic evaluation revealed one benign (benign non-teratoid, n=1) and six malignant tumors (malignant teratoid, n=2; malignant non-teratoid, n = 4). A total of 88 miRNAs were upregulated and 43 miRNAs were downregulated significantly (P<0.05) in the tumor specimens. Many of these significantly dysregulated miRNAs were known to play various roles in carcinogenesis and tumor behavior. RT-PCR validated three significantly upregulated miRNAs and three significantly downregulated miRNAs namely miR-217, miR-216a, miR-216b, miR-146a, miR-509-3p and miR-211. Many DE miRNAs that were significant in ME tumors showed dysregulation in retinoblastoma, glioblastoma, and precursor, normal and reactive human cartilage. Enriched pathway analysis suggested a significant association of upregulated miRNAs with 15 pathways involved in prion disease and several types of cancer. The pathways involving significantly downregulated miRNAs included the toll-like receptor (TLR) (p<4.36E-16) and Nuclear Factor kappa B (NF-κB) signaling pathways (p<9.00E-06).

Conclusions

We report significantly dysregulated miRNAs in intraocular ME tumors, which exhibited abnormal profiles in other cancers as well such as retinoblastoma and glioblastoma. Pathway analysis of all dysregulated miRNAs shared commonalities with other cancer pathways.     

Comparison of evening and morning dosing of travoprost 0.004%/timolol 0.5% fixed combination in 6 month period

An open label, multi-center, 6 months observational study of new fixed combination (travoprost 0.004%/timolol 0.5%), in order to evaluate both efficacy (intraocular pressure lowering) and tolerability (patient and investigator satisfaction) of two dosing regimens--evening (PM) and morning (AM). After screening for enrollment, to 40 patients (79 eyes with primary open angle glaucoma or ocular hypertension), new fixed combination travoprost 0.004%/timolol 0.5% was prescribed once a day in the evening (PM). Patients were enrolled according to each investigator decision on indication for travoprost 0.004%/timolol 0.5% fixed combination once a day, without washout period after previous medication. Intraocular pressure was measured at 9 AM at all time control points: at baseline, after 1 month, after 3 months and after 6 month. After 1 month, screening for nonresponders (criteria: 20% intraocular pressure lowering) and subjects with major side effects was performed. At second control visit, after 3 months PM dosing, intraocular pressure was measured and patients were instructed to continue once a day the same medication, but in the morning (AM) for consequent 3 months. After 1 month, reduction in mean intraocular pressure value was 21.66%. At the visit after 3 month, the mean intraocular pressure was 15.67 +/- 2.17 mm Hg (reduction 21.14%). 3 month after dosing regimen changed to AM (6 month after beginning of travoprost 0.004%/timolol 0.5% combination therapy), reduction in intraocular pressure value was 19.86%. The differences (mean +/- standard deviation) in intraocular pressure values after 1, 3 and 6 month were all highly statistically significant compared to baseline values. The tolerability was evaluated in five steps (Likert scale) ranging from unsatisfactory to excellent by both patient and investigator--taken at 3 and 6 month control visit. 95% of patients and 100% of investigators were satisfied with the possibility of choosing dosing regimen for travoprost 0.004%/timolol 0.5% fixed combination. Travoprost 0.004%/timolol 0.5% fixed combination proved sufficient intraocular pressure control dosed either PM or AM with no statistically significant difference between two dosing regimens. Possibility to choose between two dosing regimens gives each practitioner additional reassurance that glaucoma therapy will be individualised to needs of each patient.