In vitro and in vivo efficacy of SYL040012, a novel siRNA compound for treatment of glaucoma.

Glaucoma is a progressive ocular syndrome characterized by degeneration of the optic nerve and irreversible visual field loss. Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Increased IOP is the result of an imbalance between synthesis and outflow of aqueous humor (AH). Blocking ß2 adrenergic receptor (ADRB2) has shown to reduce IOP by decreasing production of AH at the ciliary body (CB). SYL040012 is a siRNA designed to specifically silence ADRB2 currently under development for glaucoma treatment. Here, we show that SYL040012 specifically reduces ADRB2 expression in cell cultures and eye tissues. The compound enters the eye shortly after administration in eye drops and is rapidly distributed among structures of the anterior segment of the eye. In addition, SYL040012 is actively taken up by cells of the CB but not by cells of systemic organs such as the lungs, where inhibition of ADRB2 could cause undesirable side effects. Moreover, SYL040012 reduces IOP in normotensive and hypertensive animal models and the effect appears to be long lasting and extremely well tolerated both locally and systemically.

Phase I clinical trial of SYL040012, a small interfering RNA targeting ß-adrenergic receptor 2, for lowering intraocular pressure.

The objective of this study was to evaluate ocular tolerance, safety, and effect on intraocular pressure (IOP) of a topically administered small interfering RNA; SYL040012, on healthy volunteers. The study was an open-label, controlled, single-center study comprised of two intervals that enrolled 30 healthy subjects having IOP below 21 mmHg. SYL040012 was administered to one eye as a single dose to six subjects during interval 1. During interval 2 two different doses of SYL040012 were administered to one eye on a daily basis to two separate groups of 12 subjects each, over a period of 7 days. The contralateral eye was evaluated but not administered and served as control for the tolerance study. SYL040012 was well tolerated locally. No local or systemic adverse events related to the product developed in response to any of the doses studied. SYL040012 was not detected in plasma at any time point. Administration of SYL040012 over a period of 7 days reduced IOP values in 15 out of 24 healthy subjects regardless of the dose used. IOP decrease was statistically significant in response to one of the doses tested and responsiveness to SYL040012 seemed to be greater in individuals with higher baseline IOP.

Silencing human genetic diseases with oligonucleotide-based therapies.

RNA interference is an endogenous mechanism present in most eukaryotic cells that enables degradation of specific mRNAs. Pharmacological exploitation of this mechanism for therapeutic purposes attracted a whole amount of attention in its initial years, but was later hampered due to difficulties in delivery of the pharmacological agents to the appropriate organ or tissue. Advances in recent years have to a certain level started to address this specific issue. Genetic diseases are caused by aberrations in gene sequences or structure; these particular abnormalities are in theory easily addressable by RNAi therapeutics. Sequencing of the human genome has largely contributed to the identification of alterations responsible for genetic conditions, thus facilitating the design of compounds that can address these diseases. This review addresses the currently on-going programs with the aim of developing RNAi and other antisense compounds for the treatment of genetic conditions and the pros and cons that these products may encounter along the way. The authors have focused on those programs that have reached clinical trials or are very close to do so.

Neuropeptide S reinstates cocaine-seeking behavior and increases locomotor activity through corticotropin-releasing factor receptor 1 in mice.

Neuropeptide S (NPS) is a recently discovered neuropeptide that increases arousal and wakefulness while decreasing anxiety-like behavior. Here, we used a self-administration paradigm to demonstrate that intracerebroventricular infusion of NPS reinstates extinguished cocaine-seeking behavior in a dose-dependent manner in mice. The highest dose of NPS (0.45 nM) increased active lever pressing in the absence of cocaine to levels that were equivalent to those observed during self-administration. In addition, we examined the role of the corticotropin-releasing factor receptor 1 (CRF(1)) in this behavior as well as locomotor stimulation and anxiolysis. CRF(1) knock-out mice did not respond to either the locomotor stimulant or cocaine reinstatement effects of NPS, but still responded to its anxiolytic effect. The CRF(1) antagonist antalarmin also blocked the increase in active lever responding in the reinstatement model and the locomotor activating properties of NPS without affecting its anxiolytic actions. Our results suggest that NPS receptors may be an important target for drug abuse research and treatment and that CRF(1) mediates the cocaine-seeking and locomotor stimulant effects of NPS, but not its effects on anxiety-like behavior.

Acidic sphingomyelinase downregulates the liver-specific methionine adenosyltransferase 1A, contributing to tumor necrosis factor-induced lethal hepatitis.

S-adenosyl-L-methionine (SAM) is synthesized by methionine adenosyltransferases (MATs). Ablation of the liver-specific MAT1A gene results in liver neoplasia and sensitivity to oxidant injury. Here we show that acidic sphingomyelinase (ASMase) mediates the downregulation of MAT1A by TNF-alpha. The levels of MAT1A mRNA as well as MAT I/III protein decreased in cultured rat hepatocytes by in situ generation of ceramide from exogenous human placenta ASMase. Hepatocytes lacking the ASMase gene (ASMase-/-) were insensitive to TNF-alpha but were responsive to exogenous ASMase-induced downregulation of MAT1A. In an in vivo model of lethal hepatitis by TNF-alpha, depletion of SAM preceded activation of caspases 8 and 3, massive liver damage, and death of the mice. In contrast, minimal hepatic SAM depletion, caspase activation, and liver damage were seen in ASMase-/- mice. Moreover, therapeutic treatment with SAM abrogated caspase activation and liver injury, thus rescuing ASMase+/+ mice from TNF-alpha-induced lethality. Thus, we have demonstrated a new role for ASMase in TNF-alpha-induced liver failure through downregulation of MAT1A, and maintenance of SAM may be useful in the treatment of acute and chronic liver diseases.

Synthetic nanocarriers for the delivery of polynucleotides to the eye.

This review is a comprehensive analysis of the progress made so far on the delivery of polynucleotide-based therapeutics to the eye, using synthetic nanocarriers. Attention has been addressed to the capacity of different nanocarriers for the specific delivery of polynucleotides to both, the anterior and posterior segments of the eye, with emphasis on their ability to (i) improve the transport of polynucleotides across the different eye barriers; (ii) promote their intracellular penetration into the target cells; (iii) protect them against degradation and, (iv) deliver them in a long-term fashion way. Overall, the conclusion is that despite the advantages that nanotechnology may offer to the area of ocular polynucleotide-based therapies (especially AS-ODN and siRNA delivery), the knowledge disclosed so far is still limited. This fact underlines the necessity of more fundamental and product-oriented research for making the way of the said nanotherapies towards clinical translation.

Safety and Efficacy Clinical Trials for SYL1001, a Novel Short Interfering RNA for the Treatment of Dry Eye Disease.

Purpose: To evaluate the efficacy and safety of SYL1001, a short interfering (si) RNA targeting the transient receptor potential cation channel subfamily V member 1 (TRPV1), for the treatment of dry eye disease (DED). Methods: This study combines a phase I and two phase II clinical trials to test different doses of SYL1001 in a total of 156 healthy subjects and patients with DED. After 10 days of treatment, the primary efficacy endpoints were the effect on (1) the scoring in the Visual Analogue Scale (VAS) and Ocular Surface Disease Index (OSDI) questionnaires, and (2) ocular tolerance evaluated by corneal fluorescein staining and conjunctival hyperemia. Secondary endpoints included the assessment of systemic and local tolerance. Results: Topical administration of SYL1001 1.125% once daily produced a significant decrease in VAS scores compared with placebo from day 4 until the end of treatment (change from baseline at day 10: -1.73 ± 0.32 vs. -0.91 ± 0.34; P = 0.013). For all treatments, OSDI scores were significantly reduced compared to their respective baseline values (P < 0.01), although no significant changes were detected between groups. Conjunctival hyperemia (quantified as normal or abnormal) significantly improved after instillation of SYL1001 1.125% compared with placebo (50% vs. 20%; P < 0.05). Excellent tolerability was reported, with no differences in the rates of occurrence of adverse events between groups. Conclusion: These trials achieved their primary endpoints of identifying the most effective dose of SYL1001 (1.125%). SYL1001 showed a large safety margin and may provide novel therapeutic opportunity for the relief of dry eye. (ClinicalTrials.gov numbers, NCT01438281, NCT01776658, and NCT02455999.).

Phosphorylation of glycosyl-phosphatidylinositol by phosphatidylinositol 3-kinase changes its properties as a substrate for phospholipases.

Phosphatidylinositol 3-kinases (PI3K) phosphorylate the 3-position of the inositol ring of phosphatidylinositol-4,5-bisphosphate to produce phosphatidylinositol-3,4,5-trisphosphate. It is not clear whether PI3K can phosphorylate the inositol group in other biomolecules. We sought to determine whether PI3K was able to use glycosyl-phosphatidylinositol (GPI) as a substrate. This phospholipid may exist either in free form (GPIfree) or forming a lipid anchor (GPIanchor) for the attachment of extracellular proteins to the plasma membrane. We demonstrate the specific PI3K-mediated phosphorylation of the inositol 3-hydroxyl group within both types of GPI by incubating this phospholipid with immunoprecipitated PI3K. The phosphorylated product behaves in HPLC as a derivative of a PI3K lipid product. To our knowledge, this is the first demonstration that PI3K uses lipid substrates other than phosphoinositides. Further, we show that this has potential functional consequences. When GPIfree is phosphorylated, it becomes a poorer substrate for GPI-specific phospholipase D, but a better substrate for phosphatidylinositol-specific phospholipase C. These phosphorylation events may constitute the basis of a previously undescribed signal transduction mechanism.

Short-interference RNAs: becoming medicines.

RNA interference is a cellular mechanism by which small molecules of double stranded RNA modulate gene expression acting on the concentration and/or availability of a given messenger RNA. Almost 10 years after Fire and Mello received the Nobel Prize for the discovery of this mechanism in flat worms, RNA interference is on the edge of becoming a new class of therapeutics. With various phase III studies underway, the following years will determine whether RNAi-therapeutics can rise up to the challenge and become mainstream medicines. The present review gives a thorough overview of the current status of this technology focusing on the path to the clinic of this new class of compounds.

Growth hormone (GH) and GH-releasing peptide-6 increase brain insulin-like growth factor-I expression and activate intracellular signaling pathways involved in neuroprotection.

Beneficial effects of GH on memory, mental alertness, and motivation have been documented. Many actions of GH are mediated through IGF-I; hence, we investigated whether systemic administration of GH or GH-releasing peptide (GHRP)-6 modulates the brain IGF system. Treatment of adult male rats with GHRP-6 or GH for 1 wk significantly increased IGF-I mRNA levels in the hypothalamus, cerebellum, and hippocampus, with no effect in cerebral cortex. Expression of the IGF receptor and IGF-binding protein (IGFBP)-2 were not affected. Phosphorylation of Akt and Bad was stimulated in areas where IGF-I was increased, with no change in MAPK or glycogen synthase kinase-3beta. This suggests that GH and GHRP-6 activate phosphatidylinositol kinase intracellular pathways involved in cell survival in response to growth factors. Indeed, the antiapoptotic protein Bcl-2 was augmented in these same areas, with no change in the proapoptotic protein Bax. IGFBP-5, also reported to be involved in neuron survival processes, was increased mainly in the hypothalamus, suggesting a possible neuroendocrine role. In conclusion, GH and GHRP-6 modulate IGF-I expression in the central nervous system in an anatomically specific manner. This is coincident with activation of intracellular signaling pathways used by IGF-I and increased expression of proteins involved in cell survival or neuroprotection.

Growth hormone-releasing peptide-6 inhibits cerebellar cell death in aged rats.

Insulin-like growth factor (IGF)-I is essential for cerebellar granule neuron survival and a decline in IGF-I is implicated in various age-dependent processes. Here we show that IGF-I mRNA levels are decreased in the cerebellum of old rats compared with young rats and this was associated with increased cell death and activation of caspases 3 and 9. Growth hormone-releasing peptide (GHRP)-6, a synthetic ligand for the ghrelin receptor, increased IGF-I mRNA levels, decreased cell death and inhibited caspase 3 and 9 activation in the cerebellum of aged rats. These results suggest that increasing IGF-I expression in the cerebellum can decrease cell death in aged rats via inhibition of caspase 3 and 9 activation.

Differential expression and localization of transient receptor potential vanilloid 1 in rabbit and human eyes.

INTRODUCTION: The superfamily of transient receptor potential (TRP) cation channels is involved in nociception. Members of this family, such as the vanilloid receptor type 1 (TRPV1) channel, are activated by a wide range of stimuli including heat (⟩43°C), low pH (⟨6.5), hypoxia, and hypertonicity. Here we report TRPV1 expression in rabbit and human eyes. MATERIAL AND METHODS: We analyzed the expression of TRPV1 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and protein by immunohistochemistry in eyes of New Zealand White rabbits and humans. RESULTS: In rabbit and human eyes, TRPV1 protein was present in all layers of the corneal epithelium, but only in the basal layer of the conjunctiva. It was also in the ciliary and lens epithelia of both species as well as in the secretory cells of the rabbit lacrimal gland. The retinal pigment epithelium was positive for this protein in both species. TRPV1 was also present in rabbit Müller cells, where it had a similar pattern of expression to vimentin intermediate filaments. Analysis by qRT-PCR showed that TRPV1 mRNA was found in all of the structures where the protein was present. The highest level was in the lens and the lowest in the retina. CONCLUSION: TRPV1 is expressed in cells that are particularly active in Ca²âº exchange as well as in cells with significant water transport activity. Because TRPV1 is a Ca²âº channel, it probably functions in the regulation of both water and Ca²âº movements in ocular tissues.

Activation of the intrinsic cell death pathway, increased apoptosis and modulation of astrocytes in the cerebellum of diabetic rats.

Poorly controlled diabetes mellitus results in structural and functional changes in many brain regions. We demonstrate that in streptozotocin-induced diabetic rats cell death is increased and proliferation decreased in the cerebellum, indicating overall cell loss. Levels of both the proform and cleaved forms of caspases 3, 6 and 9 are increased, with no change in caspases 7, 8 or 12. Colocalization of glial fibrillary acidic protein (GFAP) and cleaved caspase 3 and GFAP in TUNEL-positive cells increased in diabetic rats. Changes in GFAP levels paralleled modifications in proliferating cell nuclear antigen (PCNA), increasing at 1 week of diabetes and decreasing thereafter, and proliferating GFAP-positive cells were decreased in the cerebellum of diabetic rats. These results suggest that astrocytes are dramatically affected in the cerebellum, including an increase in cell death and a decrease in proliferation, and this could play a role in the structural and functional changes in this brain area in diabetes.

Uncoupling protein 2 protects dopaminergic neurons from acute 1,2,3,6-methyl-phenyl-tetrahydropyridine toxicity.

Oxidative stress is implicated in the death of dopaminergic neurons in sporadic forms of Parkinson's disease. Because oxidative stress can be modulated endogenously by uncoupling proteins (UCPs), we hypothesized that specific neuronal expression of UCP2, one member of the UCP family that is rapidly induced in the CNS following insults, could confer neuroprotection in a mouse model of Parkinson's disease. We generated transgenic mice overexpressing UCP2 in catecholaminergic neurons under the control of the tyrosine hydroxylase promoter (TH-UCP2). In these mice, dopaminergic neurons of the substantia nigra showed a twofold elevation in UCP2 expression, elevated uncoupling of their mitochondria, and a marked reduction in indicators of oxidative stress, an effect also observed in the striatum. Upon acute exposure to 1,2,3,6-methyl-phenyl-tetrahydropyridine, TH-UCP2 mice showed neuroprotection and retention of locomotor functions. Our data suggest that UCP2 may represent a drug target for slowing the progression of Parkinson's disease.

Liver cell proliferation requires methionine adenosyltransferase 2A mRNA up-regulation.

Regulation of liver cell proliferation is a key event to control organ size during development and liver regeneration. Methionine adenosyltransferase (MAT) 2A is expressed in proliferating liver, whereas MAT1A is the form expressed in adult quiescent hepatocytes. Here we show that, in H35 hepatoma cells, growth factors such as hepatocyte growth factor (HGF) and insulin up-regulated MAT2A expression. HGF actions were time- and dose-response dependent and required transcriptional activity. Mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-phosphate kinase (PI 3-K) pathways were required for both HGF-induced cell proliferation and MAT2A up-regulation. Furthermore, in H35 cells treated with HGF, the inhibition of these pathways was associated with the switch from the expression of fetal liver MAT2A to the adult liver MAT1A isoform. Fetal liver hepatocytes exhibited an identical response pattern. Treatment of H35 hepatoma cells with MAT2A antisense oligonucleotides decreased cell proliferation induced by HGF; this decrease correlated with the decay in MAT2A messenger RNA (mRNA) levels. Finally, growth inhibitors such as transforming growth factor (TGF) beta blocked HGF-induced MAT2A up-regulation while increasing MAT1A mRNA levels in H35 cells. In conclusion, our results show that MAT2A expression not only correlates with liver cell proliferation but is required for this process.