Alejandro Martínez-Águila

Melatonin analogue agomelatine reduces rabbit's intraocular pressure in normotensive and hypertensive conditions.

In the search for new compounds to reduce intraocular pressure (IOP), with fewer side effects, we have found that agomelatine, a melatonin analogue, can reduce IOP being, therefore, interesting for the treatment of ocular hypertension and glaucoma. In normotensive conditions, agomelatine (10μl 100μM) reduced IOP by 20.8±1.4% (n=18) with a maximal effect 180min after the compound application and 68.8±5.7% (n=8) in a hypertensive condition. Concentration-response curve depicted a sigmoid behaviour presenting a pD2 value of 9.7±0.3 which was equivalent to an EC50 of 0.19nM. The effect of agomelatine was partially antagonized by 4PPDOT (MT2 antagonist receptor. 10μl 100μM) and prazosin (MT3 antagonist receptor. 10μl 100μM) (85.6±1.6% and 87.2±1.9%, N=18 respectively.) Agomelatine hypotensive effect in normotensive condition was comparable to latanoprost (40μl) and brimonidine (40μl) and it was no so effective as dorzolamide (40μl) or timolol (40μl). These results may suggest the use of this melatonin analogue for the treatment of those ocular conditions, which involve an abnormal raise of intraocular pressure.

Involvement of carbonic anhydrases in the ocular hypotensive effect of melatonin analogue 5-MCA-NAT

Abstract:  We have previously demonstrated that melatonin and its analogue, 5‐methoxycarbonylamino‐N‐acetyltryptamine (5‐MCA‐NAT), reduce intraocular pressure (IOP) in New Zealand rabbits. More recently, we have shown that 5‐MCA‐NAT can also regulate ciliary adrenoceptor gene expression. Like adrenoceptors, carbonic anhydrase (CA) enzymes are involved in aqueous humour secretion by the ocular ciliary epithelium. Moreover, CA enzymes have been reported to be regulated by melatonin. Hence, the aim of this study was to investigate whether the hypotensive effect of 5‐MCA‐NAT is also because of a regulation of CA genes and enzymes. Time course of 5‐MCA‐NAT effect on rabbit IOP was followed for 7 hr every day for up to 144 hr (6 days). 5‐MCA‐NAT reduced IOP, maximally by 51.30 ± 2.41% (at 3 hr), and the hypotensive effect was maintained for up to 96 hr with a single application. IOP studies with 5‐MCA‐NAT plus Trusopt® and immunohistochemical analysis confirmed that CA are molecular targets of 5‐MCA‐NAT. In addition, real‐time quantitative PCR (qPCR) and immunocytochemical assays were performed to determine changes in CA2 (CAII) and CA12 (CAXII) expression in cultured rabbit nonpigmented ciliary epithelial cells (NPE) treated with 5‐MCA‐NAT. NPE cells showed a prominent decrease in both CA, at the mRNA and protein levels. These data confirm that the long‐term hypotensive effect of 5‐MCA‐NAT is also due, to a down‐regulation of CA2 (CAII) and CA12 (CAXII) expression.

Melatonin Receptors Trigger cAMP Production and Inhibit Chloride Movements in Nonpigmented Ciliary Epithelial Cells

Melatonin and its analog 5-MCA-NAT (5-methylcarboxyamino-N-acetyl tryptamine) are active compounds reducing intraocular pressure (IOP). This action is mediated through MT2 and the putative MT3 melatonin receptor, producing a transient reduction of IOP that lasts for a few hours and has not yet been characterized. The use of melatonin and its analog are causing a decrease in chloride efflux from rabbit nonpigmented epithelial cells (NPE), possibly explaining the decrease in IOP. Melatonin and 5-MCA-NAT inhibited rabbit NPE chloride release in a concentration-dependent manner, whereas the pD2 values were between 4.5 ± 1.2 and 4.4 ± 1.0, respectively. Melatonin hypotensive action was enhanced by the presence of MT2 antagonists, such as DH97 (N-pentanoyl-2-benzyltryptamine) and 4-P-P-DOT (4-phenyl-2-propionamidotetralin) and by the nonselective melatonin receptor antagonist luzindole. Prazosin (1.5 µM) partially reverses the melatonin action by acting as a selective MT3 antagonist. However, at 15 nM it acts as an α-adrenergic receptor antagonist, enhancing the melatonin effect. Regarding the intracellular pathways triggered by melatonin receptors, neither phospholipase C/protein kinase C pathway nor the canonical reduction of intracellular cAMP was responsible for melatonin or 5-MCA-NAT actions. On the contrary, the application of these substances produced a concentration-dependent increase of cAMP, with pD2 values of 4.6 ± 0.2 and 4.9 ± 0.7 for melatonin and 5-MCA-NAT, respectively. In summary, melatonin reduces the release of chloride concomitantly to cAMP generation. The reduction of Cl− secretion accounts for a decrease in the water outflow and therefore a decrease in aqueous humor production. This could be one of the main mechanisms responsible for the reduction of IOP after application of melatonin and 5-MCA-NAT.

Melatonin and Its Analog 5-Methoxycarbonylamino-N-Acetyltryptamine Potentiate Adrenergic Receptor-Mediated Ocular Hypotensive Effects in Rabbits: Significance for Combination Therapy in Glaucoma

Melatonin is currently considered a promising drug for glaucoma treatment because of its ocular hypotensive and neuroprotective effects. We have investigated the effect of melatonin and its analog 5-methoxycarbonylamino-N-acetyltryptamine, 5-MCA-NAT, on β2/α2A-adrenergic receptor mRNA as well as protein expression in cultured rabbit nonpigmented ciliary epithelial cells. Quantitative polymerase chain reaction and immunocytochemical assays revealed a significant β2-adrenergic receptor downregulation as well as α2A-adrenergic receptor up-regulation of treated cells (P < 0.001, maximal significant effect). In addition, we have studied the effect of these drugs upon the ocular hypotensive action of a nonselective β-adrenergic receptor (timolol) and a selective α2-adrenergic receptor agonist (brimonidine) in normotensive rabbits. Intraocular pressure (IOP) experiments showed that the administration of timolol in rabbits pretreated with melatonin or 5-MCA-NAT evoked an additional IOP reduction of 14.02% ± 5.8% or 16.75% ± 5.48% (P < 0.01) in comparison with rabbits treated with timolol alone for 24 hours. Concerning brimonidine hypotensive action, an additional IOP reduction of 29.26% ± 5.21% or 39.07% ± 5.81% (P < 0.001) was observed in rabbits pretreated with melatonin or 5-MCA-NAT when compared with animals treated with brimonidine alone for 24 hours. Additionally, a sustained potentiating effect of a single dose of 5-MCA-NAT was seen in rabbits treated with brimonidine once daily for up 4 days (extra IOP decrease of 15.57% ± 5.15%, P < 0.05, compared with brimonidine alone). These data confirm the indirect action of melatoninergic compounds on adrenergic receptors and their remarkable effect upon the ocular hypotensive action mainly of α2-adrenergic receptor agonists but also of β-adrenergic antagonists.

Effect of Melatonin and 5-Methoxycarbonylamino-N-Acetyltryptamine on the Intraocular Pressure of Normal and Glaucomatous Mice

Melatonin is a neurohormone that is produced not only by the pineal gland but also by several ocular structures. One of the main physiologic roles of melatonin is the reduction of intraocular pressure (IOP). Using both control C57BL/6J and glaucomatous DBA/2J mice as well as TonoLab tonometry, this study evaluated the effect of melatonin and 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) when glaucomatous pathology was fully established and compared pharmacological behavior in treated mice versus control mice. In addition, 5-MCA-NAT was tested to determine its effects on ameliorating increased IOP in a glaucoma model. The results demonstrate that melatonin and 5-MCA-NAT can reduce IOP in a concentration-dependent manner. The EC50 values for melatonin in control and glaucomatous animals were 34 µM and 50 µM, respectively. Interestingly, melatonin decreased IOP in 19.4% ± 3.7% and 32.6% ± 6.0% of control and glaucomatous mice, respectively, when the animals were studied at age 12 months. 5-MCA-NAT reduced IOP in the same manner and was able to stop IOP progression in glaucomatous mice. Use of melatonin receptor antagonists showed that hypotensive effects were blocked by the MT2 receptor antagonists luzindole and 4-phenyl-2-propionamidotetralin in the case of melatonin and by only 4-phenyl-2-propionamidotetralin in the case of 5-MCA-NAT. In conclusion, melatonin and 5-MCA-NAT can effectively reduce IOP in a glaucoma model, and their hypotensive effects are more profound in the glaucoma model than in control animals.

Ocular disorders and the utility of animal models in the discovery of melatoninergic drugs with therapeutic potential

Introduction:   The pineal indole-derived hormone melatonin is a modulator of circadian and seasonal rhythms with an important role in ocular health and disease. This could be due to specific melatonin receptors that have been identified in structures such as cornea, lens, ciliary body, retina, choroid and sclera. In addition, a local synthesis of melatonin occurs in several of these ocular tissues. Areas covered: The authors review existing literature on the most common animal models where ocular melatonin actions have been tested. The therapeutic potential of melatonin in diabetic keratopathy and retinopathy, keratitis, cataracts, glaucoma, uveitis, age-related macular degeneration and retinitis pigmentosa is discussed. Furthermore, the authors comment on the usefulness of different animal models for the development of melatoninergic drugs with therapeutic potential. Expert opinion: The use of animals for the study of ocular diseases and the potentiality of melatonin and its analogs, as future therapeutic drugs, should be performed on the basis of a rationale study. It is important to note that melatonin receptors seem to be widespread all over the eye. This strongly suggests that, in order to modify the physiology and biochemistry of malfunctioning ocular tissue, the melatonin receptors which are present in that tissue must be first identified. Second there is the need to confirm that those receptors targeted perform the desirable responses, and as a third measure, to use selective agonists (or antagonists) instead of melatonin. However, although some animals mimic ocular pathologies relatively well, and these can be used in melatonin studies, there is still a long way to go till some of the results obtained in animal models could be used for human therapy.

Regulation of ocular adrenoceptor genes expression by 5-MCA-NAT: implications for glaucoma treatment.

We have demonstrated that 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT), reduces intraocular pressure (IOP) in rabbits. In addition, we have reported a link between hypotensive effect of 5-MCA-NAT and sympathetic nervous system. Moreover, it is known that aqueous humour production is controlled by the activation of adrenoceptors (ADRs) present in the ocular ciliary epithelium. Thus, the aim of this study is to investigate if the hypotensive effect of 5-MCA-NAT is due to a regulation of ciliary ADR genes expression. To confirm this we followed the effect of 5-MCA-NAT on rabbit IOP for 144 consecutive hours. A sustained IOP reduction for up to 72 h (P<0.01) was seen. In addition, changes in ADRB2 and ADRA2A mRNA were measured in cultured rabbit nonpigmented ciliary epithelial cells. After 5-MCA-NAT treatment, a significant downregulation of ADRB2 and upregulation of ADRA2A was observed. These results provide the regulation of ADRs mRNA by 5-MCA-NAT.

Elevated intraocular pressure increases melatonin levels in the aqueous humour

To study the levels of melatonin in the aqueous humour of normotensive and hypertensive intraocular pressure (IOP) patients and to compare them to an animal model of glaucoma.

Dry Eye Treatment Based on Contact Lens Drug Delivery: A Review.

Abstract: Dry eye disease affects a substantial segment of the word population with increasing frequency. It is a multifactorial disease of the ocular surface and tear film, which causes ocular discomfort, visual disturbances, and tear instability with potential damage to the cornea and conjunctiva. Because of its multifactorial etiology, the use of different pharmacological treatment for dry eye treatment has been proposed, which include anti-inflammatory molecules, lubricants or comfort agents, and secretagogues. However, in some cases these pharmacological approaches only relieve symptoms temporarily, and consequently, eye care professionals continue to have difficulties managing dry eye. To improve pharmacological therapy that allows a more efficient and long-term action, effective ocular drug delivery of the currently available drugs for dry eye treatment is required. Contact lenses are emerging as alternative ophthalmic drugs delivery systems that provide an increased residence time of the drug at the eye, thus leading to enhanced bioavailability and more convenient and efficacious therapy. In this article, we reviewed the different techniques used to prepare contact lens-based drug delivery systems and focused on articles that describe the delivery of compounds for dry eye treatment through contact lenses.

Changes in P2Y Purinergic Receptor Expression in the Ciliary Body in a Murine Model of Glaucoma

Glaucoma is a neuropathology, often accompanied by an elevated intraocular pressure (IOP), which can lead to blindness. Since DBA/2J mice develop glaucoma, several studies of the physiopathology of glaucoma have been reported in this animal model. It is also known that purinergic receptors are involved in the pathology of glaucoma by controlling aqueous humor production and drainage and therefore controlling IOP. There are no studies on purinergic receptors in the DBA/2J model of glaucoma and their relation to the development of the pathology, so the aim of this study was to make an approach to the purinergic mechanisms involved in glaucoma. All the experiments were performed using DBA/2J and C57BL/6J mice and investigating P2Y1, P2Y2, and P2Y6 receptors. IOP measurements were made with a non-invasive rebound tonometer, and animals were instilled with diadenosine tetraphosphate (Ap4A) and the corresponding purinergic antagonists in order to see their effects on IOP. The expression of mRNA for P2Y1, P2Y2, and P2Y6 purinergic receptors was carried out by quantitative real-time PCR. Additionally, P2Y-receptor expression was performed by immunohistochemical techniques carried out on the ciliary processes. The results showed that IOP decreases when Ap4A was instilled and that the expressions of the analyzed purinergic receptors were stable throughout all the ages under study in the C57BL/6J mice (control mice). On the other hand, there were significant changes in the purinergic receptor expression in DBA/2J suggesting that elevated IOP in these animals could be related to an increase of P2Y2 expression and a decrease in P2Y1 receptors.