This activity is carried out in the Foundation’s laboratories, using pharmacological screening techniques in cell line cultures, based on a chemical library of medicines in clinical use, as shown in the following article::

Torres-Rico M, García-Calvo V, Gironda-Martínez A, Pascual-Guerra J, García AG, Maneu V. Targeting calciumopathy for neuroprotection: focus on calcium channels Cav1, Orai1 and P2X7. Cell Calcium. 2024;102928.

This activity is carried out in the Foundation’s laboratories, using pharmacological screening techniques in cell line cultures, based on a chemical library of medicines in clinical use, as shown in the following article:

Figure: Graphic representation of the medicine triad hypothesis to mitigate calcium influx via Cav1, Orai1 and P2X7. This strategy seeks to combat neuroinflammation, thus promoting neuroprotection and delaying progression of neurodegenerative diseases to more advanced stages.

The combination of isotopic techniques with other neurochemical monitoring techniques for neurotransmitter release led to the first demonstration that a high-threshold calcium channel, called L (Cav1), tightly regulated catecholamine release induced by depolarizing stimuli, as shown in the following article: 

García AG, Sala F, Reig JA, Viniegra S, Frias J, Fonteriz R, Gandía L. Dihydropyridine Bay-K-8644 activates chromaffin cell calcium channels. Nature. 1984;308:69-71.

Later, patch-clamp electrophysiological techniques were introduced, leading to the identification and characterization of several calcium channel subtypes, and their regulatory role in the neurosecretory process, as shown in the following article::

García, A. G., García-De-Diego, A. M., Gandía, L., Borges, R., & García-Sancho, J. (2006). Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells. Physiological Reviews, 86(4), 1093-1131.

With the collaboration of Alter Laboratories and the Autonomous University of Madrid, a series of dihydropyridine derivatives with cardiovascular activity were designed, synthesized, and pharmacologically characterized in various isolated organ preparations (mainly arteries and heart). After the intellectual property protection of the findings, several articles were published, the most notable of which is the following:

Sunkel CE, Fau de Casa-Juana M, Santos L, García AG, Artalejo CR, Villarroya M, González-Morales MA, López MG, Cillero J, Alonso S, et al. Synthesis of 3-[(2,3-dihydro-1,1,3-trioxo-1,2-benzisothiazol-2-yl) alkyl] 1,4-dihydropyridine-3,5-dicarboxylate derivatives as calcium channel modulators. J Med Chem. 1992 Jun 26;35(13):2407-14. doi: 10.1021/jm00091a008. PMID: 1377748.

Figure: Compound 22 exhibited a pronounced activating effect on the L-type calcium channels (Cav1). See its chemical structure in comparison with other L-channel agonists.

At the THF, we have frequently applied radioligand binding techniques to membranes and cells. The objective was the characterization of a specific receptor for a neurotransmitter. To do this, we used agonists and antagonists specific to that receptor, radiolabelled with tritium or carbon-14.

In the following article, the expression of D2 dopaminergic receptors (but not D1) in bovine chromaffin cells was characterized.

Maroto R, Lopez MG, Del Valle M, Naranjo JR, Mellstrom B, García AG. Expression of the bovine striatal D2 receptor but not of the D1 receptor in bovine adrenal medulla. Mol Pharmacol. 1995;47:40-50 | DOI: 10.1124/mol.47.1.40

Figure: This figure shows a graph from the article in question, showing the saturation, with increasing concentrations, of a radioactive ligand for dopaminergic receptors in the striatal membranes of a rat.

The Ferrer International Laboratory was developing a new anti-migraine medicine and wanted to understand its mechanism of action, which was elucidated by the THF. The medicine showed multi-calcium channel- blocking properties, of the flunarizine type.

The following article describes the pharmacological profile of this medicine, dotarizine, compared with the reference medicine, flunarizine. 

Villarroya M, Gandia L, Lara B, Albillos A, Lopez MG, García AG. Dotarizine versus flunarizine as calcium antagonists in chromaffin cells. Br J Pharmacol. 1995;144:369-376 | DOI: 10.1111/j.1476-5381.1995.tb13236.x

Over the past two decades, we have extensively studied the mechanisms involved in apoptotic cell death, as well as the cytoprotection induced by medicines which act on the signalling pathways that lead to the said cell death. 

An example of cell death induced by calcium overload entering the cell via L-type calcium channels (Cav1), and of the cytoprotection induced by Cav1 channel blockers, is documented in the following article:

Cano-Abad MF, Villarroya M, García AG, Gabilan NH, Lopez MG. Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death. J Biol Chem. 2001;276:39695-39704 | DOI: 10.1074/jbc.M104234200

At the THH, we have been accumulating experience studying various animal models of diseases, at both central and peripherical levels.  

Here is a study of cerebral infarction caused by focal cerebral ischemia, and of reduction of infarction by combining citicoline and nimodipine:

Sobrado M, Lopez MG, Carceller F, García AG, Roda JM. Combined nimodipine and citicoline reduced infarct size, attenuate apoptosis and increase bel-2 expression after focal cerebral ischemia. Neuroscience. 2003;118:107-113 | DOI: 10.1016/S0306-4522(02)00842-0

At the THF, the conjecture that medicine combinations show greater efficacy (additivity and synergy) in the rescuing of vulnerable neurons from death in stroke, nervous system trauma, and the various neurodegenerative diseases, has been extensively researched.

The following article experimentally demonstrates this challenging hypothesis:

Romero A, Egea J, García AG, Lopez MG. Synergistic neuroprotective effect of combined concentrations of galantamine and melatonin against oxidative stress in SH-SYSY neuroblastoma cells. J Pineal Res. 2010;49:141-148 | DOI: 10.1111/j.1600-079X.2010.00781.x

Figure: Administered separately, galantamine (G) and melatonin (M), at low concentrations, do not rescue vulnerable neurons subjected to an environment of oxidative stress. However, the combination of these two medicines at low doses did have a significant neuroprotective effect. The highest concentrations of the medicines administered individually clearly had a neuroprotective effect. The experiment demonstrated that the combination of threshold doses of medicines exerts synergistic neuroprotection, with a lower potential for adverse effects.

In the area of pharmacological neuroprotection — one of the most intensely explored at the THF — of great importance has been the search for new targets and for medicines targeting them; both in the repositioning programme and in programmes for the design and synthesis of new chemical entities.

The following article is paradigmatic of this strategy:

Nicolau SM, Egea J, Lopez MG, García AG. Mitochondrial Na*/Ca exchanger, a new target for neuroprotection in rat hippocampal slices. Biochem Biophys Res Commun. 2010;400:140-144 | DOI: 10.1016/j.bbrc.2010.08.028

In collaboration with medical chemists from several universities and the Higher Council for Scientific Research (CSIC: Consejo Superior de Investigacones Científicas), we are developing the area for the computational design and synthesis of new chemical entities with neuroprotective potential. This strategy complements the THF’s programme about the repositioning of medicines with neuroprotective potential.  

In recent decades, numerous joint patents have been drafted and registered, and several articles have been published about this topic. The following is an example of this line of research:

Calzaferri F, Narros-Fernández P, de Pascual R, de Diego AMG, Nicke A, Egea J, García AG, de Los Ríos C. Synthesis and Pharmacological Evaluation of Novel Non-nucleotide Purine Derivatives as P2X7 Antagonists for the Treatment of Neuroinflammation. J Med Chem. 202, Feb 25;64(4):2272-2290

This hybrid laboratory-clinical study is a curious example of ALS and its possible mechanism of propagation. It was conducted in collaboration with the Hospital Clínico San Carlos in Madrid. One of the published articles is the following:

Yañez M, Galán L, Matias-Guiu J, Vela A, Guerrero A, García AG. CSF from amyotrophic lateral sclerosis patients produces glutamate independent death of rat motor brain cortical neurons: Protection by resveratrol but not riluzole. Brain Res. 2011;1423:77-86 | DOI: 10.1016/j.brainres.2011.09.025

At the THF, we have worked and continue to work with transgenic mice as models of neurodegenerative diseases. In the APP/PS1 Alzheimer’s model — the model that concerns us here — we investigated the alterations in exocytosis with implications for the synaptic failure at the neuronal level that occurs in this disease, as shown in the figure in this article:

De Diego AMG, Lorrio S, Calvo-Gallardo E, García AG. Smaller quantal size and faster kinetics of single exocytotic events in chromaffin cells from the APP/PS1 mouse model of Alzheimer’s disease. Biochem Biophys Res Commun. 2012;428:482-486 | DOI: 10.1016/j.bbrc.2012.10.082

Figure: Immunohistochemical staining for mutated huntingtin (Htt) in chromaffin cells of the adrenal medulla of R6/1 mice in pre-disease stages (2 months of age, 2m) before the appearance of phenotypic motor alterations.

This mouse model carries the mutation that defines Huntington’s disease, with alterations in exocytosis and muscle activity. Several of these parameters were studied in the following article:

Martínez-Ramírez C, Baraibar A, Nanclares C, Méndez-López I, Gómez A, Muñoz MP, De Diego AMG, Gandía L, Casarejos MJ, García AG. Altered excitability and exocytosis in chromaffin cells from the R6/1 mouse model of Huntington’s disease is linked to overexpression of mutated huntingtin. J Neurochem. 2018;147:454-476 | DOI: 10.1111/jnc.14585

Figura: Tinción inmunohistoquímica para huntingtina mutada (Htt) en células cromafines de la médula adrenal de ratones R6/1 en etapas pre-enfermedad (2 meses de edad, 2m) antes de que aparezcan alteraciones motoras fenotípicas.

The SOD1/G93A transgenic mouse model faithfully reproduces human ALS. The following article presents a study of the effects of chronic administration of a blocker of the P2X7 receptor in the course of the symptomatology and pathogenesis of the disease:

Ruiz-Ruiz C, Garcia-Magro N, Negredo P, Avendano C, Bhattacharya A, Ceusters M, García AG. Chronic administration of P2X7 receptor antagonist JNJ-47965567 delays disease onset and progression, and improves motor performance in ALS SOD193A female mice. Dis Model Mech. 2020;dmm045732 | DOI: 10.1242/dmm.045732

Figure: Disease onset in SOD1G93A mice with ALS treated intraperitoneal (IP) route, 4 times per week with vehicle (orange) or 30 mg/kg JNJ-47965567 (green). (C) Probability of onset in the female subgroup. Analysis performed with Cox regression, considering age at first injection as a co-variate. Differences significant at P<0.05.

The rd10 mouse is a good model of inherited retinal dystrophy leading to blindness. In this model, we are studying the potential neuroprotective effect of medicines related to the P2X7 receptor and calcium channels. An example of the type of studies being conducted in collaboration with the University of Alicante is shown below..

Martínez-Gil N, Kutsyr O, Noailles A, Fernández-Sánchez L, Vidal L, Sánchez-Sáez X, Sánchez-Castillo C, Lax P, Cuenca N, García AG, Maneu V. Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy. Int J Mol Sci. 2022;23:14758 | DOI: 10.3390/ijms232314758

Figure: P2X7R expression in mouse retinas analysed by immunohistochemistry. Cross-sections of retinas from a representative C57BL/6J mouse (A,D,G) and rd10 mice at early (B,E,H) and late (C,F,I) stages, stained with antibodies against P2X7R (A–C,G–I). The nuclei were stained with TO-PRO (D–F). In C57BL/6J mice, P2X7R-immunopositive fluorescence is localised primarily in the INL and GCL. Scale bar: 50 µm.

In collaboration with the pharmaceutical company Bioibérica, the University of Granada, the University of Castilla-La Mancha, and the Autonomous University of Madrid, the THF catalysed the study of a new Bioibérica molecule, BIS014, which surprisingly proved to be highly effective against neuropathic pain in several mouse models (sciatic nerve ligation, diabetic neuropathy, and cancer chemotherapy), as detailed in the following article:

Padín, J. F., Maroto, M., Entrena, J. M., Egea, J., Montell, E., Vergés, J., López, M. G., Cobos, E. J., & García, A. G. (2023). Small Synthetic Hyaluronan Disaccharide BIS014 Mitigates Neuropathic Pain in Mice. Journal of Pain, 24(1), 68-83. DOI: 10.1016/j.jpain.2022.07.014

BIS014 is protected by several international patents, including those in the United States and Europe, in the drafting of which the THF participated.

The figure shows the comparative efficacy of the compound BIS014 with the standard reference gabapentin in a mouse model of chemotherapy-induced neuropathic pain.

The genetic model of arterial hypertension in the SHR (Spontaneously Hypertensive Rat) imitates human arterial hypertension, so it has been widely used in the search for new anti-hypertensive medicines, as well as in the study of the mechanism of action of already known anti-hypertensive medications. In the THF, this model has been used in several studies. One of these, a very interesting one, relates to the ability of the anti-oxidant resveratrol to prevent the development of hypertension, when administered chronically in the drinking water of SHR from an early age. The study is presented in the following article:

This study addressed the alterations in calcium homeostasis, mitochondria, and vascular concentration that occur with the development of a chronic hypertensive state in the spontaneously hypertensive rat (SHR):

This article was prepared in collaboration with scientists from the University of La Laguna and the University of Valladolid.

Several joint publications by scientists from the University of Valladolid, the Autonomous University of Madrid, and the THF led to the functional triad hypothesis: calcium channels, endoplasmic reticulum, and mitochondria collaborate efficiently in the generation and regulation of calcium signals that trigger exocytosis, as shown in the following figure:

Figure: Functional triads responsible for the generation of high local Ca2+ transients and catecholamine secretion in chromaffin cells: regulatory role of voltage-operated calcium channels (VOCCs), endoplasmic reticulum and mitochondria..