Drug-releasing electroactive implant for nervous system repair

Abstract

The present invention relates to a filler hydrogel which is implanted in sites of neurological damage and releases drugs with antifibrotic and neural growth-promoting activity, and to the use thereof combined with electrical stimulation and with other drugs to promote nervous system repair in vivo; as well as to compounds, methods, and procedures for preparing and implementing said implants.

Description

[0001] m DRUG-RELEASING ELECTROACTIVE IMPLANT FOR NERVOUS SYSTEM REPAIR

[0002] The present invention relates to a volumetric or tissue-filled implant that releases drugs with antifibrotic and neural growth-facilitating activity and provides electrical stimuli, and to its combined use with electrostimulation protocols and other drugs to promote regeneration of the nervous system in vivo; as well as to the compounds, methods and procedures for preparing and executing said implants, and to neural electrostimulation systems that incorporate the compounds or are co-administered with the compounds to prevent fibrosis and induce axonal or dendritic growth on or around the implanted electrodes.

[0003] BACKGROUND OF THE INVENTION

[0004] The nervous system (NS) is damaged in a wide variety of pathological processes, including trauma, hemorrhage and vascular events leading to hypoxia and ischemia, infections, inflammatory disorders, genetic, metabolic, toxic, and neurodegenerative diseases. In clinical practice, these pathologies can affect any region of the central nervous system (CNS), composed of the brain and spinal cord, or involve the peripheral nervous system (PNS), composed of nerves that carry sensory and motor information to and from all organs of the body. Functional loss secondary to a neurological injury depends on the location and severity of the injury.

[0005] Traumatic spinal cord injury is a common and well-studied type of neurological damage, but still without effective treatment. The pathophysiology of spinal cord injury (SCI), as well as the proposed strategies for its repair, are described in detail in different publications (Collazos-Castro JE. Lesión y repara de la cord spinal: aspectos neuropatológicos y funcional. In: Esclarín de Ruz A. (Ed): Lesión medular, enfoque multidisciplinar. Editorial Médica Panamericana. Madrid, 2020, pp. 235-241. Collazos-Castro JE. Biomaterial-based systems as biomimetic agents in the repair of the CNS. In: Salgado A. (Ed): Handbook of Innovations in Central Nervous System Regenerative Medicine. Elsevier, Amsterdam, 2020).In summary, SCI interrupts the axons (axotomy) that run through the affected area, and also causes death of neurons, glial, vascular and meningeal cells, and is accompanied by a series of complex responses that lead to tissue scarring without restoring the functional anatomy of the NS.

[0006] In humans, traumatic brain injury typically produces large defects in neural tissue, ranging from 1 to 10 cm in length or even larger. After the acute phase, in which glia and inflammatory cells phagocytose the devitalized tissue, cavities with a variable component of fibrotic connective tissue form. The latter are contributed by pericytes, perivascular and meningeal fibroblasts, other meningeal cells, and fibroblasts from the spinal ligaments (Fernandez E, Pallini E. Connective tissue scarring in experimental spinal cord injuries: significance of dural continuity and role of epidural tissues. Acta Neurochir. (Wien) 1985; 76: 145-8; Góritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisen J. A pericyte origin of spinal cord scar tissue. Science 2011 ; 333:238-42; Soderblom C, Luo X, Blumenthal E, Bray E, Lyapichev K, Ramos J, Krishnan V, Lai-Hsu C, Park KK. Tsoulfas P, Lee JK. Perivascular fibroblasts form the fibrotic scar after contusive spinal cord injury.J Neurosci. 2013; 33:13882-7; Alves-Sampaio A, Del-Cerro P, Collazos-Castro JE. Composite Fibrin / Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs. int J Mol Sci. 2023;24:11102). Connective tissue cavities and scars constitute a mechanical and molecular impediment to axonal regeneration and glial migration, and interfere with the presentation of topographical and molecular signals for neural cell growth. For effective treatment of neurological damage, such cavities must be filled with volumetric implants that do not increase fibrosis and that provide guidance and stimulation for neural regeneration.

[0007] Microtubules are a relevant target for attempts to enhance axonal regeneration. Patent WO 2006 / 094811 claims the use of microtubule-stabilizing drugs, such as taxol, for the preparation of pharmaceutical compounds that stimulate axonal growth after CNS injuries. Since proper microtubule dynamics are also essential for cell migration and division, drugs that stabilize microtubules significantly affect these processes and can therefore reduce fibrosis. Taxol administration decreased fibrotic tissue formation and produced some axonal growth in rats with LM (Hellal F, Hurtado A, Ruschel J, Flynn KC, Laskowski CJ, Umlauf M, Kapitein LC, Strikis D, Lemmon V, Bixby J, Hoogenraad CC, Bradke F, Microtubule stabilization reduces scarring and causes axon regeneration after spinal cord injury. Science 2011;331 :928-931).However, there is much doubt among experts regarding the possible usefulness of taxol and related drugs to facilitate the repair of neurological injuries, due to its neurotoxicity since it blocks the migration and division of glial cells and other reparative cells, leading to the formation of empty cavities in the spinal cord instead of facilitating the closure of the tissue gap (Popovich PG, Tovar CA, Lemeshow S, Yin Q, Jakeman LB. Independent evaluation of the anatomical and behavioral effects of Taxol in rat models of spinal cord injury. Exp. Neurol. 2014;261: 97-108).

[0008] While taxol stabilizes microtubules, there are drugs that have the opposite effect, that is, they increase microtubule mobility, for example, kinesin inhibitors. The latter, and in particular inhibitors of kinesin Eg5, also increase axonal growth and have been proposed for the treatment of nervous system injuries (Lin S, Liu M, Son YJ, Timothy Himes B, Snow DM, Yu W, Baas PW. Inhibition of Kinesin-5, a microtubule-based motor protein, as a strategy for enhancing regeneration of adult axons. Traffic. 2011; 12:269-86; Baas PW, Matamoros AJ. Inhibition of kinesin-5 improves regeneration of injured axons by a novel microtubule-based mechanism. Neural Regen Res. 2015;10:845-9).A preliminary study evaluated the usefulness of monastrol, a low-affinity EG5 inhibitor, in combination with peripheral nerve grafts and chondroitinase ABC in enhancing axonal regeneration in rats with SCI (Xu C, Klaw MC, Lemay MA, Baas PW, Tom VJ Pharmacologically Inhibiting kinesin-5 activity with monastrol promotes axonal regeneration following spinal cord injury. Exp Neurol. 2015 Jan;263:172-6). The peripheral nerve graft acted as a substrate and guide for axonal growth through the injured area, and chondroitinase ABC acted as a degrader of proteoglycans that inhibit axonal regeneration. Monastrol, administered as a solution for 14 days through an intrathecal catheter at the implant site, had an additional facilitating effect on axonal growth, although it did not impact functional recovery.

[0009] Several EG5 inhibitors have been developed with much higher affinity and selectivity than monastrol (Myers SM, Collins I. Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy. Future Med Chem. 2016;8:463-89), which gives them greater therapeutic potential. Several patents claim these compounds and their use as antimitotics for cancer treatment, for example, patents US8765817 and US20150352114.

[0010] However, the usefulness of Eg5 inhibitors for the treatment of fibrosis in the NS or other body organs has not been investigated.

[0011] Receptor tyrosine kinase inhibitor drugs may also have beneficial effects following neurological injury, including a reduction in fibrosis. For example, platelet-derived growth factor receptor (PDGFR) inhibitors, such as imatinib, reduce fibrosis in various animal models of skin, lung, and liver disease (Distler JH, Jün A, Huber LC, Schulze-Horsel U, Zwerina J, Gay RE, Michel BA, Hauser T, Schett G, Gay S, Distler O. Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis. Arthritis Rheum. 2007;56:311-22; Rhee CK, Lee SH, Yoon HK, Kim SC, Lee SY, Kwon SS, Kim YK, Kim KH, Kim TJ, Kim JW. Effect of nilotinib on bleomycin-induced acute lung injury and pulmonary fibrosis in mice. Respiration. 2011;82:273-87; Pesce A, Ciurleo R, Bramanti A, Armeli lapichino EC, Petralia MC, Magro GG, Fagone P, Bramanti P, Nicoletti F, Mangano K.Effects of Combined Administration of Imatinib and Sorafenib in a Murine Model of Liver Fibrosis. Molecules. 2020;25:4310). These compounds have also been proposed for the treatment of neurological injuries. For example, anti-inflammatory, neuroprotective, and antifibrotic effects have been reported for imatinib in rodents with SCI (Abrams MB, Nilsson I, Lewandowski SA, Kjell J, Codeluppi S, Olson L, Eriksson U. Imatinib enhances functional outcome after spinal cord injury. PLoS One. 2012;7(6):e38760), but independent researchers did not obtain the same results with this drug (Sharp KG, Yee KM, Steward O. A re-assessment of treatment with a tyrosine kinase inhibitor (imatinib) on tissue sparing and functional recovery after spinal cord injury. Exp Neurol. 2014 Apr 254:1-11).Although its usefulness is controversial, clinical trials are currently underway evaluating the safety of imatinib administration in people with cervical spinal cord injury (Treatment of Cervical Spinal Cord Injury With Imatinib - a Safety and Feasibility Study. ClinicalTrials.gov Identifier: NCT02363361).

[0012] From all of the above, it is evident that the control of fibrosis after NS injuries is an unsolved problem that must be addressed to aid neural repair. Complete inhibition of fibrosis or the use of neurotoxic drugs or drugs that nonspecifically stabilize the cytoskeleton, as is the case with taxol, can cause an extension of the tissue defect and a complete failure of reparative responses. The usefulness of the new selective Eg5 inhibitors in reducing fibrosis and increasing neural growth is unknown, and for one skilled in the art, it is also not obvious how these drugs could be administered and combined to have a positive effect on neural repair. Furthermore, it is believed that Eg5 inhibitors can negatively affect memory and learning (Freund

[0013] RK, Gibson ES, Potter H, Dell'Acqua ML. Inhibition of the Motor Protein Eg5 / Kinesin-5 in Amyloid β-Mediated Impairment of Hippocampal Long-Term Potentiation and Dendritic Spine Loss. Mol Pharmacol. 2016:89:552-9), so it would be necessary to administer them directly to the injured area to limit unwanted effects. Other drugs, such as imatinib, appear to be beneficial in SCI, but there is much doubt about these effects since they were not reproduced when evaluated by independent researchers.

[0014] On the other hand, another multifunctional tool that can help in the repair of various tissues is electrical stimulation, since it has been shown to increase cell migration and division, as well as the secretion of trophic factors (Rajendran

[0015] SB, Challen K, Wright KL, Hardy JG. Electrical Stimulation to Enhance Wound Healing. J Funct Biomater. 2021;12:40).

[0016] Although previous attempts have failed to promote axonal regeneration through CNS injury using electrical stimulation, they have shown that it can induce some axonal growth responses (Martin JH. Neuroplasticity of spinal cord injury and repair. Handb Clin Neurol. 2022:184:317-330). For example, in rodents, it can increase the branching of intact axons in the corticospinal tract (Brus-Ramer M, Carmel JB, Chakrabarty S, Martin JH. Electrical stimulation of spared corticospinal axons augments connections with ipsilateral spinal motor circuits after injury. J Neurosci. 2007;27:13793-801). However, there is evidence that it can also have the opposite effect, that is, inhibit axonal regeneration (Enes J, Langwieser N, Ruschel J, Carballosa-Gonzalez MM, Klug A, Traut MH, Ylera B, Tahirovic S, Hofmann F, Stein V, Moosmang S, Hentali ID, Bradke F. Electrical activity suppresses axon growth through Ca(v)1.2 channels in adult primary sensory neurons. Curr Biol. 2010;20:1154-64).

[0017] Low-frequency (20 Hz) electrical stimuli increased the speed of motor axon regeneration in the PNS (Al-Majed AA, Neumann CM, Brushart TM, Gordon T. Brief electrical stimulation promotes the speed and accuracy of motor axonal regeneration. J Neurosci. 2000;20:2602-8) but not in the spinal cord, even when peripheral nerve fragments were simultaneously implanted in the injured area (Harvey PJ, Grochmal J, Tetzlaff W, Gordon T, Bennett DJ. An investigation into the potential for activity-dependent regeneration of the rubrospinal tract after spinal cord injury. Eur J Neurosci. 2005;22:3025-35).

[0018] One strategy under exploration for neural repair is to apply electrical stimuli through electroconductive materials implanted within the injured area, which simultaneously provide substrate and guidance for neural growth. In vitro, glia and neurons grow very well on metallic or carbon electrodes coated with conducting polymers biofunctionalized with molecules that promote cell adhesion (Collazos-Castro JE, Hernández-Labrado G, Polo JL, García-Rama C. N-Cadherin and L1-functionalized conducting polymers for synergistic stimulation and guidance of neural cell growth. Biomaterials 2013;34:3603-3617). Electrostimulation through these cellular substrates can act directly on neurons or glia or indirectly influence them by modifying or electrosecreting the molecules present at the electrode / cell interface (Collazos-Castro JE, Polo JL, Hernández-Labrado G, Padial-Cañete V, García-Rama C.Bioelectrochemical Control of Neural Cell Development on Conducting Polymers. Biomaterials 2010;31:9244--9255; Collazos-Castro JE, García- Rama C, Alves-Sampaio A. Glial progenitor cell migration promotes CNS axon growth on functionalized electroconducting microfibers. Acta Biomater. 2016;35:42-56).

[0019] In this regard, previous patents (P201231969, PCT / ES2013 / 070879) have described electroconductive microfibers modified with molecules that promote axonal growth and glial migration over long distances in vitro. From a neurophysiological point of view, microfibers allow ultrasensitive recording of neuronal activity (Vara H, Collazos-Castro JE. Biofunctionalized Conducting Polymer / Carbon Microfiber Electrodes for Ultrasensitive Neural Recordings. ACS Appl Mater Interfaces. 2015;7:27016-26), and also improved electrical activation of spinal neural circuits (Vara H, Collazos-Castro JE. Enhanced spinal cord microstimulation using conducting polymer-coated carbon microfibers. Acta Biomater. 2019;90:71-86).These microfibers, incorporated into alginate hydrogels for implantation in rodents with SCI, also partially decreased fibrosis and served as a scaffold for cell growth in the cavities formed in the neural tissue, facilitating axonal regeneration and directed cell migration (Alves-Sampaio A, Garcia-Rama C, Collazos-Castro JE. Biofunctionalized PEDOT-coated microfibers for the treatment of spinal cord injury. Biomaterials 2016; 89:98-113). Despite these histological effects, implantation of microfibers in alginate hydrogel also increased the gap in the neural tissue, such that there was no possibility of functional reconnection of the cord. In an attempt to reduce the additional neural damage associated with the implantation of microfibers, recently published studies have replaced alginate with fibrin hydrogel (Escarrat V, Perez-Sanchez J, El-Waly B, Collazos-Castro JE, Debarbieux F.Composite Fibrin and Carbon Microfiber Implant to Modulate Posttraumatic Inflammation after Spinal Cord Injury. Cells. 2023 12:839; Alves-Sampaio A, Dei-Cerro P, Collazos-Castro JE. Composite Fibrin / Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs. Int J Mol Sci. 2023;24:11102). In these studies, fibrin bundles with microfibers were introduced into the spinal cord injury site in mice or pigs, and although the implant induced some guided axonal growth, no regeneration of supraspinal axons across the injury or functional recovery was obtained, and the lesion volume was significantly increased in the porcine model.

[0020] Considering the described state of the art, it is concluded that functional repair of the central nervous system is a multifaceted problem that remains unsolved. In particular, the need has been identified to develop filler implants that reduce fibrosis and simultaneously reduce cavitation and the extent of neural damage, while stimulating neural growth through the injured area and achieving functional recovery. These multiple effects are essential for successful neural tissue repair and are achieved in the present invention thanks to the combination of a neural tissue filler implant composed of biomaterials and drugs, combined with electrical stimulation.More specifically, the problem is solved by an implant that fills the cavity secondary to the neurological injury, releases antifibrotic drugs, and applies electrical stimulation to the damaged area of ​​the nervous system. This implant can also be combined with the local or systemic administration of other compounds to improve the restoration of tissue architecture.

[0021] DESCRIPTION OF THE INVENTION The present invention falls within the technical sector of the development of implants to induce regeneration of the nervous system, as well as combinations of biomaterials, drugs and electrical stimulation for neurological repair. The invention provides compounds, methods and devices to fill cavities or defects in neural tissue and control fibrosis after neurological damage, as well as to facilitate, stimulate and direct axonal growth and adhesion, proliferation, migration and differentiation of glial cells, neurons, vascular cells, connective tissue cells, and other types of cells that can contribute to the repair of neurological injuries.

[0022] More specifically, this invention relates to drug-eluting filler implants with antifibrotic and neural growth-facilitating activity, and their combined use with electrical stimulation protocols and other drugs to promote nervous system repair in vivo . This invention also relates to the compounds, methods, and procedures for preparing and executing such implants. The implant comprises a hydrogel that fills the tissue defect and contains drugs for local release, and electrodes for applying electrical stimuli. The electrodes can be incorporated within the hydrogel, usually in the form of microfibers or microwires to act as a substrate for directed cell growth, or they can be positioned adjacent to the hydrogel to electrically stimulate perilesional structures and promote neural growth through the lesion.

[0023] The combination of drugs, biomaterials, and electrical stimulation in this invention provides synergistic effects on neural regeneration that cannot be achieved by these interventions separately. The effects on tissue repair are achieved through the combined action of the hydrogel, which fills the lesion and thus prevents the accumulation of blood and cellular debris and simultaneously facilitates tissue growth; the drugs released by the hydrogel, which decrease fibrosis and increase glial migration and the extension and branching of axons; and the electrical stimulation, which increases reparative responses. Furthermore, the implant may contain microfibers or microwires that guide cells and axons through the lesion.

[0024] Therefore, this invention finds industrial application in the areas of regenerative medicine, biomedicine, biotechnology, and bioengineering and tissue engineering. More specifically, the invention has application in the medical industry to promote neural tissue repair and functional restoration after neurological injuries, and in the field of electrical neuroprostheses to stimulate or record neural electrical activity and replace lost functions. The present invention also has application in the biotechnology industry, particularly in the design of devices to induce the proliferation, migration, and selection of glial cells and neural progenitors, and axonal growth in vitro. Finally, the applications may extend to the engineering of tissues and organs other than the nervous system.

[0025] The present invention presents the following advantages that give it great potential for industrial development, including:

[0026] 1) Identify pharmacological compounds, doses, and effective delivery methods to reduce fibrosis induced by neurological injuries. The drugs can be applied locally to the injured areas, formulated in a slow-absorbing, filling hydrogel, thus avoiding systemic toxicity and without the need to implant pumps and probes for continuous release into neural tissue.

[0027] 2) Develops a new implant for the injured nervous system, in which the drug-releasing hydrogel filler is combined with electrical stimulation, inducing a cell growth response that facilitates tissue defect closure and cannot be achieved with hydrogel, drugs, or electrical stimulation alone. Electrodes can be incorporated within the hydrogel to further support and guide neural growth, or they can be positioned adjacent to the hydrogel to stimulate perilesional cells. The implant does not contain cells and therefore does not present the limitations and complications of cell therapy or tissue grafts.

[0028] 3) Develop a methodology for applying the implant to neurological injuries like those seen in humans. The implant is easily configurable for injuries of different shapes and sizes, and is therefore transferable to clinical practice.

[0029] 4) The implant can be combined with the administration of other drugs locally or systemically to improve tissue repair responses.

[0030] The tissue filler hydrogel can be manufactured from various compounds approved for clinical use, for example, fibrin, and its main pharmacological component is a compound that reduces cell proliferation without presenting neurotoxic activity in nanomolar concentration, preferably a molecule that inhibits or blocks the function of kinesins, in particular kinesin-5, also called Eg5 or KIF11. As exemplified in the present invention, Eg5 is abundantly expressed in connective tissue cells (fibroblasts, pericytes, meningeal cells) that proliferate and form a fibrotic scar that prevents tissue repair after a spinal cord injury, and drugs that inhibit it, for example, compound SB743921, decrease and even eliminate the formation of fibrous tissue and increase axonal growth in the area of ​​injury.In the present invention, EG5 inhibitor drugs are incorporated into fibrin hydrogels without losing their activity, and are released for periods of time sufficient to control fibrosis and facilitate neural tissue reparative responses. However, without electrical stimulation and elements for cell adhesion and guidance, the closure of the tissue defect and neural growth that occurs after hydrogel implantation are incomplete and disorganized, resulting in ineffective for functional repair.

[0031] To organize and improve neural growth in the area of ​​injury, induce neural activity, and achieve functional tissue repair, the implant object of this invention includes electrodes that can be of two types:

[0032] - Internal, i.e., located inside the hydrogel, in which case biofunctionalized and aligned electroconductive structures are preferred, such as carbon microfibers or nanostructures or metallic microwires coated with biomolecules that promote adhesion and directed cell growth;

[0033] - External, meaning located outside the hydrogel, usually adjacent to the hydrogel or on the connective tissue layers surrounding neural tissue, such as the meninges of the central nervous system. These electrodes can have any shape that adapts to the structures on which they are placed.

[0034] Furthermore, the complexity of neurological injuries and the inflammatory and fibrotic responses that occur with any implant in the nervous system may still require combining the implant of this invention with the administration of other drugs to achieve the desired reparative effects. As shown in the following examples, PDGFR inhibitor drugs such as imatinib are very useful for enhancing the benefits of the implant on neural tissue repair.

[0035] Therefore, a first aspect of the invention relates to a volumetric, electroactive and drug-releasing implant comprising: a) a hydrogel for tissue filling of natural or synthetic polymers selected from the list comprising: polypeptides, fibrin polymers, fibronectin polymers, collagen, proteoglycans, or glycosaminoglycans, alginate, chitosan, acrylamide, methacrylate, hydrogelatin and derivatives and combinations of these materials; preferably, a fibrin hydrogel, (the hydrogel is obtained by gemification of the aforementioned polymers), b) at least one drug incorporated into the previous hydrogel to control fibrosis and facilitate axonal growth and glial migration through the injury site, wherein one of the drugs is a compound that blocks or inhibits the function of kinesins, or that reduces their expression, preferably inhibitors of kinesin Eg5 and, more preferably, the drug SB743921;c) a set of electrodes for applying electrical stimuli to the injured tissue, wherein at least one electrode is positioned within the hydrogel or adjacent to it;

[0036] Preferably, the electrode assembly comprises at least two electrodes.

[0037] A "volumetric" implant, also known as a "filler implant," is designed to fill a tissue cavity or add volume to the tissue. Therefore, the implant of the present invention takes the shape of the area or cavity it is intended to fill.

[0038] In a preferred embodiment, at least one electrode is positioned adjacent to the hydrogel, in the damaged areas of the nervous system, in direct contact with the neural tissue or on the connective tissue coatings.

[0039] In another preferred embodiment, at least one electrode is positioned inside the hydrogel and additionally serves as a support, guide and stimulus for cell growth in the injured area.

[0040] In another preferred embodiment, the inner electrodes of the hydrogel comprise or consist of: carbon microfibers, carbon nanostructures, or metallic microwires, for example, of steel, gold, platinum, platinum / iridium, or other metals and their oxides, composites and alloys, a composite being understood as a heterogeneous mixture of these metals with other synthetic or natural non-metallic components to form a single composite. The terms “microfiber” and “microwire” refer to elongated objects with a micrometric diameter. Typically, carbon microfibers are filaments with an average diameter between 1 and 100 μm, and metallic microwires are between 1 and 1000 μm (measured, for example, by scanning electron microscopy (SEM)).

[0041] The term "carbon nanostructure" refers to carbon structures where at least one of their dimensions (on average), usually average diameter, is nanometric in size, i.e. less than 1 μm (measured, for example, by scanning electron microscopy (SEM). The term nanostructure includes, for example, nanoparticles, nanofibers, nanotubes, etc.

[0042] In another preferred embodiment, the electrodes external to the hydrogel comprise or consist of carbon electrodes, steel electrodes, iridium electrodes, platinum electrodes, platinum / iridium electrodes, gold electrodes, or electrodes made of other metals and their oxides, composites, and alloys. These electrodes can have any morphology that adapts to the structures on which they are placed.

[0043] In another preferred embodiment, the electrodes are coated with conductive polymers. Preferably, the conductive polymers are selected from the list comprising: poly(3,4-ethylenedioxythiophene) (PEDOT), polypyrrole, and polyaniline.

[0044] In another preferred embodiment, the surface of the electrode or the conductive polymer is biofunctionalized, that is, at least one molecule with biological activity has been attached to it. For the biofunctionalization of the electrodes, bridging molecules and methods described in the literature and in other patents can be used, some of which are incorporated herein by reference (WO2014096489). In a preferred embodiment, the molecule with biological activity used for the functionalization of the electrodes or the conductive polymer is selected from cell adhesion molecules, extracellular matrix molecules, growth factors, guidance factors for axons or dendrites such as netrins, ephrins, semaphorins, protocadherins or Slit or Wnt proteins, morphogens such as Sonic Hedgehog, proteoglycans, glycosaminoglycans, gangliosides, proteins, peptides or combinations thereof.

[0045] In a preferred embodiment, the drug, preferably an Eg5 inhibitor, more preferably SB743921, is at a concentration of 1 μg to 1 mg per ml of hydrogel. A second aspect of the present invention relates to the method used for the manufacture of the implant, as described above, which comprises the preparation of a drug-releasing reilene hydrogel. The mixing of the solutions required for the formation of the hydrogel (gelation) can be carried out both in vitro, that is, in tubes, molds, plates or other laboratory materials, and in vivo, that is, directly in the area of ​​tissue damage so that it conforms more precisely to the shape and size of the lesion.

[0046] In a preferred embodiment, a fibrin hydrogel is prepared by mixing:

[0047] - a first aqueous solution containing human fibrinogen and factor XIII and - a second aqueous solution containing thrombin and CaCl2, where at least one of these solutions contains the drug, preferably an Eg5 inhibitor, which is retained after gelation of the fibrin, and where at least one of these solutions optionally contains the electrode array. The electrode array is optional, as it could be positioned adjacent to the hydrogel during application of the implant in the body.

[0048] In a preferred embodiment, the mixture is made in a first solution:second solution ratio between 1:1 and 2:1 by volume.

[0049] In another preferred embodiment, the first solution contains human fibrinogen in concentrations between 0.5 and 100 mg / ml and factor XIII between 3 and 300 U / ml.

[0050] In another preferred embodiment, the second solution contains thrombin in concentrations between 3 and 300 U / ml and CaCl2 in concentrations between 0.26 and 26 mM in water, more preferably, in water with 0.9% (w / v) NaCl.

[0051] In another preferred embodiment, the drug, preferably an Eg5 inhibitor, more preferably, SB743921, is at a final concentration of 1 microgram to 1 milligram per milliliter of mixture.

[0052] In another preferred embodiment, the drug is pre-encapsulated in particles, vesicles, liposomes, or the like, preferably nanometric or micrometric in size, to increase their retention or improve their activity, and then incorporated into the hydrogel precursor solutions. In another preferred embodiment, the electrodes or at least one electrode (carbon microfibers, metal microwires, or other electroconductive elements) are incorporated aligned in the hydrogel to direct neural growth during the in vitro or in vivo gelation process.

[0053] In a third aspect, the present invention relates to the volumetric implant of the present invention for use in the repair of neural tissue or the nervous system.

[0054] In a preferred embodiment, for use in the repair of neural tissue, the following steps of a surgical method are carried out: a. cleaning the neurological injury area of ​​cellular debris, blood and fibrotic adhesions; b. implanting the drug-filled hydrogel in the cleaned neurological injury area according to step a) during gelation when it occurs in vivo, or after gelation when it occurs in vitro; c. positioning at least one electrode adjacent to the hydrogel, when no electrodes have been incorporated into the implanted hydrogel in step b, or electrodes have not been implanted in the neurological injury area prior to step b; d. delivering the electrical stimuli through the electrodes.

[0055] In a preferred embodiment, the lesion area is additionally washed in step a) with a solution containing the same pharmacological compound present in the hydrogel, and the solution is left in contact with the tissue for a few minutes to impregnate the perilesional area with the drug prior to the formation of the hydrogel.

[0056] In another preferred embodiment, the hydrogel with the pharmacological compound is formed in vitro and subsequently implanted in the clean neurological injury area according to step a).

[0057] In another preferred embodiment, the hydrogel with the pharmacological compound is formed in vitro, within a second implantable material, and the resulting composite implant is introduced into the damaged region of the cleaned nervous system according to step a).

[0058] In a preferred embodiment, the second implantable material is selected from a list of polymers comprising silicone, polyacrylamide, methacrylate, hydrogelatin, collagen, elastin, fibronectin, and derivatives and combinations of these materials.

[0059] In a preferred embodiment, the hydrogel is formed with the pharmacological compound in vivo, such that gelation occurs directly in the area of ​​neurological injury cleaned according to step a).

[0060] In another preferred embodiment, the electrode array is implanted in the area of ​​neurological damage and the hydrogel with the pharmacological compound is subsequently formed in vivo, that is, within the neural tissue, surrounding the electrodes and filling the tissue defect.

[0061] In another preferred embodiment, the hydrogel is formed in vitro with the drug compound and the electrode array, and subsequently implanted into the area of ​​neurological injury.

[0062] In another preferred embodiment, the hydrogel with the pharmacological compound is gelled in vivo within the tissue defect, and the electrode array is positioned adjacent to the hydrogel, within the nervous system or on the membranes surrounding the nervous tissue (meninges, epineurium, perineurium).

[0063] In another preferred embodiment, electrical stimuli are applied via the implant's electrode array to promote or direct tissue reparative responses. Such stimuli may be biphasic or monophasic, of positive or negative polarity, and of appropriate intensity and frequency to induce neural growth responses. Preferably, the intensity of the applied electrical current, in absolute terms, is between 1 nanoampere and 20 milliamps, and / or the frequency of the electrical stimuli is between 0.00001 Hz and 100,000 Hz.

[0064] In a final preferred embodiment, the electroactive implant, for use in neural tissue repair, is used in combination with other pharmacological compounds administered locally or systemically, which facilitate implant integration and neural repair. The pharmacological compound used in combination is preferably selected from the list comprising: an anti-inflammatory, antifibrotic, antibiotic, neuroprotective drug, or a neural growth facilitator. More preferably, the pharmacological compound used in combination is a receptor tyrosine kinase inhibitor selected from imatinib, sunitinib, sorafenib, masitinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, and tofacitinib. Most preferably, the pharmacological compound used in combination is imatinib.

[0065] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical features, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will be apparent in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. Likewise, numerous documents are cited throughout this specification of the invention and are incorporated herein by reference.

[0066] DESCRIPTION OF THE FIGURES

[0067] Fig. 1 illustrates the development of fibrotic connective tissue after spinal cord transection in rats.

[0068] A) Dorsal view of the surgical procedure performed on a rat spinal cord with excision of 2 mm of neural tissue and subsequent suturing of the dura mater. B) Development of proliferation and migration of PDGFR-positive fibrotic cells in the injured area. At 6 days post-injury (DPL), a dense fibrotic scar has formed, blocking any possibility of reconnection of the nervous system. C) The vast majority of scar cells that impede axonal growth and astrocyte migration (GFAP) also express the kinesin EG5. A negative control staining for EG5, omitting the primary antibody, is shown in the lower right panel. Tissue sections are 10 μm thick. Scale bars: B, 500 μm; C, left, 200 μm; C, right, 50 μm.

[0069] Fig. 2 demonstrates the reduction of meningeal fibroblast proliferation after exposure to compound SB743921, an inhibitor of EG5 kinesin.

[0070] A) Increase in total cell number over 48 hours in untreated or SB743921-treated cultures. Without treatment, the initial cell number increased 4-5-fold, whereas with treatment, the cell number remained constant. B) Quantification of cells that incorporated EdU, a DNA synthesis indicator for mitosis. Without the drug, 90% of cells entered mitosis over the 48 hours of follow-up; whereas only 20% did so in the presence of the drug. C) Appearance of untreated (top row) and SB743921-treated (bottom row) cells, processed for immunocytochemistry after 48 hours of culture. The treated cells remained alive but showed cytoskeletal (tubulin) alterations.

[0071] Fig. 3 exemplifies the reduction of fibrosis after spinal cord injury and systemic (subcutaneous) administration of SB743921 in rats.

[0072] A, B, C), Appearance of the lesion area at 7 DPL in 10 μm-thick histological sections processed for the indicated markers. A partial reduction in fibrosis (PDGFR) was observed after administration of SB743921, which was also more lax and allowed the growth of numerous neurofibromatosis (NF)-positive axons. As a consequence, the stumps approached each other and the neural tissue gap was reduced to less than 500 μm, as detected by labeling for GFAP (E and F) and MAP2 (H and I). Serotonergic (D and F) and noradrenergic (TH, G11) axons entered the scar area, approaching the caudal stump. Scale bars: A, B, 200 μm; C — I, 100 μm.

[0073] Fig. 4 exemplifies the reduction of fibrosis after spinal cord injury and administration of a combined treatment with SB743921 and subcutaneous imatinib in rats.

[0074] A, B) Appearance of the spinal cord in an untreated control animal. A dense PDGFR-positive scar separates the rostral and caudal stumps, creating a neural gap approximately 2 mm long. Neither axons (NF, serotonin) nor astrocytes (GFAP) extended into the scar. C, D) Appearance of the spinal cord in an animal treated with SB743921 and imatinib. The drug combination completely eliminated fibrosis (C), partially approximating the spinal cord stumps, but without complete closure of the tissue defect. The regions delimited by rectangles in C and D are magnified at the bottom (c, d) to illustrate the abundant axonal branching in both the caudal (c) and rostral stumps (d). In the absence of a substrate for their growth across the tissue gap, axons accumulated at the edge of the stumps. Scale bars: AD, 500 μm; b and d, 100 μm.

[0075] Fig. 5 illustrates tissue cavitation and fibrosis in rats with spinal cord transection and electrostimulation at the injury site, without hydrogel filler implant or drug administration.

[0076] Biphasic electrical pulses were applied to the injured area for 10 days, implanting electrodes and using the stimulation protocol with the methods described in detail in Example 5. The marrows were extracted at 14 DPL, cut at 10 μm thickness, and processed for the markers indicated in each panel of the figure. Electrostimulation did not significantly modify the fibrosis (PDGFR) that sealed the stumps, and it also increased the formation of cavities in the marrow. Neural cells (astrocytes, GFAP) and neurons (MAP2) were separated by the cavities and fibrotic tissue, which also did not allow axons (serotonergic, SER; noradrenergic, TH) to pass from one side to the other. Scale bars: 500 μm.

[0077] Fig. 6 exemplifies the implantation of in vitro formed filling hydrogel with fibrin, SB743921, electroconductive microfibers, and slow positioning of a microwire adjacent to the hydrogel for electrical stimulation at the spinal cord transection site in rats.

[0078] A) The SB743921 / fibrin hydrogel implant with microfibers was introduced into the 2 mm tissue gap; a stainless steel microwire (50.8 μm in diameter, with Teflon insulation that was removed from the tip, leaving 1 mm exposed) was then positioned rostrally and dorsally to the implant. Finally, the dura mater was sutured with 10-0 suture. The counter electrode was placed ventrally in the subcutaneous abdominal tissue. The stimulation and counter electrode cables terminated in gold pins that exited the dorsocaudal skin of the animal. B) A train of 15 biphasic electrical pulses, with a total duration of 45 ms, was applied every 2 s for 90 minutes for 10 days, beginning on the fourth post-injury day.

[0079] Fig. 7 Illustrates the reduction of cavitation, promotion of neural growth and closure of the medullary gap in rats, after implantation of the in vitro formed hydrogel with fibrin, ''SB743921 / microfibers, combined with electrostimulation.

[0080] At 14 DPL, spinal cords were removed and cut into 10-μm-thick sections, and processed for immunohistochemistry for the specified markers. The left shows the status of the cord in animals that received drug implantation but without electrostimulation, in which a cell-free cavity was produced in the lesion area. The right shows the tissue response to the implant combined with electrostimulation, which closed the tissue defect and produced numerous axonal growth (AF) into the lesion area, accompanied by astrocyte migration (GFAP) from the lesion edges. Scale bar: 250 μm.

[0081] Fig. 8 exemplifies the therapeutic synergy between the electroactive implant releasing SB743921 and systemic administration of imatinib in rats with spinal cord transection.

[0082] Histological sections through the center of the lesion, processed for GFAP and serotonin immunohistochemistry at 14 DPL, are shown. All animals received the implant and electrical stimulation as illustrated in Figures 6 and 7, substantially reducing the neural tissue gap from 2 mm to approximately 500 μm. The right shows that animals additionally treated with imatinib showed greater axonal branching and outgrowth compared with those not receiving the drug (left). Axons outpaced the astrocytic growth front (GFAP) into the lesion. Noradrenergic axons and those labeled for neurofilament (not shown) also increased outgrowth in response to imatinib. Scale bar: 200 μm.

[0083] Fig. 9 exemplifies the application of a fibrin hydrogel containing SB743921 and carbon microfibers, and the positioning of an electrode array adjacent to the hydrogel, for the treatment of neural damage caused by spinal cord contusion in pigs. Intraoperative photographs are shown corresponding to: A) the moment in which the contusion touches the porcine spinal cord; B) the appearance of the traumatized area 24 hours after the contusion; C) a longitudinal incision in the meninges, including the pia, after which a portion of the devitalized neural tissue spontaneously evacuates; D) the appearance of the area after washing it with saline solution and subsequently with a 5 pM solution of SB743921, and the introduction of a preformed fibrin hydrogel containing SB743921 and carbon microfibers coated with a conductive polymer; E) filling the rest of the cavity with the same in vivo gelled hydrogel, and positioning electrodes (stainless steel microwires) on the hydrogel.F) Suture of the dura mater.

[0084] Fig. 10 shows the neural repair obtained by implanting a fibrin hydrogel containing SB743921 and microfibers, with or without electrical stimulation via metallic microwires positioned on the hydrogel, after spinal cord contusion in pigs. The treatment was applied following the surgical procedure described in Fig. 9. For electrical stimulation (ES), the same protocol used in rats, as described in Example 7, was used, always starting each biphasic pulse from the negative (cathodal) phase. After completing the follow-up, the animals were sacrificed, their spinal cords were removed, sectioned at 10 μm thickness, and processed for immunohistochemistry of PDGFR, NF, and noradrenergic (TH) axons. A) Histology of the spinal cord in control animals, which suffered the contusion without treatment, showing the damaged area that ended in a practically empty cavity, with only a few remnants of fragmented axons.Furthermore, the spinal cord tissue showed atrophy on both sides of the lesion. The boxed area is shown enlarged in (A1). B) The same area of ​​A1 in a contiguous section stained for TH. The axons terminate at the rostral edge of the cavity (on the left), without penetrating it. C) Appearance of the spinal cord in animals with contusion plus implant accompanied by electrical stimulation (ES). Spinal cord atrophy has decreased, and a bundle of neural tissue with hundreds of axons that have regenerated in the area of ​​the microfiber implant, crossing the lesion from one side to the other, is observed. The axonal bundle is shown enlarged in (C1), and the box outlined in C1 is enlarged in (C2), combining the fluorescence images with transmitted light (LT) to show the microfibers. D) Massive regeneration of noradrenergic axons in the lesion area in animals that received the combined treatment. Scale bars: A, and C, 1 mm; A1, C1, B and D, 500 μm; C2, 100 μm.

[0085] Fig. 11 shows the recovery of motor skills obtained by implanting a fibrin hydrogel containing SB743921 and microfibers, with or without electrical stimulation via metallic microwires positioned on the hydrogel, after spinal cord contusion in pigs. The treatment was applied following the surgical procedure described in Fig. 9. The functional recovery of the pigs was evaluated using the PTIBS scale (score 1, paralysis; score 10, normal locomotion) in the different treatment groups. The implant combined with ES significantly improved (p<0.05 at 14, 21 and 28 days post-injury) motor skills in pigs with spinal cord contusion.

[0086] Fig. 12 illustrates the neural repair obtained by implanting a fibrin hydrogel containing SB743921 and microfibers, combined with electrical stimulation (ES), after spinal cord contusion in pigs. In this case, the treatment was applied in the same manner as described in Figures 9 and 10, except that the ES pulses always began in the positive (anodal) phase. A) This ES polarity also induced abundant axonal growth across the injured area, evidenced by the presence of NF-positive axon bundles, shown at higher magnification in (a). B) Noradrenergic axons also grew profusely across the injury site. Scale bars: A, 1 mm; a1 and B, 500 μm.

[0087] Fig. 13 shows the antifibrotic and neural growth-promoting effects of in vitro-formed fibrin / SB743921 hydrogel implanted with an additional scaffold in a 1-cm neural gap in the cervical spinal cord hemisection in pigs. An in vitro-formed fibrin hydrogel containing either SB743921 (SB) or without SB (No SB) was implanted within a silicone tube to provide mechanical support at the injured site. Spinal cords were harvested at 30 days postinjury to assess cellular responses and sectioned at 50 μm thickness for eriochromocyanine staining (top left) or at 10 μm thickness for PDGFR and NF immunohistochemical staining (top right and magnifications below). The hydrogel degraded only partially in both cases, leaving remains contained in the silicone tube (stars).Extensive fibrosis developed in and around the tube in the absence of SB (arrow), but fibrosis was almost completely suppressed when the implants contained SB (arrow). Without the drug, the fibrosis was impervious to axons, with degenerative axonal bulbs present right at the interface with the fibrotic tissue (arrow in the lower left panel); whereas with the drug, numerous axons were observed growing to the edge of the lesion, even when some fibrosis remained (arrow in the lower right panel). Scale bar, 5 mm.

[0088] Fig. 14 illustrates the in vitro formed fibrin hydrogel implant with SB743921 and an assembly of interconnected electroconductive microfibers, and epidural electrodes, in a spinal cord hemisection with tissue excision in pigs.

[0089] A) Carbon microfiber assembly (20 MFs, 12 mm long), interconnected by a 35N LT microwire of 12.8 pm diameter and coated with the conductive polymer PEDOT:PSS-co-MA. B) Implantation of the fibrin hydrogel (star) containing the electrically interconnected microfiber assembly into the porcine spinal cord injury. C) Suture of the dura mater and epidural fixation of auxiliary metallic electrodes (counter and reference electrodes) 5 mm from the lesion edges. The arrow points to the 35N LT microwire ending in the connector.

[0090] EXAMPLES

[0091] The invention will now be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the product of the invention.

[0092] Example 1: Development of fibrotic connective tissue after spinal cord transection in rats.

[0093] An adult male Wistar rat spinal cord transection model was used. The experimental protocols followed the regulations of the European Commission and the Spanish legislation for the protection of experimental animals (86 / 609 / EEC, 32 / 2007 and 223 / 1988) and were approved by the Ethics Committee on Animal Experimentation of the National Hospital for Paraplegics of Toledo, Spain. Briefly, animals were anesthetized by intraperitoneal (IP) injection of pentobarbital (50 mg / kg) and xylazine (10 mg / kg), with supplemental doses of 30% administered one and a half hours later as needed. Atropine (0.04 mg / kg, IP) was also administered to decrease bronchial secretions, marbofloxacin was administered as a prophylactic antibiotic, and an ophthalmic ointment was administered to prevent corneal abrasion. A dorsal laminectomy was performed on the T9-T10 vertebrae, under which the T10 and T11 spinal cord segments are located, respectively.A small diagonal cut was made in the dura mater, followed by a complete transection of the spinal cord. After a second, more rostral cut, the intervening tissue was removed, leaving a neural tissue gap of approximately 2 mm (Fig. 1A). Bleeding was controlled by introducing a piece of Espongostan® into the cavity, which was removed once hemostasis was achieved. Durorrhaphy was then performed with simple sutures using 10-0 thread; finally, the muscles and skin were sutured in layers.

[0094] Eighteen animals were used, distributed into six survival groups, ranging from 2 to 7 days postinjury. The animals were anesthetized and perfused transcardially with saline and 4% paraformaldehyde. Spinal cords were removed and immersed in 30% sucrose for 3 days, then cut into 10-µm sections for histological and immunohistochemical staining. The development of fibrotic connective tissue was assessed by immunohistochemical staining for the platelet-derived growth factor receptor (PDGFR), which is expressed by pericytes and fibroblasts. The monoclonal antibody used (Abeam [Y92] - C-terminal ab32570) was raised in rabbits against a synthetic peptide within the human PDGFR, between amino acid 1050 and the carboxyl terminus (Database link: P09619).In addition, the expression of kinesin Eg5 in fibrotic tissue (polyclonal antibody GTX30692) and the presence of astrocytes, identified by their expression of glial fibrillary acidic protein (GFAP, antibody BD Biosciences 556327), were evaluated.

[0095] For immunohistochemical staining, tissue sections were blocked for 1 h at room temperature in phosphate-buffered saline (PBS) containing 0.3% triton and 2% goat serum, and after washing, were incubated with primary antibodies overnight at 4°C. The following day, they were treated for 2 h with fluorescent secondary antibodies (labeled with Aiexa-488 or Alexa-594) dissolved in PBS. Control sections without primary antibodies were included in all cases to assess staining reliability. Cell nuclei were stained with bisbenzimide (Hoechst 33342, Molecular Probes, 1.5 mg / ml in PBS). Fig. 1B illustrates the proliferation and migration of fibrotic cells from the meninges and perivascular space (fibroblasts and pericytes), which stained very efficiently by immunohistochemistry for PDGFR in the injured area.Invasion of the spinal cord cavity by these scar cells began on day 2 and was complete by day 6 post-injury, creating a dense fibrotic tissue. In exceptional cases, some scar contraction occurred between days 4 and 6 post-injury, partially approximating the rostral and caudal stumps of the spinal cord to a distance of less than 1 mm (Fig. 1 B). However, in most cases the fibrotic scar extended 1 to 3 mm longitudinally along the cord. Axons always stopped or deviated at the lesion edge, being unable to traverse the scar. Astrocytes also remained confined to the lesion edge (Fig. 1 C). The vast majority of fibrotic tissue cells expressed Eg5 (Fig. 1 C).

[0096] Example 2: Reduction of meningeal fibroblast proliferation after exposure to compound SB743921, an Eg5 kinesin inhibitor.

[0097] After verifying that Eg5 is expressed in the fibrotic scar connective tissue that forms in the spinal cord and is generated in a large part of the meninges, the effect of the compound SB743921 on the proliferation of meningeal fibroblasts in vivo was investigated. This compound is a very potent inhibitor (Ki = 0.1 nM) of Eg5 (Myers SM, Collins I. Recent findings and future directions for interpolar mitotic kinesin inhibitors in cancer therapy. Future Med Chem. 2016;8:463-89).

[0098] Meningeal fibroblasts were cultured from adult rat dura and arachnoid mater in the presence of fetal bovine serum (FBS) following published procedures (Collazos-Castro JE, Polo JL, Hernández-Labrado G, Padial-Cañete V, García-Rama C. Bioelectrochemical Control of Neural Cell Development on Conducting Polymers. Biomaterials 2010;31:9244-9255). Twenty-four hours after cell seeding, SB743921 was added at a final concentration of 10 nanomolar (nM). Live cell behavior was studied by videomicroscopy, capturing images every 30 minutes for 48 hours. Cell proliferation was also studied in independent cultures by 5-ethynyl-2'-deoxyuridine (EdU) incorporation.Cells were cultured without or with SB743921 at 10 nM, and fixed with paraformaldehyde at 48 h, after which they were processed to fluorescently reveal EdU, and to visualize the cells and their nuclei using immunohistochemistry for tubulin and Hoechst, respectively.

[0099] Videomicroscopy studies revealed that meningeal fibroblasts treated with SB743921 arrested their division, remaining spherical for an extended period of time without completing mitosis, and subsequently acquired an aberrant heterogeneous morphology characterized by an increase in cell size, accompanied by enlargement and deformation of the nucleus, as well as flattening of the cytoplasm. However, no direct cytotoxic effect of the drug was observed. Cells treated with SB743921 survived for 48 hours but maintained their original number, whereas untreated cells multiplied 4- to 5-fold (P < 0.001; Fig. 2A). Furthermore, at 48 hours, almost 90% of control cells had incorporated EdU, that is, they had synthesized DNA to divide, compared with 20% of those treated with the drug (P < 0.001; Fig. 2B and C).

[0100] Example 3: Reduction of fibrosis after spinal cord injury and systemic administration of SB743921 in rats.

[0101] Having demonstrated that SB743921 efficiently inhibits meningeal fibroblast proliferation in vitro, its in vivo toxicity and effect on fibrosis following spinal cord injury in rats were investigated. The same spinal cord transection model described in Example 1 was used, i.e., a 2 mm neural gap was created and the dura mater was sutured without further treatment at the injured site. The drug was dissolved in water at 0.8 mg / ml and administered subcutaneously (SC).

[0102] A total of 30 animals were administered in this trial, divided into 5 groups with different treatment regimens for SB743921: a) 1 mg / kg subcutaneously, one dose every 24 hours for 2, 4, or 6 days, starting at 12 hours post-injury. b) 1 mg / kg, 5 doses total, every 12 hours, starting at 12 hours post-injury. c) 1 mg / kg, 4 doses total, every 12 hours, starting at 24 hours post-injury. d) 1 mg / kg, 4 doses total, every 12 hours, starting at 12 hours post-injury. e) 1,125 mg / kg, 4 doses total, every 12 hours, starting at 12 hours post-injury.

[0103] The marrow was removed at 7 DPL for histological study, since by that time the fibrotic connective tissue had sealed the stumps in animals that did not receive any treatment (example 1 and Fig. 1).

[0104] Neural damage, fibrosis development, inflammation and reparative responses (axonal growth, glial migration, blood vessel growth) were assessed by immunohistochemical staining for axons (neurofilament, NF, Sigma-Aldrich N0142; serotonin, ImmunoSolution IG1112; tyrosine hydroxylase, TH, Sigma AB152), neuronal cell bodies and their dendrites (microtubule-associated protein, MAP2, Sigma M1406), astroglia (glial fibrillary acidic protein, GFAP, BD Biosciences 556327); pericytes and fibroblasts (PDGFR, Abeam AB32570; collagen type IV, Meridian, T40251R), macrophages and microglia (ED1; Chemicon MAB 1435; isolectin B4, Fisher 121411), and endothelial cells (RECAI, Bio-Rad, MCA970R). The results obtained are summarized in Table 1.

[0105] Table 1. Effect of systemic administration of SB743921 in rats with spinal cord transection

[0106] As shown in Table 1, the most effective treatment regimen was regimen "b", which partially reduced fibrosis. A scar remnant of 300 to 500 microns generally formed in the rostrocaudal area of ​​the spinal cord, substantially approximating the stumps and accompanied by axonal growth and branching (Fig. 3). Axons labeled for neurofilament, serotonin, or tyrosine hydroxylase entered the fibrotic tissue and practically reached the caudal stump at 7 DPL. There were no obvious signs of neurotoxicity in the perilesional tissue, although tissue damage due to microhemorrhages was detected, with vascular alterations due to the inhibition of cell proliferation. Four of the 7 animals that received regimen "b" had died at 7 DPL with signs of enterocolitis and bacterial translocation or intestinal perforation, and the survivors already showed signs of intestinal involvement.

[0107] In conclusion, SB743921 was a partially effective drug in reducing fibrosis and increasing axonal growth when administered systemically, but at effective doses it presented a very high mortality rate in rats with spinal cord transection, making it necessary to search for safer and more effective therapeutic regimens for this purpose.

[0108] Example 4: Reduction of fibrosis after spinal cord injury and administration of a combination treatment with SB743921 and imatinib subcutaneously in rats.

[0109] Due to the high toxicity of systemically administered SB743921, the possibility of reducing fibrosis by administering a combination of SB743921 and imatinib systemically was investigated. Imatinib is a PDGFR inhibitor. This receptor is involved in the migration and proliferation of fibroblasts and pericytes during the healing of other organs (Rajkumar VS. Platelet-derived growth factor p-receptor activation is essential for fibroblast and pericyte recruitment during cutaneous wound healing. Am J Pathol 2006; 169:2254-65) and, as shown in Example 1, is abundantly expressed in the fibrotic tissue that forms after spinal cord injury.

[0110] The doses of both drugs were progressively decreased to limit their adverse effects while determining the maximum tolerated dose and effectiveness. The same spinal cord transection model and histological evaluation methods described in Examples 1 and 3 were used. Both drugs were administered by subcutaneous injection every 12 hours, starting 12 hours post-injury. A total of 15 rats were used in this trial, divided into 5 groups with the following pharmacological regimens: a) 4 doses of SB743921, 0.8 mg / kg, plus 5 doses of imatinib, 25 mg / kg. b) 4 doses of SB743921, 0.5 mg / kg, plus 5 doses of imatinib, 25 mg / kg. c) 4 doses of SB743921, 0.8 mg / kg, plus 5 doses of imatinib, 12.5 mg / kg. d) 4 doses of SB743921, 0.8 mg / kg, plus 5 doses of imatinib, 6.25 mg / kg. e) 4 doses of SB743921, 0.65 mg / kg, plus 4 doses of imatinib, 5 mg / kg.

[0111] The results obtained are summarized in Table 2.

[0112] Table 2. Effect of combined subcutaneous administration of SB743921 and imatinib in rats with spinal cord transection

[0113] As shown in Table 2, regimen "a" was highly effective, markedly reducing and even completely eliminating fibrotic connective tissue while generating abundant axonal branches that grew over the astrocytes at the lesion edge (Fig. 4). Complete inhibition of fibrosis prevented fusion of the spinal cord stumps, leaving a 1- to 1.5-mm-long cavity that eliminated any possibility of axonal regeneration. However, tissue damage was minimal, except for the presence of isolated microhemorrhages.

[0114] On the other hand, the effective regimen "a" was fatal in 100% of cases between 6 and 9 days post-injury, and the animals still presented enterocolitis, peritonitis, and mucositis. Most of the other combination regimens also caused 100% mortality and were much less effective. Only regimen "e" allowed half of the animals to survive beyond day 9 post-injury, but the antifibrotic effects were very inconsistent, with a slight reduction in some cases and an increase in inflammation and fibrosis in the bone marrow.

[0115] Taken together, Examples 3 and 4 demonstrated that a safe and effective regimen for treating fibrosis caused by spinal cord injury cannot be achieved with systemic administration of SB743921 and imatinib. Furthermore, the most effective regimens left cavities in the injured area, indicating that in any case, it will be necessary to provide a substrate and stimulus for neural growth.

[0116] Example 5: Ineffectiveness of electrostimulation alone to repair the spinal cord in rats.

[0117] To determine the effect of electrical stimulation alone on tissue repair, the stimulation protocol was applied in the absence of other pharmacological treatments or implants in the injured area. The spinal cord was transected, creating the 2 mm gap as described in the previous examples, and an electrode was implanted to deliver electrical pulses to the injured area. The electrode consisted of a stainless steel microwire (50.8 μm in diameter) with Teflon insulation (AM Systems) that was stripped off at the tip, leaving 1 mm of exposed metal. The metal tip was introduced into the subarachnoid space and fixed dorsally over the rostral edge of the lesion. The dura mater was subsequently sutured. Another electrode of the same material, but with a larger area of ​​uninsulated metal, was placed in the subcutaneous tissue of the animal's abdomen and used as a counter electrode.The ends of the cables had gold pins that exited through the dorsal skin of the animal for connection to the electrical pulse generator (Biologic VSP potentiostat / galvanostat).

[0118] Stimulation began at 4 days post-injury and continued for 10 days, so that animals were perfused at 14 DPL to extract the spinal cord and perform histological analysis as in the previous examples. An electrical pulse protocol that induces arborization of healthy axons of the corticospinal tract when the cerebral cortex is stimulated was used (Brus-Ramer M, Carmel JB, Chakrabarty S, Martin JH. Electrical stimulation of spared corticospinal axons augments connections with ipsilateral spinal motor circuits after injury. J Neurosci. 2007;27:13793-801), but stimulating for an hour and a half daily instead of 6 hours to prevent local reactions that could increase tissue damage. In summary, biphasic pulses of 200 ps / phase were applied, starting with the cathodic phase, in 45 ms bursts at 333 Hz, repeated every 2 s, hour and a half daily for 10 days.The current intensity was maintained at the threshold that induced visible contraction of the paraspinal or thoracic muscles and that was well tolerated without signs of pain by the animals, generally being 350 pA to 650 pA. The application of stimuli of 150 pA above the motor threshold usually produced discomfort and stress to the animals, so the current was always kept within this limit, with a maximum of 750 uA. For biphasic pulses of 500 pA, the ohmic drop between the electrodes was around -4 / +2 V the first day and -7 / +4 V after 10 days.

[0119] The stimulation protocol was well tolerated, and no systemic adverse events were detected. The typical state of the rat spinal cord after 10 days of electrical stimulation is shown in Fig. 5. As can be seen, this treatment alone was insufficient to promote spinal cord repair. A fibrotic scar approximately 1 mm thick sealed the spinal cord stumps, preventing the growth of ascending and descending axonal tracts and astroglial migration through the injured area. In addition, cavities formed in the cord, which were not observed in untreated animals (compare Fig. 5 with Fig. 1B).

[0120] Example 6: Implantation of fibrin hydrogel with SB743921 and electroconductive microfibers, and adjacent electrodes for stimulation of the injury site, in rats with spinal cord transection.

[0121] In Examples 3 to 5, it was concluded that systemic treatments with SB743921 and / or imatinib, or electrostimulation, performed alone, did not provide a safe and effective treatment to reduce fibrosis and neural tissue gap after spinal cord transection in rats. On the other hand, previous studies with implants of electroconductive microfibers embedded in alginate or fibrin hydrogel, without administering drugs or electrostimulation, have shown that the microfibers aid guided axonal growth through the spinal cord cavities, but their implantation is ineffective in inducing regeneration of supraspinal axons and also considerably increases tissue damage and stump separation, such that functional neural repair is not achieved (Alves-Sampaio A, García-Rama C, Collazos-Castro JE. Biofunctionalized PEDOT-coated microfibers for the treatment of spinal cord injury.Biomaterials 2016; 89:98-113; Alves-Sampaio A, Del-Cerro P, Collazos-Castro JE. Composite Fibrin / Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs. Int J Mol Sci. 2023;24:11102).

[0122] In order to avoid systemic toxicity of antifibrotic compounds, while providing substrate for neural growth, a fibrin hydrogel containing SB743921 and electroconductive microfibers was prepared in vitro and introduced into the injured area, and a microwire was implanted adjacent to the hydrogel (Fig. 6A) to apply electrostimulation as described in Example 5. The dura mater and muscles were sutured, and the gold-pinned wire terminals were pulled through the dorsal skin of the animal (Fig. 6A). The electrodes were connected daily to the potentiostat / galvanostat for application of electrical pulses (Fig. 6B).

[0123] Conducting polymer-coated carbon microfibers were prepared and biofunctionalized with cell adhesion molecules using protocols described in detail in previous patents and publications (P201231969, PCT / ES2013 / 070879; Collazos-Castro JE, García-Rama C, Alves-Sampaio A. Glia! Progenitor cell migration promotes CNS axon growth on functionalized electroconducting microfibers. Acta Biomater. 2016;35:42-56). Briefly, 7-ij.m diameter carbon microfibers were used, on which the electrosynthesis was carried out (1 pA / mm 2 for 32 min) poly(3,4-ethylenedioxythiophene) (PEDOT) doped with poly[(4-styrene sulfonic acid)-co-(maleic acid)] (PSS-co-MA), resulting in a PEDOT:PSS-co-MA coating. The carboxylic groups of PSS-co-MA were reacted with EDC / NHS, and then with the amino groups of poly-L-lysine (PLL). Heparin, basic fibroblast growth factor (bFGF), and fibronectin were added over the PLL layer.

[0124] For the preparation of the fibrin hydrogel, a solution containing human fibrinogen (Sigma F4883) and factor XIII (ciuvot, CSL Behring 78779) was mixed in a 2:1 ratio with a solution containing thrombin (Sigma-Aldrich T7009) and CaCl2 (2.6 mM, Sigma-Aldrich) in water with 0.9% (w / v) NaCl. The hydrogel with SB743921 and microfibers was prepared in vitro in 2 mm diameter tubes, adding the microfibers in layers. The final concentrations of fibrinogen, thrombin and factor XIII were 80 mg / ml, 33.3 U / ml and 33.3 U / ml, respectively. 8 pl of the solution with SB743921 was added to the hydrogel, at a final concentration of 0.6 mM.

[0125] Example 7: Reduction of cavitation, promotion of neural growth and closure of tissue gap in rats with spinal cord transection and fibrin / SB743921 / microfiber implant combined with electrostimulation.

[0126] Twenty rats were transected and implanted with fibrin hydrogel containing SB743921, biofunctionalized PEDOT:PSS-co-MA coated carbon microfibers, and electrodes adjacent to the hydrogel as in Example 6. Half of them were treated with electrical stimulation to determine whether synergies existed in the combined treatment. The animals were sacrificed 14 days post-injury to extract the marrow and perform histological analysis following the procedures described above.

[0127] The fibrin-filled hydrogel containing SB743921 allowed good local control of fibrosis with excellent animal survival, but in the absence of electrical stimulation, no reparative response was obtained. The fibrin degraded and the released drug inhibited fibrosis, but in the absence of stimulus for neural growth, it produced an empty cavity with an extension very similar to the original (approximately 2 mm, Fig. 7, left). However, after 10 days of electrical stimulation, the cavity disappeared and the neural gap was considerably reduced (Fig. 7, right), with the growth of multiple cell types, including neural cells, being observed. Numerous neurofilament-positive axons originated from both spinal cord stumps and entered the center of the lesion. Astrocytes from both stumps also aligned and migrated within the tissue gap, remaining only approximately 600 μm apart (Fig. 7, right).However, PDGFR+ connective tissue cells were still found in the injured area, albeit in smaller numbers than in the absence of treatment. They were oriented more longitudinally in the spinal cord and accompanied by axons and blood vessels. Apparently, this secondary fibrotic tissue was still partially inhibitory to axonal regeneration, preventing serotonergic axons from reaching the caudal stump of the spinal cord, at least at the time the animals were sacrificed (14 DPL).

[0128] In summary, the results of this example demonstrated that the use of a drug-eluting neural tissue-filling hydrogel (SB743921), combined with electrostimulation, can reduce fibrosis while promoting tissue gap closure and neural growth, without producing systemic toxicity or other relevant adverse effects, after spinal cord transection in adult rats.

[0129] Example 8: Therapeutic synergy between the electroactive drug-eluting implant and systemic administration of imatinib in rats with spinal cord transection.

[0130] The safety and usefulness of administering fibrin hydrogel / SB743921 / microfibers + electrical stimulation (ES) to the injured area, together with subcutaneous imatinib, was then evaluated. The same methodology described in Example 7 was used, with the animals being sacrificed at 14 DPL for histological analysis.

[0131] Imatinib was dissolved in water at 100 mg / ml and then in 0.9% (w / v) saline and injected subcutaneously. After performing assay 4 to determine drug toxicity, only 10 rats were used here; all received the implant combined with ES. They were also divided into 2 groups with the following imatinib regimens: a) 4 doses of 5 mg / kg, every 12 hours, starting 12 hours post-injury; b) Regime "a" and an additional 5 mg / kg every day until the end of the electrostimulation protocol.

[0132] Regimen "a" was completely safe for the animals when used with the implant, with no associated deaths or adverse effects. Furthermore, histologically, a decreased presence of inflammatory cells was observed in the perilesional spinal cord segments, although no significant effects were detected in terms of neural gap closure or axonal growth and glial migration. Furthermore, regimen "b" virtually eliminated the residual PDGFR+ fibrotic tissue from the lesion area, again leading to separation of the spinal cord stumps in two-thirds of cases. However, in the remaining third of animals, additional tissue gap closure with astrocyte migration and axonal growth was observed. At 14 DPL, the distance between the astrocyte front and the stumps was reduced to approximately 400 μm (Fig.8), and in some cases, the gap was completely eliminated, and both migrating astrocytes and growing axons completely crossed the lesion and reached the caudal stump. Furthermore, the electroactive implant eluting SB743921 combined with subcutaneous imatinib exhibited a clear synergy, significantly increasing axonal extension and branching (Fig. 8).

[0133] Example 9: Application of fibrin hydrogel with SB743921 and carbon microfibers, with an electrode array adjacent to the hydrogel, for the treatment of neural damage caused by spinal cord contusion in pigs.

[0134] To successfully repair human neurological injuries, it is essential to evaluate therapeutic strategies in large animals. The feasibility of clinical application of the present invention was validated in two porcine models of spinal cord injury, namely, contusion at the thoracolumbar junction (T14-T15) and cervical hemisection (C6). Contusion reproduces the patterns of neuronal loss and tissue cavitation found in more than 60% of patients with spinal cord injury (Collazos-Castro JE, Soto VM, Gutiérrez-Dávila M, Níeto-Sampedro M. Motoneuron loss associated with chronic locomotion impairments after spinal cord contusion in the rat. J. Neurotrauma 2005; 22:544-558). Cervical spinal cord hemisection models neural tissue laceration damage and is also the most common location of spinal cord injuries.When hemisection is combined with neural tissue extraction, the pathological process of tissue cavitation is also reproduced (Cerro PD, Barriga-Martin A, Vara H, Romero-Muñoz LM, Rodríguez-De-Lope Á, Collazos-Castro JE. Neuropathological and motor impairments after incomplete cervical spinal cord injury in pigs. J Neurotrauma. 2021 ; 38:2956-2977).

[0135] This example describes the methodology for producing spinal cord contusions in pigs, and illustrates the use of the present invention for the treatment of such injuries. For this test, whose histological and functional repair results are shown in Example 10, 18 female Large White English pigs (Sus scrofa domesticas) were used, 2 months old and weighing 14 to 21 kilograms at the time of injury. These pigs were divided into three experimental groups:

[0136] 1) untreated spinal cord contusion (n = 6).

[0137] 2) contusion, myelotomy at 24 hours post-injury, lavage with SB743921 solution and treatment with fibrin hydrogel, SB743921 and microfibers, with subcutaneous imatinib, but without ES (n ~ 6).

[0138] 3) the same treatment as treatment 2, positioning electrodes adjacent to the hydrogel and applying electrical stimulation for 10 days (n = 6). The ES protocol was the same as that used for the rats in examples 5, 6, and 7, but the daily duration was increased from 1.5 hours to 6 hours.

[0139] The animals were purchased from Granja Agropardal, a company specializing in raising pigs for research. The experimental procedures were approved by the Ethics Committee on Experimentation (CEEA), the Authorized Body of Toledo, and the Ministry of Agriculture and Environment of the JCCM. All surgical procedures were performed under inhalation anesthesia following the protocol described in Alves-Sampaio A, Del-Cerro P, Collazos-Castro JE. Composite Fibrin / Carbon Microfiber Implants for Bridging Spinal Cord Injury: A Translational Approach in Pigs. Int J Mol Sei. 2023;24:11102. This publication also describes in detail the automated closed-loop system used for porcine spinal cord contusion and the general animal care.

[0140] The surface that struck the spinal cord was flat and circular, with a diameter of 8 millimeters (Fig. 9A). The peak impact force was set at 30 N. After the contusion, the muscles were sutured in layers and close together to prevent seroma formation, and finally the skin was closed. A urinary catheter was placed to evacuate and quantify urine output until spontaneous urination was restored.

[0141] Twenty-four hours after the contusion, the animals were anesthetized again, the injury site was exposed (Fig. 9B), and a longitudinal incision was made in the dura and pia. After the incision, some of the devitalized tissue evacuated spontaneously due to pressure from the edema in the contusion area (Fig. 9C). The injured area was then irrigated several times with saline solution using a fine cannula, removing most of the cellular debris. The lesion was then washed with a 5 pM SB743921 solution, which was left on for 10 minutes to impregnate the perilesional area. A preformed fibrin hydrogel containing SB743921 and carbon microfibers coated with a conductive polymer was then introduced (Fig. 9D). The rest of the cavity was filled with the same type of in vivo gelled hydrogel, in this case reducing the final concentrations of fibrinogen, thrombin and factor XIII (approximately 22.2 mg / ml, 11.1 U / ml and 11.1 U / ml, respectively). Finally, electrodes (stainless steel microwires) were positioned over the hydrogei (Fig. 9E), and the dura mater was sutured (Fig. 9F).

[0142] Surgical reintervention at 24 hours post-injury was successful in all cases, with no deaths or postoperative complications associated with the procedure. In compliance with CEEA guidelines, all animals received treatment to reduce postoperative pain and inflammation of soft tissues and osteoarticular areas with meperidine (4 mg / kg SC) every 12 hours for two days, and meloxicam (0.2 mg / kg SC) for 7 days. They were also administered marbofloxacin (2 mg / kg IM) as an antibiotic for 7 days. Additionally, pigs in groups 2 and 3 received 9 doses of imatinib (1.4 mg / kg, SC), starting 6 hours after implantation, every 12 hours for the first 4 doses, and every 24 hours for the next 5.

[0143] Example 10: Improvement of neural repair and motor recovery in pigs with spinal cord contusion that received fibrin hydrogei with SB743921 and carbon microfibers, combined with cathodic-initiated electrostimulation, through the electrodes adjacent to the hydrogei.

[0144] The histological and functional effects of the procedures described in Example 9 are reported. Animals were given inhalational anesthesia to surgically expose the injured spinal cord segment and adjacent rostral and caudal segments. Subsequently, a lethal dose of pentobarbital (120 mg / kg IV) was administered, and the T13 to L3 spinal cord segments were removed. Tissue was fixed by immersion in 4% paraformaldehyde in 0.1 M PBS for 4 days. The spinal cord segments were dissected and immersed separately in PBS with 30% sucrose for 4 days and subsequently embedded in OCT® and stored at -20 °C until processing. The tissue was cut on a cryostat into 10 μm horizontal sections for immunohistochemical staining, and 50 μm sections for eriochrome cyanine and cresyl violet staining.Histological studies were performed to evaluate neural damage, tissue cavitation, axonal and glial growth, and invasion of the lesion by fibrotic connective tissue cells. For immunohistochemistry, the same procedures and antibodies described in Examples 1 and 3 were used.

[0145] The lesion was approximately 1 cm longitudinally and involved most of the spinal cord dorsoventrally, usually sparing a small portion of white matter in the ventrolateral region on one side. In control animals, the necrotic tissue was resorbed, leaving only fragmented axons and a large central cavity surrounded by fibrosis (Fig. 10A), with no neural regeneration across the cavity (Figs. 10A1 and 10B). Furthermore, the spinal cord tissue on each side of the lesion showed a considerable degree of atrophy (Fig. 10A).

[0146] Treatment with fibrin hydrogel / SB743921 / microfibers, combined with imatinib and electrical stimulation, promoted impressive neural growth in the damaged area (Figs. 10C and 10D). The perilesional spinal cord segments hypertrophied and converged, narrowing the cavity (Fig. 10C). Furthermore, a bundle of thousands of axons regenerated through the cavity, following the microfibers and connecting the two sides of the cord (Fig. 10C1 and C2). In the more ventral area, where no microfibers were implanted, neural regeneration was more limited, but serotonergic and noradrenergic supraspinal axons nevertheless branched copiously and invaded the residual cavity in a disorganized manner (Fig. 10D).

[0147] Functional recovery of pigs was assessed using the PTIBS scale described in Lee JH, Jones CF, Okon EB, Anderson L, Tigchelaar S, Kooner P, Godbey T, Chua B, Gray G, Hildebrandt R, Cripton P, Tetzlaff W, Kwon BK. A novel porcine model of traumatic thoracic spinal cord injury. J Neurotrauma. 2013;30:142-59. On this scale, a score of 1 indicates hindlimb paralysis, and 10 points denote normal locomotion. Functional recovery of the three treatment groups is presented in Fig. 11. The implant combined with ES improved motor skills in pigs with spinal cord contusion, with significant differences (p<0.05) at 14, 21, and 28 days post-injury compared to the untreated control group. All animals receiving the combination treatment had improved between 3 and 4 points at the end of 10 days of electrostimulation, while the controls improved 1 to 2 points.

[0148] Example 11: Improved neural repair in pigs with spinal cord contusion receiving fibrin hydrogel with SB743921 and carbon microfibers, combined with anodal-onset biphasic electrical pulses.

[0149] In Example 10, biphasic electrical pulses were always applied starting with the negative (cathodal) phase. An additional group of pigs was used to determine whether the neuroregenerative effect is obtained exclusively with this electrical polarity, or whether it can also be achieved with biphasic electrical pulses starting with the positive (anodal) phase. The other experimental conditions were exactly the same as those described in Examples 9 and 10. As illustrated in Fig. 12, ES starting with the anodal phase also induced hypertrophy of the perilesional neural tissue and abundant axonal regeneration through the medullary cavity, connecting one side of the spinal cord to the other. No adverse effects attributable to the treatment were recorded.

[0150] Example 12: Antifibrotic and neural growth facilitating effect of fibrin hydrogel / SB743921 implant with other support material, in a cervical medullary hemlsection with excision of neural tissue in pigs.

[0151] In severely damaged areas of the nervous system, for example, in large breaks in neural tissue due to laceration, abrasion, or massive compression, it may be necessary to use additional support materials for the drug-eluting hydrogel to reinforce its mechanical strength or provide additional guidance for tissue ingrowth, or sites to anchor the implant to host structures.

[0152] This example demonstrates that fibrin / SB743921 hydrogel formed in vitro within and implanted with a second material, in this case a silicone tube, is useful in limiting fibrosis and secondary tissue damage, and initiating axonal growth responses following open porcine spinal cord injury, similar to a laceration with loss of neural tissue.

[0153] A porcine cervical spinal cord hemisection model was used with removal of 1 cm of neural tissue from the C6 cervical spinal segment, recently described (Cerro PD, Barriga-Martín A, Vara H, Romero-Muñoz LM, Rodríguez-De-Lope Á, Collazos-Castro JE. Neuropathological and motor impairments after incomplete cervical spinal cord injury in pigs. J Neurotrauma. 2021; 38:2956-2977).

[0154] For this test, 6 pigs were used, which were distributed into two experimental groups:

[0155] 1) Spinal cord injury and implantation of silicone tube with drug-free fibrin hydrogel (n - 3).

[0156] 2) Spinal cord injury and implantation of silicone tube with fibrin hydrogel formed with SB743921 (n - 3).

[0157] Fibrin containing SB743921 gelled in vitro within a tube with a morphology similar to the C6 segment hemi-medulla, the wall of which was a flexible medical-grade silicone membrane approximately 100 μm thick. The concentrations of the compounds stated in Examples 6 and 9 were used for the hydrogel and the drug.

[0158] The implant was introduced immediately after creating the cavity in the spinal cord, and the dura mater was subsequently sutured to hold it in place. One month after injury, the animals received intravenous anesthesia to surgically expose the cervical cord. Subsequently, they were given a lethal dose of pentobarbital (120 mg / kg IV) and the C1 to T2 spinal cord segments were removed. The tissue was fixed and processed following the methodology described in Example 10. Neural damage, tissue cavitation, axonal and glial growth, and invasion of the lesion by fibrotic connective tissue cells were assessed. For immunohistochemistry, the same procedures and antibodies described in Examples 1, 3, and 10 were used.

[0159] As illustrated in Fig. 13, the implant remained in place, and the hydrogel inside the silicone tube only partially degraded in both groups. The implant without the drug produced tissue damage and marked fibrosis that was impenetrable to regenerating axons. Bundles of dense fibrotic tissue 1 to 2 mm thick, originating mostly in the meninges, formed across the spinal cord stumps and also extended around and within the silicone tube, blocking any possibility of neural growth. In some cases, contraction of the fibrotic scar apparently widened the silicone tube, causing compression of the neural tissue and further damage.

[0160] The tissue response was much better in animals that received the SB743921-eluting implant. In this case, fibrosis was markedly reduced, being eliminated in some locations and, in others, showing a residual thickness approximately 20% of that observed in animals with fibrin without the drug. Furthermore, axons reached the tissue edge and began to grow into the most peripheral portion of the hydrogel (Fig. 13). The reduced fibrotic scar did not contract, so there was no compression or tissue damage due to deformation of the silicone tube.

[0161] Example 13: Fibrin hydrogel implant with SB743921 and an interconnected assembly of electroconductive microfibers, combined with epidural electrodes in a spinal cord hemisection with neural tissue excision in pigs. Finally, 4 pigs received cervical spinal cord hemisection and 1 cm of neural tissue excision using the methodology described in Example 12, in order to evaluate the feasibility of performing the fibrin hydrogel / SB743921 implant containing an assembly of interconnected microfibers for electrical stimulation, aligned longitudinally of the spinal cord. Additionally, epidural electrodes were sutured to the dura mater, adjacent to the injury site, to complete the electrostimulation circuit and to measure the resulting electrical parameters.

[0162] Using the methodology described in Example 6, the conductive polymer PEDOT:PSS-co-MA was deposited and functionalized onto a carbon microfiber assembly interconnected by a 12.8 pM diameter metallic microwire (AXON' Cable SAS, France) (Fig. 14A). The microwire was insulated with silicones once the carbon microfibers were joined. The microfibers were trimmed to a final length of 12 mm prior to polymer electrodeposition. The electrical functionality of the assembly was evaluated before and after electrodeposition, as well as after inclusion in the fibrin hydrogel, confirming the increase in charge transfer due to the polymeric coating of the microfibers. Prior to introducing the implant into the tissue defect, the area was washed with a 5 pM SB743921 solution and left to act for 10 minutes. These pigs were also given 4 subcutaneous doses of matinib (1.4 mg / kg), every 12 hours starting 8-12 hours after the injury.

[0163] The preformed fibrin hydrogel / SB743921 / microfiber assembly was successfully implanted into the tissue defect (Fig. 14B) without losing electrical interconnections. After implantation and completion of durorrhaphy, stainless steel electrodes (3 mm diameter discs) were sutured onto the dura mater, approximately 5 mm rostral and caudal to the lesion edges (Fig. 14C). No adverse effects due to the implant were observed. Complete inhibition of fibrosis around the interconnected microfiber assembly was observed, as well as partial closure of the tissue gap 1 month after injury.

Claims

CLAIMS 1. A volumetric, electroactive, drug-eluting implant comprising: a) a hydrogel of natural or synthetic polymers selected from the list comprising: polypeptides, fibrin, fibronectin, collagen, proteoglycans, glycosaminoglycans, alginate, chitosan, acrylamide, methacrylate, hydrogelatin, derivatives and combinations of these materials; b) at least one drug incorporated into the above hydrogel, wherein one of the drugs is a compound that blocks or inhibits the function of kinesins, or that reduces their expression; c) a set of electrodes for applying electrical stimuli to the injured tissue, wherein at least one electrode is positioned within or adjacent to the hydrogel.

2. Implant according to claim 1, wherein the compound that blocks or inhibits the function of kinesins, or reduces their expression, is an Eg5 kinesin inhibitor.

3. Implant according to claim 2, wherein the Eg5 kinesin inhibitor is the drug SB743921.

4. Implant according to any of the preceding claims 1 to 3, wherein the set of electrodes (c) is located inside the hydrogel.

5. Implant according to any of the preceding claims 1 to 4, wherein electrodes positioned within the hydrogel comprise or consist of: carbon microfibers, carbon nanostructures or microwires of metals or oxides, composites or metal alloys.

6. Implant according to any of the preceding claims 1 to 5, wherein the electrodes are coated with conductive polymers.

7. Implant according to claim 6, where the conductive polymer that coats the electrodes is selected from poly(3,4-ethylenedioxythiophene) (PEDOT), polypyrrole or polyaniline.

8. Implant according to any of the preceding claims 1 to 7, wherein the surface of the electrodes is functionalized with at least one molecule with biological activity to assist neural growth responses on or around the electrode.

9. Implant according to claim 6 or 7, wherein the conductive polymer is functionalized with at least one molecule with biological activity to help neural growth responses on or around the electrode.

10. Implant according to any of claims 8 or 9, wherein the molecule with biological activity used for the functionalization of the electrodes or the conductive polymer is selected from the list comprising: cell adhesion molecules, extracellular matrix molecules, growth factors, netrins, ephrins, semaphorins, protocadherins or Slit or Wnt proteins, morphogens, proteoglycans, glycosaminoglycans, gangliosides, proteins, peptides or combinations thereof.

11. A method for preparing an implant defined according to any of the preceding claims 1 to 10 comprising preparing a fibrin hydrogel by mixing: -a first aqueous solution containing human fibrinogen at concentrations between 0.5 and 100 mg / ml and factor XIII between 3 and 300 U / ml; and -a second aqueous solution containing thrombin at concentrations between 3 and 300 U / ml and CaCh at concentrations between 0.26 and 26 mM, wherein at least one of these solutions contains a drug that blocks or inhibits the function of kinesins, or that reduces their expression.

12. Method according to the preceding claim 11, wherein the mixing of the hydrogel precursor solutions is carried out in vitro.

13. Method according to any of claims 11 to 12, wherein at least one of the hydrogel precursor solutions contains the electrode assembly or at least one electrode.

14. Method according to the preceding claim 13, wherein the drug is previously encapsulated in particles, vesicles, liposomes or the like in order to increase its retention or improve its activity, and is then incorporated into the hydrogel precursor solutions.

15. Method according to any of claims 11 to 14, wherein additionally the selected electrodes of microfibers, microwires or other elements are incorporated into the aligned electrodes in the hydrogel, during the gelation process.

16. Implant as described in any of claims 1 to 10, for use in the repair of neural tissue or the nervous system.

17. An implant for use according to claim 16, by means of a surgical procedure comprising the following steps: a) cleaning the area of neurological injury of cellular debris, blood and fibrotic adhesions; b) implanting the hydrogel in the cleaned area of neurological injury according to step a) during gelation when this occurs in vivo, or after gelation when this occurs in vitro; c) positioning at least one electrode adjacent to the hydrogel, when no electrodes have been incorporated into the implanted hydrogel in step b, or electrodes have not been implanted in the area of neurological injury prior to step b; d) supplying the electrical stimuli through the electrodes.

18. An implant for use according to claim 17, wherein the injured area is washed and impregnated, in addition to step a), with a solution containing the same pharmacological compound that blocks or inhibits the function of kinesins, or reduces their expression; present in the hydrogel.

19. Implant for use according to any of claims 17 to 18, wherein the hydrogel is formed with the pharmacological compound in vitro and is subsequently implanted in the cleaned neurological injury area according to step a).

20. Implant for use according to the preceding claim 19, wherein the hydrogel with the pharmacological compound is prepared within a second implantable material, and the resulting composite implant is introduced into the damaged region of the cleaned nervous system according to step a).

21. Implant for use according to any of claims 17 to 18, wherein the hydrogel is formed with the pharmacological compound in vivo, such that the gelation occurs directly in the area of neurological injury cleaned according to step a).

22. Implant for use according to any of claims 17 to 18, wherein the hydrogel is prepared in vitro with the pharmacological compound, and electrodes are additionally introduced during gelation, and subsequently implanted in the area of neurological injury according to step b).

23. Implant for use according to any of claims 17 to 18, wherein electrodes are implanted in the area of neurological damage conditioned in step a), and subsequently the hydrogel with the pharmacological compound is formed in vivo according to step b), surrounding the implanted electrodes.

24. Implant for use according to any of claims 17 to 18, wherein the hydrogel with the pharmacological compound is formed in vivo within the tissue defect according to step b), and electrodes are positioned adjacent to said hydrogel, within the nervous system or on the membranes that surround the nervous tissue.

25. Implant for use according to any of claims 16 to 24, wherein biphasic electrical stimuli or monophasic electrical stimuli are applied through the electrodes of the implant.

26. Implant for use according to any of claims 16 to 25, wherein electrical stimuli are applied through the electrodes that begin or consist of an electrical current of positive polarity or negative polarity.

27. Implant for use according to any of claims 16 to 26, wherein electrical stimuli are applied through the electrodes in which the intensity of the applied electrical current, in absolute value, is between 1 nanoampere and 20 milliampere.

28. Implant for use according to any of claims 16 to 27, wherein electrical stimuli are applied through the electrodes in which the frequency is between 0.00001 Hz and 100000 Hz.

29. Implant for use according to any of claims 16 to 28, wherein said implant is used in combination with additional pharmacological compounds administered locally or systemically to facilitate implant integration and neural repair.

30. Implant for use according to the preceding claim 29, wherein the pharmacological compound is selected from an anti-inflammatory, antifibrotic, antibiotic, neuroprotective drug, and a neural growth facilitator.

31. Implant for use according to the preceding claim 30, wherein the pharmacological compound is a receptor tyrosine kinase inhibitor selected from imatinib, sunitinib, sorafenib, masitinib, dasatinib, nilotinib, gefitinib, erlotinib, lapatinib, and tofacitinib.

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