MXPA01002231A - PHARMACEUTICAL COMPOSITIONS COMPRISING FACTOR XIIIa - Google Patents

Abstract

Use of Factor XIIIa for the preparation of pharmaceutical compositions for nerve healing, preferably for administration locally at a site of injury or disease.

Description

PHARMACEUTICAL COMPOSITIONS COMPRISING THE XIHa FACTOR FIELD AND BACKGROUND OF THE INVENTION The present invention relates to pharmaceutical compositions comprising as the active ingredient Factor XI I, in particular for nerve healing. The purpose of intensive research in the last two decades has been to discover the reasons why the central nervous system (CNS) of mammals do not regenerate after a white matter lesion, while CNS lesions of lower vertebrates (for example, fish) or the peripheral nervous system (SNP) of mammals have a successful recovery. As is well known, the environment of the damaged CNS is inhibitory to regeneration (1, 2), and the degeneration of damaged axons triggers processes that lead to the secondary degeneration of the neurons that escaped the initial lesion (3, 4) . It was shown that modulation of the CNS environment resulted in axonal regeneration both in vitro and in vitro (5, 8), confirming that the inability of the mammalian CNS to reach functional recovery after injury does not necessarily constitute a reflection of the intrinsic property of the neurons themselves. A prerequisite for the recovery of any damaged tissue is effective communication between damaged tissue and the immune system (9, 10). A growing body of evidence indicates that the interaction between the immune system and the CNS is restricted under normal physiological conditions, and that this restriction manifests itself from the earliest stages after CNS injury (11). There does not appear to be a similar restriction in the peripheral nervous system (PNS) of mammals or in the central nervous system of lower vertebrates. The observations of the inventors herein in accordance with the above findings are that regeneration in the nervous system of fish correlates with the activity of inflammatory cells and their products (12). It is thought that among the activities that are associated with inflammation is an enzyme of the transglutaminase family (TGase) (13). Factor XIII of blood coagulation (hereinafter FXIII). which is also known as fibrin stabilizing factor, fibrinase and Laki-Lorand factor, is found in plasma as a tetrameric protein and consists of two types of subunits (subunits a2b2), of which subunits (a2) are potentially active (hereinafter FXIIIa). FXIII lacks subunits-b and is a homodimer (a2) of subunits-a. The FXIII plasma circulates in association with its precursor substrate, fibrinogen. In the last stage of the coagulation processes, prothrombin is activated towards thrombin by its segmentation, which catalyzes the segmentation of both fibrinogen and FXIIIa, which makes them more susceptible to complex formation. The dissociation of subunits a and b of FXIII leads to the exposure of the active site originally buried in the free subunits. Immediately thereafter, the a2 subunit dissociates from the b2 dimer to form the active FXIIIa enzyme that catalyzes the crosslinking of fibrin in the presence of Ca2 +. The α-subunit of FXIIIa belongs to a family consisting of several genes that encode enzymes with a common structure of active sites, well conserved between species (17). It is widely known that FXIII plays a role in the coagulation of blood (14-16). FXIII deficiency produces a clinical hemorrhagic diathesis. FXMI is an essential component of fibrin sealant that is used clinically in the surgical procedure for wound healing and tissue repair. Cumulative evidence points to an additional function of FXIIIa in the proliferation of connective tissues through its expression by invading macrophages (18, 19).

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention it was discovered that some characteristics of the α-subunit of FXIIIa in fish plasma are different from those of their counterparts in mammals. The 55-kDa form in which the FXIIIa enzyme is constitutively expressed in the plasma and nervous tissues of fish indicates the presence of activation and expression regulation states other than those of the 80-kDa form in which the FXIIIa it is expressed in mammals. Variations induced by lesions in the subunit-a expression of FXIIIa in the regeneration and non-regeneration of nerve tissues were also investigated. The present invention relates to the use of Factor Xllla for the preparation of a pharmaceutical compound useful in the healing of nerves. FXIIIa can be the natural enzyme available commercially and purified from human plasma or recombinant FXIIIa. The pharmaceutical composition can be presented in any form suitable for the administration of Factor Xllla, preferably in the form of sterile, freeze-dried powder, suspended in a sterile, pharmaceutically acceptable carrier, such as water or a saline solution with pH regulated with phosphate ( PBS), for local injection at the site of damage or illness. The amount of FXIIIa that should be administered will depend on the type of injury or illness, as well as the age and condition of the patient, which will be determined by a qualified physician, but preferably will be in the range of 0.1 -UU / kg of body weight. . The invention further relates to a method for nerve healing consisting of administering a therapeutically effective amount of Factor Xllla in the CNS of a patient requiring it, preferably at the site of the injury or disease.

DESCRIPTION OF THE DRAWINGS Figures 1A-1B depict a Western blot analysis of FXIIIa in fish and rats. 1A. Optical nerves of fish and rat sciatic nerves were incubated in a culture medium for 1.5 hours. The collected culture media was analyzed by SDS-PAGE and its immunoreactivity was examined with rabbit polyclonal antibodies raised against the subunit of FXIIIa in humans. 1 B. The immunoactivity of the α-subunit of FXIIIa in white blood cells (GB) of fish and in monocytes (M) was compared with that of white blood cells and plasma of rats. Note the 55-kDa protein detected in the fish against the 80-kDa protein in the rats. The product of the cleavage (55-kDa protein) of plasma FXIIIa in rats is similar in size to the protein detected in fish. Figure 2 depicts the loss of the immunoreactivity in the FXIIIa subunit-a and formation of precipitates in conditioned culture media of fish optic nerve by gradually adding thrombin. A conditioned culture medium of fish optic nerve was prepared according to the description. Conditioned culture media were subjected to complex formation in the presence of increasing amounts of human thrombin (Omrix Biopharmaceutical, Brussels, Belgium) for 1 hour at room temperature, and to centrifugation for 15 minutes at 4 ° C. Floating particles were collected and subjected to the SDS-PAGE test, and to the Western blot analysis using antisubunit-a antibodies. Note the reduction in the 55-kDa immunoreactive protein level in the presence of high concentrations of thrombin. The upper band is not the result of immunoreactivity, but of an overload of human thrombin, since it also appears in the Ponceau stain in the absence of conditioned culture media (data not shown). Figures 3A-3B depict the immunoreactivity of the α-subunit of FXIIIa and crosslinking activity in the plasma of rats and fish. Plasma (P) and serum (S) samples from fish and rats were obtained, obtained as described in the materials and methods section in search of the presence of FXIIIa immunoreactivity subunit-a by Western blotting according to SDS-PAGE in non-reducing conditions (3A). The linkage activity of FXIIIa in plasma and serum was measured by the incorporation of [3H] of putrescine into casein N, N-dimethylated (3B). Figure 4 depicts the crosslinking activity of plasma and conditioned culture media of nerves as a function of thrombin concentration. Culture media conditioned by optic nerve of fish or sciatic nerve of rats with or without heparin, as well as plasma and serum from fish or rats were analyzed for their cross-linking activities in the absence or presence of thrombin (10 U / ml). Note the low cross-linking activity of rat plasma relative to fish plasma in the absence of exogenous thrombin. Figures 5A-5C depict characterization of rat nerve FXIIIa. The subunit-a of immunorectivity of FXIIIa in the various preparations of nerves, as a function of incubation in nerve time in the preparation of conditioned culture media, is illustrated in panel (5A). The optic nerve conditioned culture medium of fish, the rat optic nerve conditioned culture medium and the TGase-purified rat tissue were subjected to the SDS-PAGE test under reducing conditions, and to the Western blot analysis by means of rabbit polyclonal antibodies against the FXIII subunit-a or rabbit polyclonal antibodies and purified against rat tissue type TGase (5B). In order to conform the specificity of the antibodies directed against FXIlla, the analysis of plasma and rat serum and of fibrogamine-p were also made as FXIII of human plasma (Behring, Marburg, Germany) under the same experimental conditions (5C). Figures 6A-6B depict the immunoreactivity of subunits a and b of FXIII in intact and injured sciatic nerve in rats. Rat sciatic nerves were excised and crushed on different days after the injury. Culture media conditioned by intact or damaged nerves (post crushing, PT) were analyzed by SDS-PAGE, and immunoreactivities were tested on subunit-a (6A) and subunit-b (6B). Note that the levels of the subunit-a are higher in the intact nerve, whereas the subunit-b was detected mostly one day after the injury. Fibrogamine-P as FXI 11 from human plasma was used as a positive control. Figures 7A-7B represent the comparative analysis of the crosslinking activity of FXIIIa in culture media conditioned by rat or fish nerves after axonal injury. Sciatic and optic nerves of rats and optic nerves of fish were excised and crushed on different days after the injury. Culture media conditioned by these nerves was analyzed to establish cross-linking activity as described. 7A. Sciatic and optic nerve in rats. 7B optic nerve in fish.

DETAILED DESCRIPTION OF THE INVENTION Recent findings have changed the traditional concept of nerve recovery, including the notion that injured nerves, like any other damaged tissue, require the help of cells and blood-derived factors to heal. In the present application, the inventors of the present show that factor Xllla (FXIIIa, the active a2 subunit of Factor XIII), an enzyme that participates in the coagulation of blood by stabilizing the fibrin clot, is also active in the nervous system where it plays a decisive role in the healing of injured tissue. It is also shown that plasma, macrophages and fish nerves contain a 55 kDa form of transglutaminase (TGase) that cross-reacts immunologically with the α-subunit of FXIII in mammals (80 kDa). The fish enzyme in plasma, unlike its counterpart in mammals, is active, and indicates a difference in the control of the coagulation pathway in both species. The analysis of the expression of FXIIIa in mammalian neural tissues and its response to injury revealed high levels of the enzyme in culture media conditioned by peripheral nerves, in comparison with culture media conditioned by nerves of the central nervous system. Furthermore, similarity was observed in the post-injury behavior of FXIIIa as the nervous tissues (peripheral nervous system) of the mammal and central nervous system of fish regenerated). This indicates that the level after the injury of Factor Xllla in the nervous system can be associated with the regenerative capacity of the tissue, and that FXIIIa can, therefore, constitute a link that supports a possible association between the blood coagulation processes and nerve healing. Until very recently, the CNS was considered as a unique tissue, and it was not thought that the rules that governed the healing of tissues in general were applicable to the CNS. A growing knowledge of the processes involved has led to this concept being modified. For example, the accumulation of a body of information suggests that the components of the inflammatory response and blood coagulation can play a crucial role in the healing of the CNS, as well as in the healing of other tissues. In accordance with the present invention, it is demonstrated that FXIIIa, a component of the coagulation process, is present in the nervous tissue, and evidence is provided to establish a correlation between the appearance after the injury and the activation of the enzyme and the regenerative capacity of the tissue. It also shows that the activation of the coagulation pathways and wound healing factors is regulated differently in fish and rats.

FXIII is an inactive tetrameric complex consisting of subunits a2 and b2, which may be associated in circulating blood. The levels of subunits b2 are higher than those of subunits a2, in such a way that the formation of free FXIIIa, susceptible to activation by Ca2 + and / or thrombin, is prevented. Thrombin, a product of prothrombin cleavage, is considered to play a central regulatory role in the formation of blood clots by concomitant activation of fibrinogen and FXIIIa. Here it is illustrated that FXIIIa is undoubtedly inactive in rat plasma, unless triggered by thrombin to initiate the process of complex formation. In contrast, FXIIIa in fish appears to be active not only in the presence but also in the absence of exogenous thrombin. It was observed that the dominant subunit of the FXIIIa immunoreactive protein in the fish had a molecular size of 55 kDa. Its presence in monocytes, white blood cells and fish plasma is similar to that reported for the α-subunit of FXIIIa in mammals (16, 20, 24). The low molecular weight of the enzyme in fish seems to reflect, at least partially, the state of activation of the blood coagulation route in fish relative to that of mammals. Therefore, it appears that fish constitutively express a form of low molecular weight FXIIIa, which is in a higher activation state than the 80-kDa protein in the FXIIIa of mammals required for cross-linking activity. However, as illustrated in this application, the fish enzyme can be activated by thrombin. This phenomenon can occur due to the nature of the regulatory mechanism in fish. The formation of clots in vitro certainly occurs faster in the blood of fish than in the blood of mammals. It is also possible that other members of the TGase family contribute to the crosslinking activity in the plasma. If so, it would be expected that there were high levels of cross-linking activity in the serum, which does not happen. The high activation condition of the blood coagulation protein in fish may reflect the evolutionary transition from an open blood system to a closed one in vertebrates, or adaptation to environmental conditions. A disadvantage of said response regulation in fish is that it is not limited to the region of the lesion, but is systemic and, therefore, increases the susceptibility for the random formation of clots, a process that is fatal. In addition to its role in blood coagulation, FXIIIa appears to consist of the extracellular TGase that participates in the healing of wounds (16, 19, 20). The tissue of the peripheral nervous system of mammals (PNS), according to the present invention, seems to respond very effectively to the lesion, as illustrated, for example, by the fact that FXIIIa becomes fully active ( example, it could not be further activated by the addition of thrombin), as a result of the excision of the sciatic nerve. This finding follows the line of other studies that indicate the presence and activation of thrombin in the nervous system of mammals as a response to injury. In sites of the same, thrombin regulates the expression and development of neurons and astrocytes (25, 26). However, it was shown that high concentrations of thrombin were toxic for both astrocytes and neurons (27). The cellular source of FXIIIa found in nerve tissue may consist of macrophages residing in normal nervous tissue (eg, microglia and / or invading macrophages after injury). The lack of correlation between the levels of the a2 subunit and the b2 subunit during wound healing in the peripheral nervous system of rats suggests that the enzyme found in the conditioned media of the tissue is derived only partially from the blood and that part This may consist of a local product of invasive resident cells or that the subunit-a experience intense consumption immediately after the injury. The optic nerve of the rat, as part of the central nervous system in mammals (CNS), does not regenerate after the injury, and axons that may have escaped the primary lesion eventually become victims of the hostile environment that formed axons in degeneration. Previous studies have shown that the 55-kDa enzyme belonging to the TGase family is present in the regenerating nerves of fish and that it plays a decisive role in recovery when injected into the transversally cut optic nerve of mammals ( 29). FXIIIa of the nerve may have some function in the cross-linking of fibrin and other components (14, 16, 17), thereby stabilizing the extracellular matrix and making it more effective by creating local concentrations of trophic factors, growth factors and cytokines. . Tgasa of the nerve may be effective in cytokines and in cross-linking growth factors. The following non-limiting examples will now illustrate the invention.

EXAMPLES Materials and methods Preparation of conditioned media of nerves. Carps (Cypirinus carpio, 800-1200 g) were anesthetized with 0.05% 3-aminobenzoic acid ethyl ester (Sigma, Israel), and rats (Wistar, 8-week old males) were anesthetized with 0.2 ml of mixed ketamine (Rhone Mericux). with 0.2 ml of 2% xylazinc (Vitamed, Israel). Optical means and optic nerves and sciatic nerves of rats (30 seconds) were crushed with forceps. The nerves were excised at different times after damage and incubated in a serum-free medium for 1.5 hours at room temperature. The resulting conditioned media were collected, centrifuged at 15,800xg for 5 minutes to remove tissue fragments, and the floating particles were collected and stored at 20 ° C.

Plasma preparation The peripheral blood of the rats' heart was removed with a 10 ml syringe coated with heparin (5000 u / ml, Carbiochem, La Jolla, CA, USA) containing 100 μl of heparin. In the fish the carotid artery was cut and the peripheral blood was collected from the eye cavity using a Pasteur test tube coated with heparin (10000 u / ml), in 2 ml tubes with 2 or 3 drops of heparin. The tubes were placed on ice, after centrifugation at 15,800 xg for 15 minutes at 4 ° C and the floating particles were collected.

Preparation of the serum The serum was prepared by the procedure described for the plasma, but without heparin. The samples were incubated at room temperature for 15 minutes (fish) 30 minutes (rats), placed on ice for the same period of time, centrifuged at 15,800 xg for 15 minutes at 4 ° C, and the floating particles were collected.

Purification of white blood cells Peripheral white mononuclear cells were obtained by fractionation of one-step Percol gradients (Pharmacia). The blood was removed as described for the plasma preparation, diluting it to 1: 1 with saline with pH regulated with warm phosphate, incubated for 5 minutes at room temperature, and then subjecting it to Percol fractionation (1077 g / ml ) (Pharmacia, Sweden). The blood mixture of Percol was processed in a Sorvall spinning centrifuge at 800xg 25-30 ° C for 25 minutes. The enriched fraction of monocytes was isolated from the interface, washed twice with saline with pH regulated by phosphate to remove the perforation traces. For the preparation of white blood cell homogenates, the cells were harvested by centrifugation, resuspended with pH regulation by extraction (10 mM Tris-7.5, 150 mM NaCl, 1% Triton, 1 mM EDTA pH 8.0, and protease inhibitors) and incubated for 2 hours at 4 ° C. After centrifugation for 5 minutes at 15,800xg at 4 ° C, the floating particle was collected. For the preparation of monocyte homogenates, the cells were resuspended in L15 medium and incubated in a 3 cm petri dish for 1 hour at room temperature. Adherent cells were washed several times with saline with pH regulated with phosphate and incubated with 500 μl of regulated pH by extraction for 2 hours at 4 ° C. After centrifugation for 5 minutes at 15,800xg at 4 ° C, the supernatants were collected and stored under freezing.

TGase activity tests The activity of TGase was checked by measuring the incorporation of putrescine in N, N-dimethylated casein. The reaction mixture contained 50 mM Tris-HCl, pH 8.0 or pH 9.0 for culture media of rats and fish respectively, 5 mM DTT, 5 mM DTT, 5 mM CaCl2, 0.075 μM putresin [3H] (38.7 Ci / mmole ) (DuPont NEN) and 4 mg / ml of N, N-dimethylated casein (Sigma). The reaction mixture was incubated for 0.5 hour or 1 hour at 37 ° C, and then placed on ice. Cold trichloroacetic acid (TCA) was added to a final concentration of 5% for 15 minutes. Samples were centrifuged (14000xg for 5 minutes at room temperature and the pellet was washed twice with 1 ml of 5% TCA and once with 100% ethanol.) Samples were resuspended and resuspended in 200 μl of 0.1 N NaOH. was measured in 10 ml of scintillation fluid (40% lumax, 60% xylene) All activity tests were performed at least three times, each experiment in triplicate Each activity is presented on average ± SEM of an experiment.

Western blot analysis The various preparations (detailed in the examples) were subjected to SDS-PAGE using 10% gel slices with acrylamide. After electrophoresis, the proteins were transferred to a nitrocellulose membrane for 2.5 hours at 200 mA (in Tris-glycine). The immunological reaction was carried out in the following manner: the spot was incubated at 4 ° C | overnight in phosphate buffered saline containing 5% milk thinner, and then with the antibodies (diluted 1: 1000 in saline with pH regulated with phosphate containing 5% milk thinned for 2 hours at 37 ° C. Afterwards, different washings were carried out with phosphate-buffered saline solution containing 0.05% Tween-20, incubation with 1: 1000 of goat anti-rabbit IgG (Jackson) conjugated with alkaline phosphatase for 1 hour at room temperature, several washes with phosphate buffered saline containing 0.05% Tween-20, and developed by an ECL detection system (Amersham) for 1 minute.Each Western blot analysis was repeated at least three times.

Antibodies For the immunodetection of the subunit-a protein, polyclonal rabbit antibodies directed to the human subunit of FXIH have been used (Centeon Pharma GmbH, Marburg, Germany) (20); for the immunodetection of the subunit-a rabbit polyclonal antibodies directed to the human subunit of FXIII (Calbiochem) have been used.

EXAMPLE 1 A 51-kDa protein was shown to be an active cleavage product of the human FXIIIa Factor subunit-a in vivo (21). To test the possibility that a 55-kDa protein, purified from the optic nerve in fish regeneration and suggested as a member of the TGase family (13. PCT Publication WO 94/03059), as a form of FXIIIa subunit-a , the inventors of the present have analyzed a conditioned optical culture medium of fish for their reactivity with antibodies directed to the subunit of FXIIIa. As shown in Figure 1A, Western blot analysis of the optic nerve conditioned medium in the fish showed a single immunoreactive band of 55 kDa, instead of the expected 80 kDa protein in the sciatic nerve conditioned medium of the rat. Monocytes and platelets, which are potential sources of FXIIIa in the plasma and in the injured tissue (20,22,23), were also analyzed for the presence of this immunoreactive protein.

Western blot analysis revealed the presence of these 55 kDa immunoreactive proteins in the extract derived from both monocyte media and white blood cells in the blood (Fig. 1 B). No immunoreactive signal was obtained when the conditioned media of the optic nerve of the fish were subjected to Western blot analysis by polyclonal rabbit antibodies, in contrast to TGase-type fish tissues (the data are not shown), and further suggest that The 55 kDa protein is the dominant form of the enzyme TGase in the conditioned medium of the optic nerve of fish. Under the same experimental conditions, white blood cells and rat plasmas exhibited, as expected, the known 80 kDa subunit of FXIIIa. In the rat plasma a 55 kDa protein was also detected that had an immunological response with the antibodies against the subunit-a, perhaps as a result of a second thrombin cleavage.

EXAMPLE 2 To find out whether the 55 kDa protein in the conditioned media of fish participates in fact in the formation of clots and, therefore, if it is a cross-linking enzyme, its amount was examined after adding thrombin and the resulting formation of precipitates. The addition of increasing amounts of thrombin in the conditioned media resulted in a gradual decrease in the amount of 55 kDa immunoreactive protein in the supernatant (Fig. 2), with the concurrent appearance of visible precipitates. The findings point to the formation of a precipitable complex that involves the 55 kDa protein. This situation is evocative of the coagulation of the plasma, where the resulting serum is devoid of coagulation components. These results therefore support the suggestion that the 55 kDa protein is a form of FXIIIa subunit-a and that thrombin can potentially activate it to a cross-linking activity.

EXAMPLE 3 To continue examining the possibility that the 55 kDa protein found in the conditioned medium by the optic nerve of fish is a form of existence of FXHIa in fish plasma (more than the 80 kDa form in other species), we compared the subunit -a, detected by immunoreactivity, in plasma and serum. Analysis by SDS-PAGE, in the absence of boiling and reduction conditions, with subsequent Western blot analysis (Fig. 3A), showed the presence in the plasma of fish of a shorter form of the immunoreactive protein of the subunit-a of FXIIIa, of molecular weight similar to that found in the preparation of the optic nerve of fish, is also present in fish plasma. Under non-reducing conditions, the molecular size of the complex in the fish is -240 kDa, more than 320 kDa as in the rat. It is expected that in the amounts of both the subunit-a and the complex (which probably consists of a2b2 subunits), it would have decreased in the fish serum samples, presumably due to the consumption of FXIIIa in the coagulation process (Fig. 3A) . Detection of a-subunit in the monomeric form requires complex activation; in the plasma, this process is blocked by the presence of heparin. In our experiment, the α-subunit of FXIIIa, which represents the active enzyme, was detected in the plasma of fish but not in that of rats. The 160 kDa in the plasma of rats represent dimer a2 in a non-active state. After activation of the blood agglomeration components, the a2 form of the dimer is no longer detectable as in the serum. The different intensity of the immunoreactive bands between the plasma of rats and fish appears to be a consequence of specificity in the antibodies. These results suggest that the 55 kDa protein is the main form of the α-subunit in the plasma FXIIIa of fish. They also suggest that if the 55 kDa protein is a segmentation product of an 80 kDa precursor, its formation occurs immediately at the time of production and / or secretion.

The cross-linking activity characteristic of FXIIIa was examined in the serum of fish and rats. Virtually no crosslinking activity could be detected in the plasma of rats under physiological conditions obtained by the addition of heparin. Activity levels in rat serum, which is thought to contain only very low levels of blood coagulation components were, as expected, even lower. In contrast, intense activity was observed in the plasma of the fish (Fig. 3B). The addition of thrombin, known for its function of activating FXIIIa, led to a 10-fold increase in enzyme activity in the plasma of the rats (Fig. 4A), while the increase in the plasma of the fish was only three times (Fig. 4B). This finding suggests that the lower activity in rat plasma in the absence of added thrombin can be attributed to the presence of the enzyme in an inactive form, which may not happen in the case of fish (Fig. 4). Interestingly, the addition of thrombin did not cause any elevation in activity in the conditioned medium by rat sciatic nerve, suggesting that in this medium thrombin is present or the enzyme exists in some form, since, unlike plasma, it does not require additional activation with thrombin (Figure 4). The above findings suggest that FXIlla activity is regulated differently in fish and rats.

EXAMPLE 4 Expression of FXIII after lesions of the nervous system The differences in form and regulation of FXIII observed between the plasma of fish and that of rats promoted the examination of the amount and activity of FXIIIa in the nervous system in response to the injury. The sciatic and optic nerve conditioning devices were prepared as described in the materials and methods section, and followed by Western blot analysis for the detection of the FXIIIa subunit. As shown in figure 5A, a subunit-a of immunoreactive protein was detected in conditioned media of both optic nerves and sciatic nerves. Its molecular size in both preparations was similar to that of FXilla that was found in mammalian plasma, for example 80 kDa (more than 55 kDa in the optic nerve of fish). It is interesting to note that the amount of enzyme in the sciatic nerve of the rats was considerably greater than in the optic nerve of the rats. The variation in the incubation period of between 1.5 and 4 hours did not affect the amounts of the enzyme in the conditioned media (Fig. 5A). To eliminate the possibility that the immunoreactivity of the FXIIIa subunit-a observed in rat nerve preparations is due to cross-reactivity with TGase-like tissue, known for its partial homology to the FXIIIa subunit-a, antibodies were used rabbit polyclonal specific for rat TGase type tissues. As illustrated in Figure 5B, antibodies contrasted with the FXIIIa subunit of α do not cross-react with TGase-like tissue, nor do antibodies to the TGase tissue type cross-react with the FXIIIa subunit-a. Moreover, the antibodies recognized the enzyme in the nerve conditioned medium in rats and in the plasma but not in the serum, with a molecular weight similar to that obtained in human plasma, which suggests the specificity of the subunit antibodies. -a of the FXIIIa (Fig. 5C).

EXAMPLE 5 To determine whether the FXIIIa that was found in the nerve tissue is derived exclusively from the plasma, or if at least in part it is produced locally by the nervous tissue itself, the inventors of the present performed Western blot analysis by antibodies against the subunit- b of FXIIIa, in addition to those against the subunit-a of FXIIIa itself. It is known that the b-subunit is associated with FXIII by the link between two homodimers, a2 and b2. If all the FXIIIa found in the nerve is derived from the plasma, the ratio between the a2 and b2 subunits in the nerve should be the same as in the plasma. It was observed that the immunoreactivity of the subunit-a decreased after the lesion, with the most acute gout on day 1 (Fig. 6A), day in which the amount of the b-subunit was higher (Fig. 6B) . It seems probable therefore that FXIlla is, at least partially, a product of nervous tissue.

EXAMPLE 6 To continue examining whether possible variations of damage induced in nerve-derived FXIIIa can be associated with the ability of the nerve to regenerate, we compared the activity of FXIIIa on nerves in regeneration and non-regeneration processes after injury of axon The analyzes revealed that cross-linking activities were greater in media conditioned by non-injured sciatic nerves than in media conditioned by non-injured mammalian optic nerves. Immediately after the injury there was a marked decrease in the activity of the enzyme. By day 4 after the injury, when the activity of FXIIIa in the sciatic nerve was greater than on day 1, the activity of the optic nerve in rats was still scarce (Figure 7A). This difference in FXIIIa levels between nervous tissue of the central nervous system and the peripheral nervous system of mammals, during the first days after injury, can be related to tissue performance, either for recovery or for regeneration. A good correlation was found between the immunoreactivities and the enzyme crosslinking activities of the two nerve preparations tested at all time phases after the injury. In the optic nerve of fish the cross-linking activity was elevated after the injury (Figure 7A). In the optic nerve of fish the cross-linking activity was elevated after the injury (Fig. 7B). It is interesting to note that these two preparations showed a similar behavior after the injury from day 1, and only differed with respect to the values of non-injury. This difference could be a reflection of the activation mode of FXIIIa in fish and rats.

EXAMPLE 7 Use of the human factor Xllla for healing of the transverse cross-sectional optic nerve Adult SPD rats, 8 to 10 weeks old, with an average weight of 300 g, are deeply anesthetized (xylazine with 5 mg / kg of xylazine and 35 mg / kg of ketamine) and their left optic nerves are exposed. Through a small opening in the meninges, the nerve fibers are cut transversely in their entirety 2 to 3 mm from the globe, without damage to the vasculature of the nerve and with minimal damage to the meninges. Immediately after the cross section of the nerve, 2 μl of the Xllla Factor is applied to the cutting site by means of a specially designed glass microprobe. In control animals, 2 μl of phosphate-buffered saline solution is applied at the site of the lesion. To test the axonal regeneration, the retrograde marking of the cells of the retinal ganglia was performed as follows. Neuroindicator fluorescent dye crystals were applied at the site of the cross section at the time of cutting and the application of 4-Di-10-Asp (Molecular Probes, The Netherlands). After 10 to 12 weeks, Fluoro-Gold (Fluorochrome) was applied 2 mm away from the cut site. The retinas were removed 4 days after the application of the second dye, and were examined under a fluorescence microscope with two filters, fluorescin for visualization of 4-Di-10-Asp and UV for Fluoro-Gold. By making a double mark of the cell bodies, this avoids the possibility of confusing the axons that had escaped from the cross section with the regenerating axons. The first dye, which is applied to the transversely cut nerve at the site of the lesion immediately after cutting, is absorbed by the severed axons and this classifies the retinal ganglion cells of the severed axons. This means that the cell bodies marked in the applications of the first and second dyes belong to regenerating axons that have already been cut a second time (for example, the first cut consists of the same injury and the second is the cut made to distance from the first in order to apply the dye). It should be noted that the dye transport capacity of regenerating axons may differ from that of the intact axons cut for the first time. In addition, as some retinal ganglion cells are likely to die during the period between the application of the second dye and the retinal cut, the doubly labeled cells should be considered as the minimum number of regenerating axons. It should be noted that each dye has its own stain efficiency and hence the different marking efficiencies must also be taken into account.

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Claims (4)

NOVELTY OF THE INVENTION CLAIMS

1. - A pharmaceutical composition for nerve healing comprising the factor Xllla and a pharmaceutically acceptable vehicle.

2. The pharmaceutical composition according to claim 1 in a form of local administration at the site of the injury or disease. 3.- The use of the factor Xllla for the preparation of a pharmaceutical composition for the healing of nerves. 4. The use according to claim 3 wherein the pharmaceutical composition is in a form for local administration at the site of the injury or disease