Patent Application
20060083734
1. Field of the Invention
The invention relates to a composition and a method for repairing a nerve damage and enhancing functional recovery of a damaged nerve.
2. Description of the Related Art
Nerve damage is usually caused by trauma or ischemia, and is difficult to repair. A vertebrate having nerve damage may suffer from motor deficits, paralysis, or even death.
Many strategies have been developed to repair nerve damage, one of which is the use of neurotrophic factors. Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β (TGF-β) superfamily, is considered the most potent neurotrophic factor that promotes survival and neurite outgrowth of dopaminergic neurons and motoneurons as well as peripheral sensory and sympathetic neurons. See Science, 260:1130-1132 (1993); Science, 266:1062-1064 (1994); Nature, 373:289-290 (1995); Nature, 373: 335-339 (1995); Nature, 373: 341-344 (1995); and Nature, 373: 344-346 (1995).
Middle cerebral artery (MCA) occlusion causes not only a wide range of infarction but impairment in motor performance. The neuronal damage in the territory of MCA, e.g., caudate putamen and striatum, will cause motor deficits. See Stroke, 28:2060-2066 (1997). GDNF gene expression was shown to be transiently induced in the ipsilateral cortex and caudate at the early stage of focal brain ischemia. See Brain Res., 776:230-234. Furthermore, the effect of GDNF on the changes of infarct size, brain edema, DNA fragmentation, and immunoreactivities for caspases after permanent MCA occlusion in rats has been investigated. See Stroke, 29:1417-1422 (1998). It is also reported that the topical application of GDNF on ischemic brain surface significantly reduced the size of infarction and the brain edema of reperfused rat brain after acute FCI injury. See Neurosci Lett., 231:37-40 (1997).
However, the topical application of GDNF is shown to reduce infarction size in a time-dependent manner, and the therapeutic time windows was shorter than other chemical compounds, such as MK-801, an NMDA (N-methyl-D-aspartate) receptor antagonist, and the free radical scavenger, alpha-phenyl-tert-butyl-nitrone (PBN). See Brain Res., 903:253-256 (2001).
The direct application of GDNF is not effective for some nerve damages, especially chronic nerve damages. For example, GDNF has not been effective against chronic FCI injury. See Hum Gene Ther. 13:1047-1059 (2002).
GDNF is reported to be able to be slowly released in physiologically relevant amounts over a long-acting period of time. See Exp Brain Res., 104:199-206 (1995); and Cell Transplant., 7:53-61 (1998). Nevertheless, slow release of GDNF cannot successfully treat chronic nerve damages, or enhance the functional recovery of nerves.
There remains a need for a means for effectively repairing nerve damages and in furtherance, enhancing the functional recovery of damaged nerves.
The purpose of the invention is to provide a method for effectively repairing nerve damages and in furtherance, enhancing the functional recovery of damaged nerves.
It is surprisingly found that the purpose of the invention can be fulfilled with a fibrin glue composition which comprises an effective amount of nerve growth factor and/or nerve repair enhancer, fibrinogen, aprotinin and divalent calcium ions; wherein the nerve repair enhancer is selected from the group consisting of a steroid, a cytokine, a chemokine, a proteinase, an extracellular matrix molecule, a guidance molecule, an anti-angiogenic factor, a neuroprotective agent, and a Nogo gene polypeptide and antibodies that specifically bind to the polypeptide.
Accordingly, in the first aspect, the invention provides a fibrin glue composition for repairing nerve damages which comprises an effective amount of nerve growth factor and/or nerve repair enhancer, fibrinogen, aprotinin and divalent calcium ions; wherein the nerve repair enhancer is selected from the group consisting of a steroid, a cytokine, a chemokine, a proteinase, an extracellular matrix molecule, a guidance molecule, an anti-angiogenic factor, a neuroprotective agent, and a Nogo gene polypeptide and antibodies that specifically bind to the polypeptide.
In a further aspect, the invention provides a fibrin glue composition for enhancing the functional recovery of nerves which comprises an effective amount of nerve growth factor and/or nerve repair enhancer, fibrinogen, aprotinin and divalent calcium ions; wherein the nerve repair enhancer is selected from the group consisting of a steroid, a cytokine, a chemokine, a proteinase, an extracellular matrix molecule, a guidance molecule, an anti-angiogenic factor, a neuroprotective agent, and a Nogo gene polypeptide and antibodies that specifically bind to the polypeptide.
In an even further aspect, the invention provides a method for repairing nerve damages, which comprises topically applying to a damaged nerve the fibrin glue composition of the invention.
Moreover, the invention also provides a method for enhancing the functional recovery of nerves, which comprises topically applying to a damaged nerve the fibrin glue composition of the invention.
The invention relates to a fibrin glue composition for repairing a nerve damage, and/or enhancing the functional recovery of a damaged nerve which comprises an effective amount of nerve growth factor and/or nerve repair enhancer, fibrinogen, aprotinin and divalent calcium ions; wherein the nerve repair enhancer is selected from the group consisting of a steroid, a cytokine, a chemokine, a proteinase, an extracellular matrix molecule, a guidance molecule, an anti-angiogenic factor, a neuroprotective agent, and a Nogo gene polypeptide and antibodies that specifically bind to the polypeptide. The invention also relates to a method for repairing nerve damages, and/or enhancing the functional recovery of a damaged nerve which comprises topically applying to a damaged nerve the fibrin glue composition of the invention.
The nerve growth factor used in the composition of the present invention is selected from, but not limited to, a glial cell line-derived neurotrophic factor, transforming growth factor-beta, fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, vascular endothelial growth factor (VEGF), and neurotrophin (such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), NT3, NT4 and NT5. More preferable, the growth factor is fibroblast growth factor, including acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Most preferably, the growth factor is glial cell line-derived neurotrophic factor.
The term “fibrin glue” as used herein refers to a biocompatible and biodegradable product formed by fibrinogen and other reagents.
The term “effective amount” as used herein refers to an amount of the active ingredients of the fibrin glue composition of the invention, which, when administered to a subject suffering from nerve damages, attains a desired effect, i.e., repairs nerve damages in the subject, and/or enhances the functional recovery of damaged nerves. The effective amount can be readily determined by persons of ordinary skills of the art.
According to the invention, the concentration of nerve growth factor in the fibrin glue composition of the invention may preferably be in the range of about 1 to 1000 μg/ml of the composition, more preferably 50 μg/ml.
The nerve repair enhancer according to the invention is selected from the group consisting of a steroid, e.g. methylprednisone; a cytokine; a chemokine; a proteinase, e.g. a metalloproteinase; an extracellular matrix molecule, e.g. laminin or tenascin; a guidance molecule, i.e. a molecule that attracts or repels the migration of a cell, e.g. netrin, semaphorin, neural cell adhesion molecule, cadherin, thioredoxin peroxidase or Eph ligand; an anti-angiogenic factor, e.g. angiostatin, endostatin, TNP-470 or kringle 5; a neuroprotective agent, e.g. NMDA, a non-NMDA antagonist, a calcium channel blocker, nitric oxide synthase (NOS), an NOS inhibitor, peroxynitrite scavenger or a sodium channel blocker; and a Nogo gene polypeptide and antibodies that specifically bind to the polypeptide.
According to the invention, the concentration of fibrinogen in the fibrin glue composition of the invention may preferably be in the range of about 10 to 1000 mg/ml, more preferably about 100 mg/ml.
According to the invention, the concentration of aprotinin in the fibrin glue composition of the invention may preferably be in the range of about 10 to 500 KIU/ml of the composition, more preferably 200 KIU/ml.
According to the invention, the divalent calcium ions can derive from any calcium ion sources, such as those provided by calcium chloride or calcium carbonate. The concentration of the calcium chloride in the fibrin glue composition of the invention may preferably be in the range about 1 to 100 mM, more preferably 8 mM.
The fibrin glue of the invention can be conveniently prepared by mixing the ingredients. For instance, nerve growth factor and/or nerve repair enhancer and fibrinogen is mixed with aprotinin solution and then the resultant mixture is further mixed with a calcium source. The resultant composition can be stored before use. Alternatively, the composition can also be freshly prepared right before use. Preferably, the fibrin glue is freshly prepared before application.
The fibrin glue composition of the invention is suitable for use in repairing all kinds of nerve damages and enhancing the functional recovery of the damaged nerves in all nerve systems, including the central nervous system, peripheral nervous system, sympathetic nervous system, and parasympathetic nervous system. Preferably, the nerve is a central nervous system nerve. In an embodiment of the invention, the nerve damage is caused by focal cerebral ischemia (FCI). In another embodiment, the nerve damage is caused by chronic focal cerebral ischemia.
As used herein, the term “repairing a nerve damage(s)” refers to an improvement in the pathological conditions of the subject suffering from nerve damages, and the term “functional recovery of nerves” refers to restoration of the physical function of damaged nerves. For example, for nerve damages caused by FCI, the reduction in total infarction volume and motor deficits are signs of the repair of the damaged nerves and restoration of the function thereof. In an animal model according to the invention, the reduction in motor deficits comprises improvement in balance, coordination and grasping strength.
The fibrin glue composition of the invention can be topically applied to damaged nerves. The topical application may be practiced during a surgery.
The method according to the invention exerts a long-term effect needed for repairing chronic nerve damages and enhancing the functional recovery of the damaged nerves. In addition, topical application of the composition of the invention to damaged nerves for only once can sufficiently achieve the desired effect.
Not wishing to be bound by theory, it is believed that the effect of the fibrin glue of the invention in repairing nerve damages and/or enhancing functional recovery of nerves does not result from the slow release of nerve growth factor, but from other unknown mechanisms.
In view of the effects on damaged nerves, the compositions and/or methods of the invention can effectively treat nerve damages-related diseases, including, but not limited to, ischemic brain disease. Preferably, the ischemic brain disease comprises stroke, thrombosis and embolization of MCA.
As shown in the examples, infra, while topical application of the fibrin glue composition of the invention to ischemic brain significantly decreases the infarction volume in MCA occluded rats and also significantly improves the motor performance at the end of 4th week after chronic FCI injure, the control group did not produce neuroprotective effect in rats after chronic focal cerebral ischemia. The data suggest that the fibrin glue composition of the invention is effective in treating the cerebral ischemia from MCA occlusion and enhancing recovery of motor function.
The following Examples are given for the purpose of illustration only and are not intended to limit the scope of the present invention.
Animals: The example conforms to the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication NO. 85-23, revised 1996). Male Long-Evans rats (National Lab. Animal Breeding and Research Center) weighing 300-350 g were used in this study. These animals were housed in a room with controlled temperature (24±1° C.) and humidity (55±5%) under a 12:12 h light-dark cycle. They were allowed free access to food and water.
Surgical procedure: The technique was a modification of the method of Huang et al (Huang S S, Tsai S K, Chih C L, Chiang L Y, Hsieh H M, Teng C M, Tsai M C. Neuroprotective effect of hexasulfobutylated C60 on rats subjected to focal cerebral ischemia. Free Radic Biol Med. 2001;30:643-649). In brief, each male Long-Evans rat was anesthetized by inhalation of a nitrous oxide/oxygen/halothane (69%:30%:1%) mixture during surgical preparation. Body temperature was maintained during surgery at 37±0.5° C. with a heating pad servo-controlled by a rectal probe. The ventral tail artery was cannulated for continuous monitoring of heart rate and mean arterial blood pressure (MABP) by Statham™ P23 XL transducer and displayed on a Gould RS-3400 physiological Recorder (Gould®, Cleveland, Ohio, USA) and the pH, Po2 and Pco2 in the blood were tested using blood sampling with Blood Gas Analyzer (GEM-5300 I.L. CO®, USA). Measurements were performed before, during and right after unilateral MCA occlusion.
Focal ischemic infarcts were produced in the right lateral cerebral cortex in the territory of the MCA. Both common carotid arteries were exposed by midline anterior cervical incision. The animal was placed in a lateral position, and a skin incision was made at the midpoint between the right lateral canthus and the anterior pinna. The temporal muscle was retracted, and a small (3-mm diameter) craniectomy was made at the junction of the zygoma and squamosal bone using a drill (Dremel™ Multipro+5395, Dremel com®. USA) cooled with saline solution. Using a dissecting microscope (OPMI-1, ZISS®), Germany), the dura was opened with fine forceps, and the right MCA was ligated with 10-0 monofilament nylon ties. Both common carotid arteries were then occluded by microaneurysm clips for 1 hr. After removing the clips, return of flow was visualized in the arteries.
Experimental groups: After FCI injury, rats were assigned in randomized sequence to one of four treatment groups (n=6 each): (a) topically applied GDNF (RBI, Natick®, MA, USA)-fibrin glue group: GDNF (1 μg) was mixed with fibrinogen plus aprotinin solution. (b) topically applied GDNF-only group: GDNF (1 μg) was dissolved in HBSS solution. (c) Control group: the fibrin glue was made by adding HBSS solution. (d) Sham group: animals underwent the same described surgical procedures except MCA ligation.
Fibrin glue (Beriplast P™, Germany) used as adhesive agent in CNS tissue and was customarily prepared before use by mixing the fibrinogen (100 mg/ml) with aprotinin solution (200 KIU/ml). This solution was further mixed with calcium chloride (8 mM) in the surgical area to form a glue cast. The final volume for locally application to infarcted brain tissue was 20 μl.
Osmotic applied GDNF group: 1 μg GDNF (RBI, NatickR, MA, USA) was dissolved in HBSS solution. (n=6). Rats were implanted with guide cannulae for the drug infusion. Rats were anesthetized and a 21-gauge stainless steel cannula was implanted in the right lateral ventricle of the rat brain. The cannula was held in place with rapid-setting dental acrylic (Lang Dental®, Wheeling, Ill., USA) anchored to the skull by an aluminum protective cap and steel screws. The minipump was implanted s.c. as described [referece osmotic] with minor modification. Briefly, a small cut was made behind the ears of the rats and the subcutaneous space was expanded with a hemostatic forceps. HBSS solution or GDNF (1 μg in HBSS solution) was filtered through a 0.2-mm sterile syringe filter (Sterile Acrodisc) and was then used to fill an osmotic minipump (Alzet™ 2004, Alza®, Palo Alto, Calif., USA). The minipump was implanted and connected directly to the cannula via 6-cm long PE-60 polyethylene tubing. The infusion rate was 0.25 μl per h for 28 days. The incision on the back was closed with cyanoacrylate glue, and dental acrylic was layered on top of the polyethylene tube.
Infarct volume analysis. After focal cerebral ischemia for 1 hr and reperfusion for 4 weeks, then the rats were anesthetized and killed by rapid decapitation. Brains were removed, inspected visually for the anatomy of the MCA and for signs of hemorrhage or infection, immersed in cold saline solution for 10 minutes, and sectioned into standard coronal slices (each 2-mm thick) using a brain matrix slicer (JACOBOWITZ™ Systems, Zivic-Miller Laboratories INC®, Allison park, USA). Slices were placed in the vital dye 2,3,5-triphenyltetrazolium chloride (TTC, 2%; Sigma, USA) at 37° C. in the dark for 30 minutes, followed by 10% formalin at room temperature overnight. The outline of right and left cerebral hemispheres as well as that of infarct tissue, clearly visualizable by TTC staining (Chen S T, Hsu C Y, Hogan E L, Maricq H, Balentine J D. A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke. 1986;17:738-743), was outlined on the posterior surface of each slice using an image analyzer (color image scanner, EPSON™ GT-9000), connected to an image analysis system (AIS software, Imaging research INC®, Canada) running on a personal computer, AMD™ K6-2 3D 400. The area of infarction was measured by subtracting the area of the non-lesioned ipsilateral hemisphere from that of the contralateral side. Infarct volume was calculated as the sum of infarct area per slice multiplied by slice thickness. Both the surgeon and image analyzer operator were blind to the treatment given to each animal.
Reproducible brain infarcts were obtained from territory of right MCA occlusion in the GDNF-fibrin glue group, the GDNF-only group and the control group. The infarct volume at the end of 4th week after FCI injury in the GDNF-fibrin glue group was significantly reduced (69.1±12.4 mm3, P<0.05) but not in the GDNF-only group (108.0±12.1 mm3), as compared with the control group (124.0±20.0 mm3) (
The results indicated that topically applied GDNF mixing in fibrin glue at infarct brain tissue after FCI injury significantly reduced the total infarcted volume by 44.3% and 36%, respectively compared to that of control group and GDNF-only group.
Behavioral tests: Behavioral measurements were performed by the rotarod test and the grasping power test at the end of 1st, 2nd, 3rd, and the 4th week after focal FCI injury.
Rotarod Test:
The accelerating rotarod was used to assess motor deficit following ischemic injury in rats (Hamm R J, Pike B R, O'Dell D M, Lyeth B G, Jenkins L W. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J Neurotrauma. 1994; 11:187-196). The rats were placed on rungs of the accelerating rotarod and the time the animals remained on the rotarod was measured. The speed was increased slowly from 4 rev/min to 40 rev/min over the course of 5 minutes. The time in seconds at which each animal fell off the rungs was recorded. Each animal received three consecutive trials.
The results of rotarod test at the four groups are shown in
Grasping Power Test:
The grasping power test was a modification of the method of Bertelli et al (Bertelli J A, Mira J C. The grasping test: a simple behavioral method for objective quantitative assessment of peripheral nerve regeneration in the rat. J Neurosci Methods. 1995;59:151-155). For the assessment of grasping strength, a bar of wires was connected to an ordinary electronic balance. Both forepaws were tested, the untested forepaw was temporarily prevented by wrapping it with adhesive tape, and the tested forepaw was kept free. Rats were allowed to grasp the bar while being lifted by the tail with increasing firmness until they loosened their grip and the grasping power was scored.
The effects on grasping power test of the four groups are shown in
The mean value of grasping powers were 78.7%, 71.7% and 101.2% (p<0.05 vs. control group and GDNF-only group) of baseline respectively in the control, GDNF-only and GDNF-fibrin glue groups at the end of 1st week after FCI injury but 89.6%, 97.6% and 120.7% (p<0.05 vs. control group) of baseline at the end of 4th week after FCI injury.
This result showed that topical application of GDNF-fibrin glue is associated with improvement of grasping strength of rats after FCI injury.
Statistics. Data are expressed as mean±standard error of mean (S.E.M.). Statistical analysis of differences in volume of infarcts and the behavioural deficit score of rats were carried by unpaired, two-tailed t tests, and by two-way analysis of variance (ANOVA) for combined data to evaluate the differences between the control and the treatment groups. A value of P<0.05 was considered to be statistically significant.
While embodiments of the present invention have been illustrated and described, various modifications and improvements can be made by persons skilled in the art. It is intended that the present invention is not limited to the particular forms as illustrated, and that all the modifications not departing from the spirit and scope of the present invention are within the scope as defined in the appended claims.
