JPH07504205A - Use of fibroblast growth factors as neuroprotective and neuromodulatory agents - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Use of fibroblast growth factors as neuroprotective and neuromodulatory agents The present invention provides to protect neural tissues that undergo reperfusion after cerebral ischemia. Fibroblast growth factors (acidic and basic FGF as well as non-mitogenic substances) Regarding the use of polypeptides belonging to the group of advanced acid vGF) and the group of derivatives thereof and their use as substances with neuromodulatory effects on motor functions. related.

Spanish patent application No. P9102 filed on September 6, 1991 under the same applicant name 007 is especially suitable for all clinical emergencies requiring pharmacological dilation of blood vessels. Fibroblasts as vasorelaxants in the context Covers the use of cell growth factors and their derivatives. Biotechno In the application of logy Re5search As5ociates, J, V. Patent document 189100,198 filed on July 6, 1988 Covers the synthesis and manipulation of growth factor analogs, which can be used to treat wounds, fractures, and burns. , facilitate the treatment of damaged myocardial tissue, degenerated neurological tissue, and other trauma. It is useful for achieving However, fibroblast growth factor, a similar protein to the potential use of compounds derived from proteins or similar proteins as vasodilators. There is no mention of that.

The fibroblast growth factor protein group is found in cells derived from mesoderm and neuroectoderm. (Gimenez-Ga) is better known as a broad-spectrum mitogen for llego G, etal, 5science +985;320:1385; ThorNls K, N & G1ll1enez-Gal lego G,, srends Bioche nsci, 1986; II:I). However, basic and acidic fibroblasts Non-mitogenic functions of growth factors have already been described (Baird A, e. tal, , Proc, Natl, Acad, Sci.

USA 1985;82:5545;Ba1rd A, & Hsueh J. Regulation Peptides, 1986; 16F243).

The central nervous system is considerably rich in fibroblast growth factors (Thomas A, et al, , Proc.

Natl, Acad, Sci, USA 1985;82:6409), The functions of these proteins in the central nervous system are still unknown. . Various experimental studies have shown that fibroblast growth factor has neurotrophic abilities. It is a substance (Helti F,, in: C1ane D, B,.

Grippa D, Trabucchi Kluonu G, Hollow ski R, (eds,), Parkinson anп@Aging, Raven Press Ltd, New Yark 1989 Near 9; Schawaber J, S, et al, J, Comp, Neural, 1X91; 309 Ni a9) The ability to maintain the survival of neural tissue and the ability of damaged nerve tissue to survive when administered exogenously. It has been shown to have the ability to induce Ron's regeneration. fibroblast growth factor Exogenous administration of angiogenesis) (Cuevas P, et al., in: Gag l1ari R, Benvenutti L, (edsA).

Controversies in EIAB for cerebral i schemia, Monduzzi, Florence　1X88;731; Purimala et al, Bratn Res, 1991.5 58:315) and can prevent neuron death after axotomy. (Lipton S, A5. Arch, Neurol, 1989; 46:12 41) It has been confirmed that. Based on these facts, fibroblast growth factor has been used clinically. It can be considered a drug with the above benefits. In a previous presentation, neuro have shown the use of fibroblast growth factor as a neuroprotective agent after ischemia ( Yamda K, et al.

Cereb, Blood Flow Metabol, 1991; 11:4 72), the use of the factor according to this method poses difficult clinical challenges that cannot be carried out without risk. with specific applications. In fact, according to this study, basic fibroblast growth factor Intravenous injection may be difficult to repeat in clinical experiments due to permanent arterial occlusion. necessary to obtain neuronal protection in the model. European patent website 0 388226 connects FGF to an Alzet mini-pump. Neuroprotection in an ischemia/reperfusion model when injected into the lateral lateral chamber through the neurules. describes the use of FGF for European patent EP 0357240 is FGF The use of FGF for neuroprotection in an ischemia/reperfusion model using intraperitoneal administration of FGF. The purpose is stated. However, the protection is much worse than that of EGF. Yes, and no protection was seen at higher concentrations (1/kg, 10/kg).

Furthermore, although FGF application was started before ischemia occurred, it was not possible in most clinical cases. In , application of the drug will occur after ischemia.

The subject of the invention is particularly in the setting of cerebral ischemia which is temporary and followed by reperfusion. Fibroblast growth factors, similar The use of compounds derived from proteins or similar proteins. Preferably, the Application of the drug occurs after ischemia has occurred. Acidic and basic fibroblast growth factors as well as appropriate formulations of non-mitogenic acidic FGF for temporary cerebral vascular accidents. Surgical recanalization (bypass) of cerebral or carotid arteries in symptomatic situations, mechanical Neuroprotection after physical communication (angiogenesis) or pharmacological communication (fibrinolysis) neuroprotection in all emergency clinical situations that require It can be used as a protective medicine.

In another aspect of the invention, as a neuroprotective agent in the case of circulatory arrest of hypothermia. Examples include the use of FGF. Pediatric cardiac surgery and aortic arch and thoracic aorta procedures, complex neurological surgeries, resection of renal cell carcinomas as well as large traumatic arteriovenous dissections. In hypothermic circulatory arrest (OCA) performed in the case of ligation, the circulatory arrest time is 1 hour. with neurological sequelae (HCA-induced brain damage can occur) is up to 4x children (Tharion J, et al, , J, Thorac, Cardiovasc, Surg, 1982.8W:66 -72), up to 15% of adults (Davis E, A, et al, Ann , Thorac, Surg, 1992;53:10X-114)).

Indeed, there are limits to safe time for improved methods of cerebral protection, especially in HCA. Gives surgeons more flexibility in approaching complex cardiac and non-cardiac defects. The associated neurological complications during the standard time of HCA, if extended to allow It is required to reduce the clutter. According to the present invention, FGF, similar proteins or or similar protein-derived compounds to be used in these cases. Can be done.

As a further aspect of the invention, motor function or aggressive The use of FGF as a medicine in cases where the ess) is impaired. cerebrum At 14.5X in the case of ischemia, motor function impairment manifests as motor function hyperactivity. The fact that Mentia in the USA, 1992), the present invention has expanded from Injected subcutaneously in cases of motor function (hyperactivity and motor agitation) and aggression. fibroblast growth factor, similar proteins or compounds derived from similar proteins. It extends to the use of things. This novel neuromodulatory function of fibroblast growth factor Analyzed in 1 rat. Pharmaceutical compositions containing FGF can be used to treat psychomotor agitation, childhood Lateral movement syndrome, severe agitation, paranoid schizophrenia, alcohol hypersensitivity, advanced Dementia aggressiveness, epileptic aggressiveness, abnormal motor hyperactivity, acute confusional state, psychiatry Clinical conditions requiring reduction of agitation and aggression, such as occurs in disease aggressiveness, etc. It can be used in certain situations.

In a further aspect of the invention, a neuroprotective or neuromodulatory agent as described. Pharmaceutical compositions intended for use as a.

Brief description of the diagram Figure 1 shows the dorsal seam of a gerbil that underwent 5 minutes of cerebral ischemia followed by 7 days of reperfusion. FIG. 2 is a coronal section of a horse. The mouse was given PBS 50 containing 0.1% heparin. μm was injected intravenously. The rectangle in the figure is the hippocampal C, where the number of pyramidal neurons was counted. The area of the AL site is shown.

Figure 2 shows a more enlarged view of the rectangular area in Figure 1, in which tissue destruction and and neuron necrosis.

Figure 3 shows 5 minutes of cerebral ischemia followed by 7 days of reperfusion, 20 seconds after the start of reperfusion. Between, a, Coronal section of the dorsal hippocampus of a gerbil injected intravenously with 2.6 μg of FGF. The figure is shown below.

The protein was injected into PBS 50111 containing 0.1% heparin. length in the diagram The square indicates the region of the CA1 region of the hippocampus where the number of pyramidal neurons was counted.

FIG. 4 corresponds to an enlarged view of the rectangular area in FIG. pyramidal neurons can be observed to be retained in a layered manner: the degenerated cells (arrows) are Only a few exist.

Figure 5 shows rats undergoing sham surgery (normal in the figure) and after 5 minutes of intracerebral ischemia. , rats that underwent 7 days of reperfusion and were then treated with FGF and those that were not treated with FGF. Normal neurons counted in the CA1 region of the dorsal hippocampus of rats This is a bar graph of the average number of .

Figure 6 shows three bar graphs representing the effects of aFGF on horizontal, vertical and open behavior. Indicates the These three types of exercise were performed at different concentrations (lμg/kg, 10 30 minutes after subcutaneous injection of FGF (μg/kg, and 100 μg/kg), Analyzed for 0 minutes.

Figure 7 shows the effects of bFGF on horizontal, vertical and open behavior. , shows three bar graphs. Movement analysis and bFGF treatment in Figure 6 It was the same.

Figure 8 shows rats killed 2 hours after intravenous injection of C''-a, FGF O, lμg. Fig. 2 shows an autoradiograph of a coronal section of the CA1 region of the dorsal hippocampus. A is natural C” -aFGF. B is heat-denatured Cl4-aFGF. natural a in the hippocampus Although an effective enrichment of FGF can be recognized, heat-denatured C''-aFG I cannot recognize F.

The examples are merely illustrative and the invention is not limited thereto in any way. The invention will be described in more detail below by means of several examples with the understanding that .

Example The FGFs used in the following experiments were prepared according to the previously published procedure (Gene 1992, 311:231-238). 139 residue type aFGF and 14 A 6-residue type bFGF was used. The vasoactive, non-mitogenic aFGF is (Science 1991, 254: 1208-1210) Manufactured in

Example: Analysis of neuroprotective effect of FGF in cerebral ischemia/reperfusion model 36 males and females Two gerbils were used. Its weight ranged from 45 to 50 g. This sardine An advantage of using a tooth is that it is possible to remove the osseous artery from the peristaltic artery, which is an artery branching from the carotid artery. These features include fairly clearly demarcated vascular regions. Kettler ( ketolar) (2.5mg/ml), atrobin (0.1mg/ml) ) and barium (valiumX2111g/ml) solution 3ml/kg Anesthetized by intracavitary injection. Expose both common carotid arteries and ligate them using silk threads for 5 minutes. The ligature was loosened and reperfused. 10 gerbils, The same type of surgery was performed, but both arteries were treated without being occluded, resulting in a sham operation. . For 20 seconds after starting reperfusion, 13 mice were treated with phosphate buffer containing 0.1% heparin. 2.6 μg of acidic fibroblast growth factor dissolved in buffer solution (PBS) was administered intravenously (right jugular). (intravenous) injection. The amount of solution injected was 50 μm. Another 13 had 0. 50 μm of PBS containing 1% heparin was injected for 20 seconds after the start of reperfusion. hand Post-operatively, the rats were kept for 7 days, after which the ischemic lesions were confirmed to be present after this period of ischemia and relapse. Areas localized in perfused rats (Gill R, & Woodrr if G, N, Eur, J, Pharmacol, 1990; 1.76: 143) for histological evaluation of normal neurons in the CA1 region of the dorsal hippocampus. The patient was perfused via the left ventricle. The brain was removed and buffered with 10% formaldehyde. Soak in the solution for 3 days. The brain was then placed in a buffer solution containing 30% sucrose. placed.

The next day, the brain was frozen and placed in a freezing microtome. Serial sections (14μ) were cut out using the following procedure. Hematoki serial sections of the frontal It was stained alternately with Syrin/Eosin and Krenl Violet. -1 from bregma , 4 to -2an sections were obtained. Cone cells with distinct nuclei are considered normal. I saw it. The altered cells showed intense nuclear pyknosis and a highly abnormal shape. That's it The survival rate of pyramidal neurons after ischemia and reperfusion in these rats was determined by comparing the same cells to 2 Four randomly selected samples are separated by more than 200 μm from each other to avoid frequency counting. Similarly, in the section of the horizontal part of the CAI region of each dorsal hippocampus, normal cones in a limited area of 0.5 [1 (Figs. 1 and 3) Estimates were made by counting cells.

Sham-operated rats and 7 days of reperfusion after 5 minutes of ischemia were treated with aFGF or of surviving neurons counted in rats treated with either vehicle. The average numbers are summarized in Figure 5. Transient cerebral ischemia (5 minutes) followed by 7 days of reperfusion (9) treated with non-mitogenic aFGF (2.6 μg) at the time of reperfusion. Similar results were obtained for 10 gerbils. In short, aFGF is It is highly effective in preventing brain damage caused by post-ischemic reperfusion, and this effect is due to its does not depend on the mitogenic function of

Example 2: Analysis of the neuromodulatory effect of FGF on motor function Male Wistar rats were used. Place 4 to 5 of them in each cage. , controlled temperature and light-dark schedule (light period 07.00-19.00 h). I kept it below. The rats were given food and water ad libitum. Proper and accurate experimentation Controls and maximum standardization of experimental procedures were performed as follows.

Digiscan Animal Activity Monitoring (Activity Cage) Model RXY ZCK TAO (Omnitech Electronics, Inc.) The activity of the rats was measured using a microcomputer (Columbus, OH, USA).

Briefly, the device consists of a square area (40.6 x 40.6 cm). , in which a plastic animal cage (of the same size) is placed. this place The place is a completely new environment for U-Soto. It's 15 horizontal red lines It has two vertical arrays of outside lines and two vertical light (infrared) screens.

Each beam interruption produces an electrical shock that is counted by an internal electrical meter. Jiru. Horizontal activity, vertical activity and open-ended behavior exhibited by rats. was selected for the purpose of this study.

Rats were given FGF (acidic or basic) or appropriate excipients (1% BSA and Inject 0.5 ml subcutaneously (PBS containing heparin) and immediately place in an active cage. , for 60 minutes (test time from 09.00 to 14.00). Activity parameters was recorded every 5 minutes.

Analyzes of variance (ANOVA) are used to analyze data, where appropriate, to determine whether causality is false. (posthoe) following a number of comparative tests by Newman Keuls. The results were analyzed by analysis of variance (ANOVA). 6 and 7, the horizontal distance, At the end of the total time, as reflected by vertical activity and open count The behavioral response of the active cage corresponding to 30 minutes is shown. Measured values are average + standard error (S EM), and a significant difference is defined as P<0.05.

As summarized in Figures 6 and 7, acid tuGF (aFGF) or base t4J By systemically administering either GF (bFGF) to normal rats, lOμ excipients depending on the dose from 1 to IO μg/kg, with a similar effect to g/kg. Locomotion activity of injected rats decreased.

ANA analysis (D results in horizontal direction) Movement F (3, 27) = 31.99 p <0.0001 bFGF and FC3,32)-30,243p<0.0001 aFGF; vertical motion F(3,27)=8.14 p<0.0005 b FGF and F(3,32)=7.82 p<0.0004 aFGF and open syndrome Count(3,27)=18.99 p<0.0001. bFGF and F(3 , 32) = 25.094 p < 0.0001 For aFGF, significantly less treatment showed the influence of Subsequent post-hoc comparative studies were carried out using the three bFG There was a significant effect between doses of F or aFGF (p<0.00 for each 1 and p<0.01).

Table 1 clearly shows data showing that: i) the formulation When hydrolyzed by treatment with ii) the three-dimensional structure of the protein is unique to FGF; Still mitogenic when twisted by fusion with the C-terminus of cocci autolysins demonstrated the ability to alter rat behavior despite the state of drug and vasoactive effects. Therefore, the behavior regulation function of the preparation depends on the three-dimensional structure of the protein. ;1ii) The aFGF type provides a non-mitogenic agent through protein engineering and is highly active in rats. The ability of FGF to alter rat activity is therefore Do not rely on the cleft promoting function.

Example 3: a, FGF injection into the brain Radioactive aFGF was added to bacteria transformed with aFGF-expressing Hector. Produced by culturing in minimal medium containing Cl4-glucose as a source Ta. Uniformly labeled C”-aFG, either native or heat-denatured FO, I μg was injected intravenously into Wistar rats (average weight 250 g). 2 After an hour, the rats were killed, their brains were removed and cryosectioned, and the sections were cut for one month. subjected to autoradiography. Figure 8 shows that natural aFGF effectively enters the brain. Two autoradiographs are shown showing that.

Table 1 aFGF, non-mitogenic (+011gAglaFGF, digested (dmste d) (IOμg/kg) Continuation of Table 1 As can be seen from the comparison in the table above, acidic fibroblast growth factor administered intravenously The pharmaceutical composition containing the present invention immediately protects neural tissue from damage after cerebral ischemia and reperfusion. Ru.

By comparing and analyzing the data and accompanying drawings, it was determined that single porous (si) FGF injected subcutaneously in the form of The number of horizontal, vertical and open behaviors in rats being It can be concluded that

In summary, fibroblast growth factor is a potent neuroprotective agent after transient cerebral ischemia. Moreover, it is a neuromodulatory agent that causes decreased motor function.

Normal aFGF+PBs PBS+heno<1nonX+SD ischemia-re-employment fluid Stone p < 0.001 vs Fig, 5 Fig, 6 111 saline bFGF bFGF bFGFl 10 100 pg/kg Physiological saline bFGF bFGF bFGFl 10 100 pg/kg Physiological saline bFGF bFGF bFGFl 10 100 μg/kg Fig, 7

Claims (16)

[Claims] 1. Preparation of a pharmaceutical composition for systemic application with the function of neuroprotection in cerebral ischemia acidic and/or basic fibroblast growth factors (FGFs) and / or non-mitogenic acidic FGFs, or members or derivatives of said groups. use. 2. Use according to claim 1, characterized in that cerebral ischemia is followed by reperfusion. 3. Claim 1 or 2, wherein the pharmaceutical composition is formulated for intravenous injection. is used as described in 2. 4. Temporary cerebral vascular accidents and subsequent cerebral or carotid arterial symptoms, Surgical recanalization (bypass), mechanical recanalization (angioplasty) or pharmacological recanalization In all urgent clinical situations requiring protection in general (fibrinolytic phenomena) Claims 1 to 3 for the manufacture of a pharmaceutical composition for use as a neuroprotective agent. Use of listed. 5. Claim 1, characterized in that the pharmaceutical composition is for the treatment of stroke. Use as described in ~4. 6. The pharmaceutical composition may be used as a neuroprotective agent in cases of circulatory arrest due to hypothermia. 5. Use according to claims 1 to 4, characterized in that it is for the purpose of 7. A pharmaceutical composition for the treatment or prevention of damage in nervous tissue, the composition comprising: The wound is caused by cerebral ischemia and acidic and/or basic fibroblasts Growth factors (FGFs) and/or non-mitogenic acidic FGFs, or members of the group or derivatives, and pharmacologically acceptable carriers, diluents and/or or the pharmaceutical composition characterized in that it contains an excipient. 8. The pharmaceutical composition according to claim 7, characterized in that cerebral ischemia is followed by reperfusion. 9. Claim 7 or 8, characterized in that it is formulated for intravenous injection. Pharmaceutical composition. 10. For use as a neuroprotective agent in cases of circulatory arrest in hypothermia The pharmaceutical composition according to any one of claims 7 to 9, characterized in that: 11. Acidic and basic fibers for the production of pharmaceutical compositions with locomotor function Fibroblast growth factors (FGFs) and non-mitogenic acidic FGFs, or the aforementioned Use of group members or derivatives. 12. Claim 11, characterized in that the pharmaceutical composition is formulated for subcutaneous injection. Uses as described in. 13. Psychomotor agitation, childhood hypermotor syndrome, severe agitation, paranoid schizophrenia disease, alcohol hypersensitivity, dementia-related aggression, epileptic aggression, abnormal luck. Excitement and Pharmaceutical composition for use in all clinical situations requiring reduction of aggressiveness 13. Use according to claim 11 or 12 for the manufacture of. 14. For use in clinical situations where a drug with locomotor function is required A pharmaceutical composition comprising acidic and/or basic fibroblast growth factors (FGF). s) and/or non-mitogen type acidic FGF, or members of said groups; contains derivatives and pharmacologically acceptable carriers, diluents and/or excipients. A pharmaceutical composition characterized by: 15. Pharmaceutical composition according to claim 14, characterized in that it is formulated for subcutaneous injection. A product. 16. Psychomotor agitation, childhood hypermotor syndrome, severe agitation, paranoid schizophrenia disease, alcohol hypersensitivity, advanced dementia aggression, epileptic aggression, abnormal luck. Excitement and Claim 1 for use in all clinical situations requiring reduction of aggression 16. The pharmaceutical composition according to 4 or 15.