WO1994012201A1

WO1994012201A1 - Use of fibroblast growth factors as neuroprotective and neuromodulatory agents

Info

Publication number: WO1994012201A1
Application number: PCT/EP1993/003234
Authority: WO
Inventors: Guillermo GIMÉNEZ GALLEGO; Pedro Cuevas SÁNCHEZ
Original assignee: Boehringer Ingelheim Espana S.A.
Priority date: 1992-11-23
Filing date: 1993-11-19
Publication date: 1994-06-09
Also published as: EP0624094A1; JPH07504205A; ES2061380B1; CA2127537A1; ES2061380A1

Abstract

The invention relates to the use of fibroblast growth factor and its derivatives as neuroprotective agents against the damage produced by ischaemia and cerebral reperfusion, and as agents with a neuromodulatory effect on motor activity, and of all compounds related thereto, in those clinical situations which require neuronal protection following cerebral or carotid ischaemia. The pharmacological use of acidic and basic fibroblast growth factor and all compounds relating thereto is also justified in clinical situations which require a decrease in agitation and aggressiveness.

Description

USE OF FIBROBLAST GROWTH FACTORS AS NEUROPROTECTIVE AND NEURO ODULATORY AGENTS

The present invention relates to the use of polypeptides belonging to the family of fibroblast growth factors (acidic and basic and the non-mitogenic form of acidic FGF), as well as their derivatives, as substances that protect the nervous tissue following episodes of transient cerebral ischaemia followed by reperfusion, as well as to their use as substances with a neu- s romodulatory effect on motor activity.

Spanish Patent Application No. P9102007, submitted on 6th September 1991 in the name of the same applicant, covers the use of fibroblast growth factors and their deriva¬ tives as vasorelaxants, especially in all those emergency clinical situations which require a pharmacological dilatation of the vessels. The patent document WO 89/00,198 in the name o of Biotechnology Research Associates, J.V., submitted on 6th July 1988, covers the synthesis and manipulation of fibroblast growth factor analogues which are useful for achieving an accelerated healing of wounds, bone fractures, burns, damaged myocardial tissue, degenerated neurological tissue and other traumas. However, no mention is made of the possible use of fibroblast growth factor, analogue proteins or compounds derived from s the latter as vasodilators.

The family of fibroblast growth factor proteins is better known as broad- spectrum mitogens for cells derived from the mesoderm and the neuroectoderm (Gimenez- Gallego G. et al, Science 1985; 320:1385; Thomas K.A. & Gimenez-Gallego G., Trends Biochem. Sci. 1986; 11:1). However, non-mitogenic activities of basic and acidic fibroblast 0 growth factor have been described (Baird A. et al, Proc. Natl. Acad. Sci. USA 1985; 82:5545; Baird A. & Hsueh J., Regulatory Peptides. 1986; 16:243). Although the central nervous system is very rich in fibroblast growth factors (Thomas A. et al, Proc. Natl. Acad. Sci. USA 1985; 82:6409), the function performed by these proteins in the central nervous system is yet to be determined. Various experimental studies have demonstrated that fibro- s blast growth factors are substances with neurotrophic capacity (Helti F., in: Clane D.B., Grippa D., Trabucchi M., Lonύ G., Horowski R. (eds.), Parkinson and Aging. Raven Press Ltd. New York 1989: 79; Schawaber J.S. et al, J. Comp. Neurol. 1991; 309: 79), capable of maintaining the survival of nervous tissue and of inducing, when administered exoge- nously, the regeneration of damaged neurones. It has been verified that the exogenous 0 administration of fibroblast growth factors is capable of inducing the formation of new blood vessels (angiogenesis) under normal and pathological conditions (ischaemia) (Cuevas P. et al, in: Gagliari R., Benvenutti L. (eds.), Controversies in EIAB for cerebral ischemia. Monduzzi. Florence 1988; 731; Purimala M. et al, Brain Res. 1991, 558: 315), and of

SUBSTITUTE SHEET preventing neuronal death following an axotomy (Lipton S.A., Arch. Neurol. 1989; 46: 1241). These circumstances enable fibroblast growth factors to be considered to be medicinal products of clinical interest. Although a prior publication exists regarding the use of fibroblast growth factors as neuroprotective substances following neuronal ischaemia (Yamada K. et al, Cereb. Blood Flow Metabol. 1991; 11: 472), its use according to this procedure involves a difficult clinical application which is not without its risks. In effect, according to this study, intracerebral infusion of basic fibroblast growth factor is necessary in order to obtain neuronal protection in a model of permanent arterial occlusion, a difficult situation to replicate in clinical practice. EP 0 388 226 describes the use of aFGF for neuro- protection in an ischaemia reperfusion model wherein aFGF was injected into the brain lateral ventricles through a cannula connected to an Alzet's mini-pump. EP 0 357 240 de¬ scribes the use of FGF for neuroprotection in an ischaemia/reperfusion model applying the drug intraperitoneally. However, protection was significantly worse as compared to EGF, and no protection was observed at higher concentrations (1 mg/kg, 10 mg/kg). Further- more, FGF application started prior to the ischaemic event, while in most clinical cases the application of the drug will take place after such an event.

The subject of the present invention is the use of fibroblast growth factor, ana¬ logue proteins or compounds derived from the latter as intravenously administered neuro¬ protective substances in cases of cerebral ischaemia, especially when being transient and followed by reperfusion. Preferably, the application of the drug takes place after an ischaemic event has happened. Adequate formulations of acidic and basic fibroblast growth factors and the mon-mitogenic form of acidic FGF can be used as neuroprotective medicinal products in all emergency clinical situations which require protection, e.g. in the case of stroke, in situations of transient cerebral vascular accident and following surgical recanali- sation (bypass), mechanical recanalisation (angioplasty) or pharmacological recanalisation (fibrinolysis) of a cerebral or carotid artery.

Another aspect of the present invention is the use of FGF as a neuroprotective agent in the case of hypothermic circulatory arrest. Hypothermic circulatory arrest (HC A) used in pediatric cardiac surgery and procedures of the aortic arch and thoracic aorta, com- plex neurological operations, excision of renal cell carcinomas and ligation of large trauma¬ tic arteriovenous fistulas is accompanied with neurological sequelae when the period of circulatory arrest is extended beyond 1 hour (HCA-induced brain damage can occur in up to 4% of children (Tharion J. et al., J. Thorac. Cardiovasc. Surg. 1982, 88: 66-72) and 15% of adults (Davis E. A. et al., Ann. Thorac. Surg. 1992; 53: 109-114)). Clearly, improved me- thods of cerebral protection are required to reduce the attendant neurologic complications during standard periods of HCA, particularly if the safe time limit for HCA is to be exten¬ ded to allow surgeons a greater latitude in the approach to complex cardiac and noncardiac

SUBSTITUTE SHEET defects. According to the present invention, FGF, analogue proteins or compounds derived from the latter can be used in these cases.

A further aspect of the present invention is the use of FGF as a pharmaceutical in cases of disturbed motor activity or aggressiveness. In view of the fact that, in 14.5% of cases of cerebral ischaemia, there is a disturbance of motor activity, manifesting itself in hyperkinesia (Framigham Study. Prevalence of dementia in the USA. 1992), the present invention extends to the use of fibroblast growth factor, analogue proteins or compounds derived from the latter, administered subcutaneously, in situations in which there is exagge¬ rated motor activity (hyperkinesia and motor agitation) as well as in situations of aggres- siveness. This novel neuromodulatory activity of fibroblast growth factor has been analyzed in 200 rats. Pharmaceutical compositions containing FGF can be used in all clinical situa¬ tions which require a decrease in agitation and aggressiveness, as occurs in: psychomotor agitation, infantile hyperkinetic syndrome, maniacal agitation, paranoid schizophrenia, alcoholic irritability, aggressiveness, of the advanced phases of dementia, epileptic aggres- siveness, abnormal hyperkinesia, acute confusional states, psychotic aggressiveness, and the like.

Further aspects of the present invention are pharmaceutical compositions inten¬ ded for use as neuroprotective or neuromodulatory agents as described.

SUBSTITUTE SHEET Brief description of the drawings

Figure 1 is a picture of a coronal section of the dorsal hippocampus of a gerbil subjected to cerebral ischaemia for 5 minutes followed by reperfusion for 7 days. The animal received intravenously 50 μl of 0.1% heparin in PBS. The rectangle in the figure delineates the sector of the CA1 area of the hippocampus in which the pyramidal cells were counted.

Figure 2 shows at higher magnification the delineated area of Figure 1, in which the disor¬ ganisation and neuronal necrosis can be appreciated.

Figure 3 presents a coronal section of the dorsal hippocampus of a gerbil subjected to cere¬ bral ischaemia for 5 minutes followed by a 7 day reperfusion that received intravenously 2.6 μg of aFGF, 20 seconds after the reperfusion onset. The protein was injected in 50 μl of PBS containing 0.1% of heparin. The rectangle in the figure delineates the sector of the CA1 area of the hippocampus in which the pyramidal cells were counted.

Figure 4 corresponds to an enlargement of the rectangle in Figure 3. The preservation of the stratified appearance of the pyramidal neurons can be observed; there are only a few degenerate cells (arrow).

Figure 5 is a histogram of the average number of normal neurons counted in the CA1 area of the dorsal hippocampus of sham operated animals (normal in the figure) and those, FGF- treated and untreated, that suffered a brain ischaemia of 5 minutes ensued by a reperfusion of 7 days.

Figure 6 shows 3 histograms which reflect the effect of aFGF on horizontal, vertical and stereotyped motor behaviour. These three types of movement were analysed for 30 minutes after 30 minutes had elapsed following the subcutaneous injection of FGF at different con¬ centrations (1 μg/kg, 10 μg/kg and 100 μg/kg).

Figure 7 shows 3 histograms in which the effect of bFGF on the horizontal, vertical and stereotyped motor behaviour can be observed. Movement analyses and bFGF treatments were as in Figure 6.

Figure 8 presents autoradiographs of coronal sections of the CA1 areas of the dorsal hip¬ pocampi of rats which were sacrificed two hours after intravenous injection of 0.1 μg C¹⁴- aFGF. A: native C¹⁴-aFGF. B: heat-denatured C¹⁴-aFGF. Effective enrichment of native aFGF within the hippocampus can be appreciated, while heat-denatured aFGF fails to do so.

SUBSTITUTE SHEET The invention will next be described in greater detail and by means of a few examples, in the understanding that they are merely explanatory and in no way limiting in nature.

Examples

The FGF's used for the following experiments were prepared according to a previously published procedure (Gene 1992, 311: 231-238). The 139 residues form of aFGF and the 146 one of bFGF were used. The vasoactive non-mitogenic form of aFGF was prepared as described (Science 1991, 254: 1208-1210).

Example 1: Analysis of the neuroprotective effect of FGF in a cerebral ischaemia/reper¬ fusion model Thirty six gerbils of both sexes were employed. Their weight fluctuated between

45 and 50g. The advantage of using this rodent consists in the property it possesses of ha¬ ving a fairly clearly demarcated vascular region arising from the vertebral arteries of the ar¬ terial region originating from the carotid arteries. Anaesthesia was induced by an intraperi- toneal injection of 3 ml/kg of a solution of ketolar (2.5 mg/ml), atropine (0.1 mg/ml) and valium (2 mg/ml). Both common carotids were exposed and occluded for 5 minutes by means of a knot using a silk thread. Reperfusion is obtained by untying the knot. Ten gerbils were sham operated by treating them in an identical form but without occluding both caro¬ tids. Twenty seconds after the reperfusion onset, 13 animals received intravenously (right external jugular vein) 2.6 μg of acidic fibroblast growth factor dissolved in phosphate-buf- fered saline solution (PBS) containing 0.1% of heparin. The volume of solution injected was 50 μl. Another 13 animals received 50 μl of 0.1% heparin/PBS twenty seconds after the reperfusion onset. After the surgery, the animals were allowed to live for 7 days, being then perfused via the left heart ventricle for a histological evaluation of the normal neurons of the CA1 area of the dorsal hippocampus, a zone in which the ischaemic lesion is localised in this animal subjected to this period of ischaemia and reperfusion (Gill R. & Woodrriff G.N., Eur. J. Pharmacol. 1990; 176: 143). The brains were isolated and immersed in 10% buffered formaldehyde solution for 3 days. They were then placed in a buffered solution containing 30% sucrose. The next day, the brains were frozen and serial sections (14 μ) were cut using a freezing microtome. The frontal serial sections were stained alternately with haematoxylin/eosin and with cresyl violet. Sections were obtained between -1.4 and -2 mm from the bregma point. Those pyramidal neurons which possessed an apparent nucleus were considered to be normal. The degenerate cells exhibited an intense pyknosis and very irregular shape. Survival rate of pyramidal neurons after ischaemia and reperfusion in each

SUBSTITUTE SHEET animal was estimated in four randomly selected sections of the horizontal portion of the CA1 sector of each dorsal hippocampus, separated more than 200 μm from each other in order to avoid taking into account twice the same cell, by counting the normal pyramidal neurons in a defined area of 0.5 mm identically located in each section (Figures 1 and 3). The average number of surviving neurons counted in sham-operated animals and those sub¬ jected to 7 days of reperfusion after 5 minutes of ischaemia, either treated with aFGF or vehicle are summarized in Figure 5. Similar results have been obtained with ten gerbils sub¬ jected to transient cerebral ischaemia (5 min) ensued by 7 days of reperfusion, treated with non-mitogenic aFGF (2.6 μg) at the time of reperfusion. In summary, aFGF protects very efficiently against brain damage caused by reperfusion ensuing ischaemia, being this effect not dependent on its mitogenic activity.

Example 2: Analysis of the neuromodulatory effect of FGF on motor activity Adult male Wistar rats withing 200-250 g were used. They were housed in groups of 4-5 per cage and kept under controlled temperature and light-dark schedule (lights on between 07.00 and 19.00 H). The animals received food and water ad libitum. Appropriate and rigorous experimental control and maximal standardization of experimental procedure was followed. A Digiscan animal activity monitor (activity cage) model RXYZCM, TAO (Omnitech Electronics, Inc., Columbus, OH, USA), was used to assess the activity of the animals. Briefly, the apparatus consists of a square area (40,6 x 40,6 cm) in which a plastic animal cage (of the same dimensions) is placed. This place constitutes a completely novel environment for the animals. It contains two perpendicular arrays of 15 horizontal infrared beams and two vertical light screens (infrared). Each interruption of the beam generates an electric impulse counted by an internal electronic counter. The horizontal activity, vertical activity and stereotypy counts displayed by the animals were selected for the purpose of this study.

Rats were injected subcutaneously with FGF (acidic or basic) or the appropriate vehicle (PBS containing 1% BSA and heparin) in a volume of 0,5 ml and immediately intro- duced in the activity cage for a 60 min session (testing time between 9.00 and 14.00 a.m.). The activity parameters were recorded every 5 min.

Data were analyzed with analysis of variance (ANOVA) followed when appro¬ priate by a post hoc Newman Keuls multiple comparison test. Figures 6 and 7 show the behavioral responses to the activity cage corresponding to the last 30 min of the total ses- sion, as reflected by the horizontal distance, vertical activity and stereotypy counts. Data are expressed as mean + SEM and significance is taken as P < 0.05. As summarized in Figures 6 and 7, systemic administration of either acidic FGF (aFGF) or basic (bFGF) to normal rats decreased locomotor activity with respect to animals injected with the vehicle, in a dose de-

SUBSTITUTE SHEET pendent manner between 1 and 10 μg/kg being as effective as that of 10 μg/kg. ANOVA analysis of the results indicates a significant treatment effect for the horizontal activity F(3,27)=31,99 pO.OOOl bFGF and F(3,32)=30,243 pO.OOOl aFGF; vertical activity F(3,27)=8,14 p<0.0005 bFGF and F(3,32)=7,82 p<0.0004 aFGF and stereotypy counts F(3,27)=18,99, pO.OOOl bFGF and F(3,32)=25,094 p<0.0001 aFGF. Subsequent post-hoc comparison test showed a significant effect among the three bFGF or aFGF doses employed (ρ<0.001 and pθ.01 respectively).

Table I collates in a clear way the data demonstrating: i) that the behavior-modi¬ fying activity is specific to FGF since, when the preparation is hydrolysed by treatment with proteases, that preparation becomes inactive; ii) that the behavior modulating activity of the preparation depends on the spatial conformation of the protein since, when its three-dimen¬ sional structure is distorted by fusion with the c-terminus of the Streptococcus pneumoniae autolysin, this protein significantly loses its capacity of modifying the rat behavior, on spite of still being mitogenic and vasoactive; iii) that the capacity of FGF to modulate the activity of the animals does not depend on its mitogenic activity, since a form a FGF rendered non- mitogenic by protein engineering, significantly modifies the activity of the rats.

Example 3: Entrance aFGF into the brain Radioactive aFGF was produced by growing bacteria transformed with an aFGF expression vector in minimal medium with C¹⁴-glucose as carbon source. 0.1 μg of uniform¬ ly labeled C¹⁴-aFGF, either native or heat-denatured, were injected intravenously into wistar rats (250 g average weight). After two hours, the animals were sacrificed, the brain extrac¬ ted and cryosectioned, and the sections autoradiographed for one month. Figure 8 shows two autoradiographs demonstrating that native aFGF effectively enters into the brain.

SUBSTITUTE SHEET Table I

Treatment Activity Number of movements ANOVA Mean SD

aFGF, non-mitogemc (10 μg/kg)

aFGF, digested (10 μg/kg)

aFGF-autolysin (40 μg/kg, purified by DEAE)

Table I, cont'd

Treatment Activity Number of movements ANOVA Mean SD

aFGF-autolysin (120 μg/kg, purified by DEAE)

aFGF-autolysin (40 μg/kg, purified by heparin)

As demonstrated by a comparison of the figures presented, pharmacological compositions containing acidic fibroblast growth factor injected intravenously immediately protect neuro¬ nal tissue against the damages following cerebral ischaemia and reperfusion.

Comparing and analyzing the data and accompanying figures, it can further be concluded that FGF administered subcutaneously in the form of a single bolus significantly reduces the number of horizontal, vertical and stereotyped movements in rats living under normal conditions.

In conclusion, fibroblast growth factor is a potent neuroprotective agent follo¬ wing transient cerebral ischaemia and is likewise a neuromodulatory active agent producing motor hypoactivity.

SUBSTITUTE SHEET

Claims

1. Use of acidic and/or basic fibroblast growth factors (FGFs) and/or the non-mitogenic form of acidic FGF, or any member or derivative of the said family, for the manufac- ture of pharmaceutical compositions with neuroprotective activity in cerebral ischae¬ mia, said compositions being formulated for systemic application.

2. Use according to claim 1, characterized in that the cerebral ischaemia is followed by reperfusion.

3. Use according to claims 1 or 2, characterized in that the pharmaceutical composition is formulated for intravenous injection.

4. Use according to claims 1 to 3 for the manufacture of pharmaceutical compositions for use as neuroprotective agents in all emergency clinical situations which require protection, in situations of transient cerebral vascular accident and following surgical recanalisation (bypass), mechanical recanalisation (angioplasty) or pharmacological recanalisation (fibrinolysis) of a cerebral or carotid artery.

5. Use according to claims 1 to 4, characterized in that the pharmaceutical composition is intended for the treatment of stroke.

6. Use according to claims 1 to 4, characterized in that the pharmaceutical composition is intended for use as a neuroprotective agent in the case of hypothermic circulatory arrest.

7. Pharmaceutical composition for the treatment or prevention of damages in neuronal tissues, said damages being caused by cerebral ischaemia, characterized in that it con¬ tains acidic and/or basic fibroblast growth factors (FGFs) and/or the non-mitogenic form of acidic FGF, or any member or derivative of the said family, and a pharmaceu¬ tically acceptable carrier, diluent and/or excipient.

8. Pharmaceutical composition according to claim 7, characterized in that the cerebral ischaemia is followed by reperfusion.

9. Pharmaceutical composition according to claims 7 or 8, characterized in that it is for¬ mulated for intravenous injection.

SUBSTITUTE SHEET

10. Pharmaceutical composition according to one of claims 7 to 9, characterized in that it is intended for use as a neuroprotective agent in the case of hypothermic circulatory arrest.

11. Use of acidic and basic fibroblast growth factors (FGFs) and the non-mitogenic form of acidic FGF, or any member or derivative of said family, for the manufacture of pharmaceutical compositions with hypomotor activity.

12. Use according to claim 11, characterized in that the pharmaceutical composition is formulated for subcutaneous injection.

13. Use according to claims 11 or 12 for the manufacture of pharmaceutical compositions for use in all clinical situations which require a decrease in agitation and ag¬ gressiveness, as occurs in: psychomotor agitation, infantile hyperkinetic syndrome, maniacal agitation, paranoid schizophrenia, alcoholic irritability, aggressiveness of the advanced phases of dementia, epileptic aggressiveness, abnormal hyperkinesia, acute confusional states, psychotic aggressiveness, and the like.

14. Pharmaceutical composition for use in clinical situations where an agent with hypo- motor activity is needed, characterized in that it contains acidic and/or basic fibroblast growth factors (FGFs) and/or the non-mitogenic form of acidic FGF, or any member or derivative of the said family, and a pharmaceutically acceptable carrier, diluent and/or excipient.

15. Pharmaceutical composition according to claim 14, characterized in that it is formula¬ ted for subcutaneous injection.

16. Pharmaceutical composition according to claims 14 or 15 for use in all clinical situa¬ tions which require a decrease in agitation and aggressiveness, as occurs in: psycho- motor agitation, infantile hyperkinetic syndrome, maniacal agitation, paranoid schizo¬ phrenia, alcoholic irritability, aggressiveness of the advanced phases of dementia, epi¬ leptic aggressiveness, abnormal hyperkinesia, acute confusional states, psychotic ag¬ gressiveness, and the like.