Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO 94/12201 2 ~ 2 7 ~ 3 7 PCT/EP93/03234
~,:
USE OF FIBROBLAST GROWTH FACTORS AS NEUROPROTECTIVE AND NEUROMODULATORY
AGENTS :
The present invention relates to the use of polypeptides belonging to the family of fibroblast
growth factors (acidic and basic and the non-mitoger~ic form of acidic FGF), as well as their
derivatives, as substances that protect the nervous tissue following episodes of transient
cerebral ischaemia followed by reperfilsion, as well as to their use as substances with a neu-
5 romodulatory effect on motor activity.
Spanish Patent Application No. P9102007, submitted on 6th September 1991 inthe name of the same applicant, covers the use of fibroblast growth factors and their deriva-
tives~as vasorelaxants, especially in all those emergency clinical situation~ 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 mar~ipulation of fibroblast growth factor analogues which are usefill for
achieving an accelerated healing of wounds, bone ~actures, burns~ damaged myocardial
tissue, degenerated neurological tissue and other traumas. However, no mention is made OI
the possib}e use of fibroblast growth factor, analogue proteins or compounds derived ~om
s the latter as vasodilators.
The fiamily of fibroblas~ growth factor proteins is betta known as broad~
spectmm mitogens for cells derived ~om the mesodenn and the neur~ectoderm (Giménez-
Gallego G. et al., Science 198~; 320:1385; Thomas K.A. & Gimé~ Gallego G., Trends
Biochem. Sci. 1986; 11:1). However, non-mitogenic activities of basic and acidic fibroblast
20 growth factor have been described (Baird A. et a~.r Proc. Natl. ~cad. Sci. USA 1985;
82:5S45; Baird A. & Hsueh J., Regulatory Peptides. 1986; 16:243). A}though the cen2ral
nervous system is very rich in fibroblast growth factors (Thomas A. et al.9 Proc. Natl Acad.
Sci. IJSA 1985; 82:6409~, the fimction performed by these proteins in the central nervous
system is ye~ to be dete~ed. Various expe~nental studies have demonstrated that fibr~
25 blast g~owth factors are substances with neurotrophic capacity (Helti F., in: Clane D.B.,
Grippa D., Trabucchi M., Lonu 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 nelvous tissue and of inducing, when administered exoge-
nously, the regeneration of damage~ neurones. It has been verified that the exogenous
30 administration of fibroblast growth factors is capable of inducing the formation of new
blosd vessels (angiogenesis) under normal and pathological conditions ~ischaemia~ (Cuevas
P. ef al., in: Gagliari R., Benvenutti L. (eds.), Controversies in EIAB for cerebral isrhemia.
Monduz~. Flore ce 1988; 731; Purimala M. e~ aL, Brain Res 1991, 558: 315), and of
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wo 94/12201 2 1 2 7 r~ 3 ~ PCT/EP93/032~S4
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; 1 1: 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 infi sion 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 aF&F for sleuro-
o 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/reperfiusion mc~del applying the
drug mtraperitoneally. However, protection was significanely 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 ischaemJa, especially when being transient and
20 _followed by reperfusion. Preferably, the application of the drug takes place ~er 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 clinicaJ situations which require protection~ e.g. in the case of
stroke, in situations of transient cerebral vascular accident and following surgical recanali-
25 sation (bypass), mechanical recanalisa~ion (angioplasty) or pharmacological recanalisation
(fibrinolysis3 of a cerebral or carotid arte~.
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 (HCA)
used in pediatric cardiac surgery and procedures of the aortic arch and thoracic aorta, com~
30 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, irnproved m~
35 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
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WO 94tl~201 2 :~ 2 7 c~ 3 7 PCT/EP93103234
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 F&F as a pharmaceutical
in cases of disturbed motor activity or aggressiveness. In view of the fact that, in 14.5% of
s cases of cerebral ischaemia, there is a disturbance of motor activity, manifesting itself in
hyperkinesia (Framigharn Study. Prevalence of dementia in the USA. 1992), the present
invention extends to the use of fibroblast grow$h factor, analogue proteins or compounds
derived from the latter, administered subcutaneously, in situations in which there is exagge-
rated motor activity (hyperkinesia and motor agitation) as well as in situations of aggres-
o siveness. This novel neuromodulatory activity of fibroblast growth factor has been analyzedin 200 rats. Pharmaceutical compositions containing FGF can be used in all clinical situa-
tions which require a decrease in agi$ation 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-
15 siveness, abnormal hyperkinesia, acute confusional states, psychotic aggressiveness, and thelike.
Further aspects of the present invention are pharmaceutical compositions inten-
ded for use as neuroprotective or neuromodulatory agents as descr.~ed.
20 "
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SUBSTITUTE SHEET
WO 94/12201 2 . 7 ;~ .3 7 PCT/EP93/03D4
Brief description of the drawings
Figure I is a picture of a coronal section of the dorsal hippocampus of a gerbil subjected to
cerebral ischaemia for 5 minutes followed by reperfilsion for 7 days. The animal received
5 antravenously 50 ~1 of 0.1% heparin in PBS. The rectangle in the figure delineates the sector
of the CAl area of the hippocampus in which the pyramidal cells were counted.
Figure 2 shows at higher magnification the delineated area of Figure I, 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 S minutes followed by a 7 day reperfilsion that received intravenously 2.6 ~
~g of aFG~, 20 seconds a~er the reperfilsion onset. The protein was injected in 50 ~1 of -.
P PBS containing 0.1% of heparin. The rectangle in the figure delineates the sector of the
Is CAl area ofthe 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 fiew .. .
degenerate cells (arrow). -
_ ~
Figure 5 is a }~stogram of the average number of normal neurons counted in the CAl area -
of the dorsal hippocampus of sham operated animals ~nonnal in the figure) and those7 FGF- ~
treated and untreated, that su~ered a brain ischaemia of ~ n~inutes ensued by a reperfi~sion .-
of 7 days.
Figure 6 shows 3 histograrns which reflect the e~ct of ~GF on hori~ontal, vertical and
stereotyped motor behaviour. These three types of movement were analysed for 30 minutes
a~er 30 minutes had elapsed following the subcutaneous injection of FGF a$ dif~erent con- :
centrations (1 ~lg/kg, 10 ~lg/kg and 100 ,ug/lc~. ~
Figure 7 shows 3 histograms in whieh the e~ect of bFGF on the horizontal, vertieal and
stereotyped motor behaviour can be observed. Movement analyses and bFGF treatments
were as in Figure 6.
35 Figure 8 presents autoradiographs of coronal sections of the CAl areas of the dorsal hip-
pocampi of rats which were sacrificed two hours a~er intravenous injection of 0.1 ,ug C14~
aFGF. A: native Cl4-aFGF B: hea~ena~ured Cl4-aFGF. Effective enrichment of native ~--
aFGF within the hippocampus can be appreciated, while heat-denatured aFGF fails to do so. ~.
SUBSTITUTE SHEET
- wo 94/12201 2 ~ ~ 7 ~ .~ 7 PCTlEPg3/03234
S
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
o 146 one of bFGF were used. The vasoac~ive non-n~itogenic fonn of aFGF was prepared as
desclibed (Science 1991, 254: 1208-12103.
Example 1: Ana~ysis of ~he neuroprotective e~fect of FGF in a cere~ral ischaemia/reper-
fusion model
Thilty six gerbils of both sexes were employed. Their weight fluctuated between
45 and 50g. The advantage of using this rodent consists in the pr~perty it possesses of ha-
ving a fairly clearly demarcated vascular region a~ising ~om the vertebral ar~enes of the ar-
terial region originating from the carotid arteries. Anaesthesia was induced by an isltrapen-
toneal injection of 3 ml~lcg of a solution of ketolar (2.5 mglml), atropine (0.1 mg/ml) and
~o valium (2 mglml). Both common carotîds were exposed and occluded for 5 minutes by
means of a knot using a sillc thread. Reperfi~sion is obtained by untyin~ the knot. Ten gerbils
were sham operated by treating them in an identiGal form but without occluding both car~
tids. Twenty seconds after the reperfiusion onset, 13 snimals received intravenously (ri~ht
extemal jugular vein) 2.6 ,ug of acidic fibroblast growth factor dissolved in phosphate-buf-
75 fered saline solution (PBS~ eontaining 0.1% of heparirl. The volume of solution injected was50 ~1. Another 13 ar~imals received 50 ,ul of O.l~/o heparin/P13S twenty seconds af~er the
reper~sion onset. A~er the surgery, the animals were allowed to live for 7 days, being then
perfi~sed via the le~ heart ventFicle for a histologicai evaluation ofthe nonnal neurons of the
CAI area of the dorsal hippocampus, a zone in whieh the ischaemic lesion is loealised in this
30 animal subj~ted to this period of ischaemia and reperfi~sion (Gill R. & Woodrriff G.N.,
Eur. J. Pharmacol. 1990; 176: 143). The brains were isolated and immersed in 10% bu~red
fo~naldehyde solution for 3 days. They were then piaced in a bu~ered solution containing
30% sucrose. The next day7 the brains were frozen and serial sections 114 ,u) were cut using
a freezing microtome. The frontal serial sections were staîned altemately with
35 haematoxylin/eosin and with cresyl violet. Sections were obtained between -1.4 and -2 mm
~om the bregma point. Those pyrals~idal neurons which possessed ~n apparent nucleus were
considered to be normal. The degenerate cells exhibited an intense pyknosis and very
irre~gutar shape. Survival rate of pyramidal neurons aflLer ischaemia and reperfusion in each
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WO 94/12201 PCTIE:P93/03~34
2127~ 6
animal was estimated in four randomly selected sections of the horizontal portion of the
CAl sector of each dorsal hippocampus, separated more than 200 ~lm 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 I and 3)
The average number of surviving neurons eounted in sham-operated animals and those sub-
jected to 7 days of reperfi~sion 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-
jec~ed to transient cerebral ischaemia (5 min) ensued by 7 days of reperfi~sion, tre~ted with
non-mitogenic aFGF (2.6 ~lg) at the time of reperfi~sion. In summary, aFGF protects very
o efficiently against brain damage caused by reperfilsion ensuing ischaemia, being this effect
not dependent on its mitogenic activity.
E~cample 2: Analysis of ~he neuromodulatory effect of FGF on motor act~vity
s 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 m~imal s~andardization of experimental
procedure was ffillowed. A Digiscan animal activity monitor (activity cage) model
20 _RXYZCM, TAO (Omnitech Electronics, Inc., Columbus, OH, USA), was used to assess
the activity of the animals. ~riefly, 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
honzontal infrared beams and two vertical light screens (ir~ared~. Each int~ruption of the
beam generates an electric impulse counted by an internal electronic counter. The horizontal
activity, vertical activity and stereotypy counts displayed by the ar~imals 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 a~ivity cage for a 6~ min session (testing time between 9.00 and 14.00 a.m.).
The activity parameters were recorded ~very 5 min.
Data were analyzed with analysis of variance (ANOVA) followed when appro-
priate by a pos~ hoc Newman Keuls multiple comparison test. Figures 6 and 7 show the
behavioral responses to the activity cage colTesponding 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 ~o a~mals injected with the vehicle. in a dose de-
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WO 94/12201 212 7 ~ 3 7 PCT/EP93tO3234
pendent manner between 1 and 10 llg/kg being as effective as that of 10 llglkg. ANOVA
analysis of the results indicates a significant treatment effect for the horizontal activi~y
F(3,27)=31,99 p<0.0001 bFGF and F(3,32)=30,243 p<0.0001 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
5 F(3,27)=18,99, p<0.0001 bFGF and F(3,32)=25,094 p<0.0001 a~G~. Subsequent post-hoc
comparison test showed a significant ei~ect among the three bFGF or aFGF doses employed
(p<0.001 and p<0.01 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
o proteases, that preparation becomes inactive; ii) that the behavior modulating activity of the
preparation depends on the spatial conforrnation of the protein since, when its three-dimen-
sional structure is distorted by filsion 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
15 of the anirnals does not depend on its mitogenic activity, since a forrn a FGF rendered non-
rnitogenic by protein engineering, significantly modifies the activity of the rats.
E~ample 3: Entrance aFGF into ~he bram
20 ., Radioactive aFGF was produced by gro~nng bacteria tran~formed with an aFGF
expression vector in ~al medium with Cl4-glucose as carbon source. 0.1 llg of uniform-
Iy labeled Cl4-aFGF~ either native or heat-denatured, were injected intravenously into wistar
rats (2S0 g average weiBlt). Af'cer two hours, the animals were sacrificed, the brain extrac-
ted and cryosectioned, and the sections autoradiographed for one month. ~igure 8 shows
25 t~wo autoradiographs demonstrating that native ~GF effectively enters into the brain.
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WO 94tl2201 : PCTIEP93/03234
2 1 ~ 7, 3 7 8
Table I -~
:
Treatment Activity Number of movements ANOVA
Mean SD F P
aFGF, non-mitogenic (10 llglkg) . ~:
_ . _
Hori~ontal
Control 1343.6 76.6 24.340.0005
Treated 502.5 162.1 , :
Vertical ::
Control 108.7 35.85
Treated 12.0 7.54 5.970.033 :-
Stereotyped
Control 560 31.66
Treated 227.83 81.71 16.1740.002 :~
. .
aFGF~ digested (10 ,~lg/kg) ~ `
Horizontal ~ .
Control137~.29575.B0 -~
Treated1125.20281.84 0.121 0.7347
Vertical - ` :
Control 111.29 61.88
. Treated 127.60 48.71 0.0370.8507 -::
Stereotyped ~:
Control 639.29 247.43
Treated 430.40 110.35 0.4520.5164
aFGF-autolysin (40 llg/kg, purificd by ~EAE)
_ . _ .
Horizontal .
Control 1476.00 438.46
Treated 1125.20 281.X4û.453 0.51~6
Vertical
Control 120.00 78.50
Treated 127.60 4~.710.007 0.9364
Stcreotyped
Control 545.80 156.51
Treated 430.40 110.350.363 0.5632
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WO 94/12201 2 1 .~ 7 j ~ 7 PCrlEP93103234
Talble I, cont'd
_ _ ~
Treatment Ac~ivity Number of movements ANOVA
Mean SD F P
aFGF-autolysin (120 ~,Ig/kg, purified by DEAE)
Horizontal
Control 1125.20 281.74
Treated 984.50 569.30 0.056 0.8192
Vertical
Cont~ol 127.60 48.71
Treated 28.25 16.60 3.025 0.1255
Stereotyped
C~ntrol 430.40 110.3S
Treated 471.00 287.09 0.021 0.8892
_ , ~
aFGF-autolysin (40 ~g/kg, purified by heparin)
_ ~
Horizontal
Control 1017.00 287.25
Treated 723.33 379.50 0.361 0.5644
Vertical
Control 210.22 115.0S
Treated 28.~0 10.61 1.60 0.2276
~,tereotyped
Control 441.67 108.40
Treated 311.33 170.99 0.461 0.5088
:,
As demonstrated by a comparison of the figures presented, phalmacological compositions
containing acidic fibroblast growth factor injected intravenously immedi~ely protect neuro-
nal tissue against the damages following cerebral ischaen~ia and reperfilsion.
Comparing and analyzing the data and accompanying figures, it can filrther be
~o concluded that FGF administered subcutaneously in the form of a single bolus significantly
reduces the number of horizontal, vertical and stereotyped movements in ra~s living under
norrnal conditions.
In conclusion, fibroblast growth factor is a potent neuroprotective agent follo-- wing transient cerebral ischaemia and is likewise a neuromodulatory active agent producing
15 motor hypoactivity.
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