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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
Interaational Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Gassification ^ 
A61K 37/02 



A2 



(ll)IntematioDal Publication Number: 
(43) International Publication Date: 



WO 93/09802 

27 May 1993 (27.05.93) 



(21) International Application Number: PCT/US92/09974 

(22) International Filing Date : 20 November 1 992 (20. 1 1 .92) 



(30) Priority data: 
240696 



22 November 1991 (22.1 1.91) NZ 



(71) Applicants (for all designated States except US): GENEN- 

TECH, INC. [US/US]; 460 Point San Bruno Boulevard, 
South San Francisco, CA 94080 (US). AUCKLAND 
UNISERVICES LIMITED [NZ/NZ]; 20 Symonds 
Street, Level 6, Private Bag 92019, Auckland 1 (NZ). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only) : GLUCKMAN, Peter 
[NZ/NZ]; 78 Lucerne Road, Remeura, Auckland 1 
(NZ). NIKOLICS, Karoly (HU/USl; 450 South Road, 
Belmont, CA 94002 (US). WILLIAMS, Christopher 
[NZ/NZl; 5D Boyle Crescent, Grafton, Auckland 1 
(NZ). . 



(74) Agents: JOHNSTON, Sean, A. et al.; Genentech, Inc., 460 
Point San Bruno Boulevard, South San Francisco, CA 
94080-4990 (US). 



(81) Designated States: CA, JP. US, European patent (AT, BE, 
CH, DE, DK, ES, PR, GB, GR, IE, IT. LU, MC, NL, 
SE). 



Published 

With declaration under Article J 7(2Xa). 

Without abstract: title not checked by the International 

Searching Authority. 



(54) Title: TGF-BETA TO IMPROVE NEURAL OUTCOME 



Hippoc, CAl-2 
Hippoc. CAS 
Hippoc. CA4 
Dentate Gyrus 
Pyriform Cortex 

Lateral Cortex 
Vehicle 



TGF-)Sj 
0.05/ug 




I 1 1 1 \ 1 — 

0 20 ^° % ^0 ®Q 

Neuronal loss score 

x+/-aem 



FOR WE PUSPOSES OF INFORMATION ONLY 

Cbdes us«l lo identily Siaies party to .he PO" on the front pages of pamphlets publishing international 

appHcaiions* under the PCX. 



AT Atotfiu 

AU Australia 

BB Barbados 

BE Belgium 

BF Burkina Faso 

BC Bulearia 

BJ Benin 

BR Brazil 

CA Canada 

CF (*4;niral African Republic 

CC Congo 

CU Swiuerland 

CI t'olc dlKoirc 

CM Cumcrcxin 

CS < /xfhoslovak'w 

CZ (Vccb Kcpufalit 

DE Cicmian> 

DK Denmark 

£S Spain 

PI f^inland 



FR Kraoce 

GA Gabon 

G6 United Kingdom 

ON Guinea 

CR Greece 

HU Hungary 

IE Ircbnd 

IT Italy 

JP Japan 

KP DeOMKRiiic Pcople'a Republic 

oT Korea 

KR Republic of Korea 

KZ Kayallwlan 

[J Liechtenstein 

LK Sri 1 ,inka 

1,U Luxembourg 

MC Monaco 

MC MadngaM;ar 

Ml. Mali 

MN Mongolia 



MR 


Mauritania 


MW 


Malawi 


NL 


Netherlands 


NO 


Norway 


NZ 


New Zealand 


PL 


Poland 


PT 


Portu^l 


RO 


Rumania 


RU 


Ku&ian Federation 


SD 


Sudan 


SE 


Sweden 


SK 


Slovak Republic 


SN 


Senegal 


SU 


Soviet Union 


TD 


Chad 


TC 


Togo 


UA 


Ukraine 


US 


United Sialci of America 


VN 


Viet Nam 



wo 93/09802 PCr/US92/09974 

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TGF-BETA TO IMPROVE NEURAL OUTCOME 

FIELD OF THE INVENTION 
This invention relates to methods end pharmaceutical compositions for the treatment or 
5 prevention of central nervous system (CNS) damage and relates particularly to methods of 

treatment comprising increasing the concentration of transforming growth factor beta 1 (TGFv?1) 
and/or analogues thereof in the central nervous system of the patient to treat an in^ry or disease 
that causes damage to ceils of the CNS. 

10 BACKGROUND OF THE INVENTION 

After asphyxlal, traumatic, toxic, infectious, degenerative, metabolic, ischemic or hypoxic 
insults to the central nervous system (CNS) of man a certain degree of neural damage may result. 
For example, such neural damage can occur in cases of perinatal asphyxia associated with 
intrapartum fetal distress such as following abruption, cord occlusion or associated with 

15 intrauterine grovirth retardation; perinatal asphyxia associated with failure of adequate resuscitation 
or apnea; severe neural insults associated with near miss drowning, carbon monoxide inhalation, 
ammonia of other gaseous intoxication, cardiac arrest, collapse, coma, meningitis, hypoglycemia, or 
status epilepticus; episodes of cerebral asphyxia associated with coronary bypass surgery; 
cerebral anoxia or ischemia associated with stroke, hypotensive episodes, hypertensive crises; 

20 cerebral trauma; or cerebral degenerative diseases such as Alzheimers disease and multiple 
sclerosis. 

Such neural damage can involve several different cell types of the CNS. For example, 
periventricular leucomalada, a lesion which affects the periventricular oligodendrocytes is generally 
considered to be a consequence of hypoxicischemic injury to the developing preterm brain. Bejar, 

25 et al., Am. J. Obstet. GvnecoL. 159:357-363 (1988); Sinha, et al.. Arch. Pis. Child.. 65:1017- 
1020 (1990); Young, et al., Ann, Neurol.. 12:445-448 (1982). Further cholinergic neuronal cell 
bodies are absent from most regions of the cortex in primates (Mesulam, et al., Neurosci.. 12:669- 
686 (1984)) and rats (Brownstein, et al., in Handbook of Chemical Neuroanatomv. Classical 
Transmitters in the CNS. pp. 23-53 (Elsevier, 1984)). Damage to the cerebral cortex by trauma, 

30 asphyxia, ischemia, toxins or infection is frequent and may cause sensory, motor or cognitive 
deficits. Glial ceils which are non-neuronal cells in the CNS are necessary for normal CNS 
function. Infarcts are a principle component of hypoxic-ischemia induced injury and loss of glial 
cells is an essential component of infarction. Multiple sclerosis is associated with loss of myelin 
and oligodendrocytes, simQarly Parkinson's disease is associated with loss of dopaminergic neurons. 

35 Several growth factors have been reported to be induced after transient hypoxic-ischemia in 

the brain. After postasphyxial seizures, the proto-oncogene c-fos is induced in surviving neurons 
and in glial cells from infarcted regions. Gunn, et al.. Brain Res.. 531:105-116 (1991). Nerve 
growth factor (NGF) synthesis is increased after hypoxia or seizures in the hippocampus and 



SUBSTITUTE SHEET 



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cerebral cortex, inrpr «t al.. Neurosri. Lett.. 98:339-344 (1989); Gall, et al., SeiOES- 245:758- 
761 (1989). However, little is known of the role of cytokines in brain injury. Glial cells have been 
shown to produce a number of cytyokines including interieukin 3 (IL-S) and interleukin 6 (IL-6). 
Interieukin 1 (IL-1) has been reported to be elevated in cerebrospinal fluid after head injury in 

5 humans. Mrri«m «t J. Lab. Clin. Med.. 110:48-54 (1987). 

Transforming growth factor beta (TGF-;ff) is another example of a cytokine and is a 
multifunctional polypeptide implicated in the regulation of cellular or tissue response to injury or 
stress. For a general review of TGF-iS and its actions, see Spom, et al., Scjence, 233:532-534 
(1986); Spom et aL, J. Cefl BioL. 105:1039-1045 (1987); Spom, et al.. Nature. 3232:217-219 

10 (1988); and Spom, et aL. in Peptide Grovirth F ^rtnrs unrf Their Receptors I. pp.419472 (Springer- 
Verlag, 1990). TGF-;ff is found in various mammalian tissues, such as bone, platelets, and 
placenta, and methods for purifying the polypeptide from such natural sources, as weO as for 
producing it in recombinant cell culture, have been described. See, for example, Assoian, et al., A 
BioL Chem.. 258:7155-7160 (1983); Frolik. et al., Proc. Nat. Acad. Sd., 80:3676-3680 (1983); 

15 Heimark, et al.. Science. 233:1078-1080 (1986); Spom, et al., U.S. Patent No. 5,104,977; 
Derynck, et al.. Nature. 21B:701-* • * (1985); Derynck, at al., U.S. Patent No. 4,886,747. 

There are several molecular forms of TGF-)ff, including those forms which are commonly 
referred to as TGF-)?1 (Derynck, et aL, {Mature, 316:701-* * * (1985)), TGF-^ (deMartin. et al., 
EMBO J.. 3673-* * * (1987); Madison, et al., DM, 2:1-8 (1988)), TGF-yK (Jakowlew, et al., 

20 Mol. Endocrin.. 2:747-755 (1988); Ten Dijke, et aL, Proc. Nat. Acad, Sci., 85:47154719 (1988); 
Derynck, et al.. EMBO J.. 2:3737-* * * (1988)), TGF-/ff4 (Jakowlew, et aL, MoL ^ndocrin. , 
2:1186-1195 (1988), and TGF-A5 (Kondaiah, et aL, J- BioL Chem.. 265:1089-* * * (1990). 

It is an object of the invention to prowde methods and pharmaceutical compositions for 
treating or preventing CNS injury or damage. The invention Is based upon the inventors' successful 

25 research into the role and effects of TGF-^ in the CNS. 



■<;ilM |UlflRY OF THE INVENTION 
Accordingly, in a first aspect the invention consists In a method of treating neural damage 
suffered after a CNS insult characterized in that it comprises the step of increasing the active 
30 concentrationis) of TGF-^1 andfor analogues of TGF-;ff1 (such as other moleculer forms of TGF-^ 
in the CNS of the patient. Preferably, the concentration of TGF-jffl in the CNS of the patient is 
increased. 

The term "treat" when used herera refers to the effecting of a reduction in the severity of 
the CNS damage, by reducing infarction, and loss of glial cells, non cholinergic neuronal cells, or 
35 other neuronal cells, suffered after a CNS insult. It encompasses the minimizing of such damage 
following a CNS insult. 

Preferably, TGF->?1 and/or analogues thereof are administered to the petiont directly. 
Aheraatively, a compound may be administered which upon adnunistration to the patient, increases 



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the active concentration of ^Gf'fi^ or naturally occurring analogues of TGF-)?1 in the CNS of the 
patient. For example, positively regulating binding proteins of or naturally occurring 

analogues thereof may be administered. 

Preferably, the pharmaceutical compositions described herein are administered in the period 
5 from the time of injury to 100 hours after the CNS insult and more preferably 0.5 to 8 hours after 
the CNS insult. 

In one embodiment of the invention, said and/or an analogue or analogues thereof is 

administered by lateral cerebro ventricular injection into the brain of a patient in the inclusive 
period from the time of the CNS insult to 8 hours thereafter. 
10 in another embodiment, TGF-j?1 and/or an analogue or analogues thereof is adnnnistered 

through a surgically inserted shunt into the cerebro ventricle of a patient in the inclusive period 
from the time of the CNS insult to 8 hours thereafter. 

In yet another embodiment, TGF-;?1 and/or an analogue or analogues thereof is administered 
peripherally into a patient for passage into the lateral ventricle of the brain in the inclusive period 
15 of from the time of the CNS insult to 8 hours thereafter. 

Preferably, it is TGf 0], rtself, that is administered by way of lateral cerebro ventricle injection or 
by use of the surgically inserted shunt. 

Preferably the pharmaceutical compositions are administered according to the pattern of 
injury or time lapsed after a CNS insult. 
20 Preferably the dosage range administered is from about 0.0001 to WO/jq of IGf-fi] or said 

analogue or said compound that elevates the concentration thereof per 100 grams of body weight. 

lGf'0] may be used alone or in conjunction with other therapeutic agents, including other 
growth factors designed to ameliorate against loss of CNS cells such as glia and non*choKnergic 
neurons. 

25 By "prevent" is meant a reduction in the severity of CNS damage suffered after a CNS 

insult and may also include inhibition of the symptoms of CNS damage. 

In yet a further aspect, the invention relates to the use of TGF-^1 and/or analogues thereof 
in the preparation of pharmaceutical compositions for treating CNS damage. 

Additionally, the invention comprises the use of a compound which, upon administration to a 
30 patient, increases the active concentration of TGF-)91 and/or naturally occurring analogues thereof 
in the CNS of the patient in the preparation of pharmaceutical compositions for treating injury to 
the CNS. 

The invention also provides pharmaceutical compositions suitable for treating CNS damage 
suffered after a CNS insult comprising TGF-)?1, and/or analogues thereof optionally provided in a 
35 pharmaceutically acceptable carrier or diluent. 

The pharmaceutical composition for treating CNS damage may also comprise a compound 
which, upon administration to the patient suffering CNS damage, increases the active concentration 
of IGF-1 and/or naturally occurring analogues thereof in the CNS of said patient. 



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4. 

RRIff DESCPIPTinN OF DRAWINGS 
Figure 1 shows composite drawings. Illustrating the distribution of TGF-Bl mRNA, following 

ischenu'c hypoxia for Example 1. 

Figure 2 is a histogram fflustrating the neuronal loss for TGF-Bl treated and control rats in 

5 Example 2. 

Rgure 3 is a histogram fliustrating the neuronal loss for TGF-Bl treated ami control rats in 
' Example 3. 

nFSRRIPTiON OF THF PRFFFRRED EM HODIMENTS 
10 The invention relates to a method of treating CNS damage suffered after a neural insult. 
For example, the patient may have suffered perinatal asphyxia or cerebral asphyxia or ischemia 
associated with a stroke or other non Bmiting examples of neural insults having been described 
eariier herein. In these instances, it is desirable to reduce or eliminate the symptoms of neural 
damage. 

15 CNS damage may for example be measured by the degree of permanent neural defidt 

cognitwe function, end/or propensity to seizure disorders. 

It is desirable that the concentration of TGF-Bl and/or analogues thereof in the central 
nervous system and in the brain of the patient in particular should be increased in order to treat 
the neural damage. Accordingly, TGF-Bl and/or analogues thereof can be administered directly to 

20 the patient. By TGF-Bl is meant transfomraig growth factor beta-1. By "analogues" (or 
-biologicany active analogues"! of TGF-Bl is meant compounds which exert a similar brological 
effect to TGF-Bl and includes naturally occurring analogues (eg. TGF-B2, TGF-B3, TGF-^4, TGF-^) 
or any of the known synthetic analogues of TGF-Bl. These compounds can be derived from 
humans or other animals. TGF-Bl and analogues can be purified from natural sources or produced 

25 by recombinant DNA techmques. 

Alternatively, compounds can be administered which, upon adnunistratton to the patient, 
increase the active concentration of TGF-Bl and/or naturally occurring analogues thereof m the 
central nervous system. By "actWe concentration" is meant the biological concentration of TGF-Bl 
and/or analogues in the central nervous system of the petient able to exert an effect on neural 

30 damage. 

TGF-Bl, its analogues, and compounds which elevate the active concentrations thereof can 
be administered centrally or systematically. Desirably the compositions are admirastered directly to 
the CNS of the patient and in particular to the region where the greatest damage has occurred. 
Accordingly, the compositions are adnunistered directly into the brain or cerebrospinal fluid by 
35 techniques including lateral ventricular through a burriiole or anterior fontanelle, lumbar or dstemal 
puncture or the Dke. The composhions also are administered by intravenous, intra-cerobrosplnal, 
irtrathecal, or intrasynovial routes. In addition, they may be administered with other agents or 
grovirth factors, for example, insulin-nke grovrth factor-1 (IGF-1). 



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For the prevention or treatment of CNS injury, the appropriate dosage of or one of 

its analogues or a compound capable of elevating the physiological concentrations of TGF-)?1, will 
depend on the type of injury to be treated, as defined above, the severity and course of the injury, 
whether such TGF*)?1 compositions are administered for preventive or therapeutic purposes, 
5 previous therapy, the patient's clinical history and response to the TGF-^1 compositions, and the 
discretion of the attending physician. The TGFv?1 compositions are suitably administered to the 
patient at one time or over a series of treatments. 

The foregoing examples show that the expression of TGF-B1 after a neural insult 
follows a specified time course and occurs in specified areas of the body. Accordingly, the 
10 compositions should be administered according to the pattern of CNS damage and time lapsed 
subsequent to an insult so as to produce the most de^rable results. 

The compositions may for example be administered about 0.5 to 100 hours after an insult. 
Alternetivdy, the composition may be administered prior to a potential CNS insult (e.g. prior to 
cardiac bypass surgery) so as to prevent or reduce the degree of neural damage suffered after 
15 insult. 

A suitable dosage range may for example be between about 0.0001 to 100//g of TGF-B1 
and/or analogues or compounds that elevate the concentration thereof per lOOgm of body weight 
where the composition is administered centrally. 

The invention also provides pharmaceutical compositions for treating neural damage suffered 

20 after an insult. The pharmaceutical compositions comprise T6F-B1 and/or analogues thereof or a 
compound which elevates the concentration of TGF-B1 in the CNS. TGF-B1, its analogue, and 
compounds that elevate the concentration thereof can be manufactured by recombinant DNA 
techniques such as those described in U.S. Patent No. 4,886,747. Alternatively, such substances 
an be isolated from natural sources. Optionally, such pharmaceutical compositions are provided in 

25 a pharmaceutically acceptable carrier or diluent that are inherently nontoxic and nontherapeutic. 
Examples of such carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum 
proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, 
potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts, or 
electrolytes such as protamine sulfate, disodium hydrogen phosphate, polysaccharides such as 

30 cellulose or methylcellulose, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal 
silica, magnesium trisQicate, polyvinyl pyrrolidone, and polyethylene glycol. Suitable diluents include 
sterile aqueous solutions comprising one or more of such carriers. TGF-)91 is typically formulated 
at an addic pH at which it is biologically active 

The invention is supported by the following experimental data. In the studies desi^bed in 

35 the following Examples, it was found that: 

1) TGF-B1 mRNA is expressed after a neural insult over a defined time course in specific 
regions of injury and TGF«)?1 itself can be detected by immunocytochemistry. 

2) Alterations in central nervous system levels of TGF-B1 can alter neural outcome 



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resulting as a consequence of a standardized neural insult 

3) Lower doses of TGF-Bl improve its efficacy in treating neural damage. 
These Examples, however, are offered by way of illustration only, and are not intended to limit the 
invention in any manner. AO patent and literature references cited throughout the specification are 
5 expressly incorporated. 

EXAMPLE 1 

Thp nbfectivB of this study was tn study the e x nrassion nf TGF-S1 in the central nervous 

system after a neural insult. 

10 Twenty one day old rats were subjected to unilateral carotid figation followed by inhalational 
asphyxia under defined conditions to produce either mild or severa neuronal loss on the figated side. 

M9d or severe neuronal loss was induced in 21 day rates as follows: The right carotid 
artery was ligated under fight halothane anaesthesia. They were then placed in an Incubator at 
34'C and 85% humidity. The inspired gases were replaced by 8% Oj In nitrogen for 15 mimites 

15 (mad) or 90 minutes (severe) then returned to eir. At various times after hypona |1 hour, 5 hours, 
3 and 5 days) the animals were anaesthetized with pentobarbitone (Nembutal), the brains removed 
and snap frozen on dry Ice for in situ hybridization. For histology, rats were sacrificed 5 days 
after hypoxia and then perfused with 0.9% saline followed by formaldehyde-acetic arid-mathanol 
(1:1:8). 

20 At defined times after the a^hyxia the rats were sacrificed for histology. After 90 minutes 

asphyxia (severe) neuronal loss was assessed by thionine/acid fudisin stain was widespread witWn 
the figated cortex. There was severe loss of neurons in the naddle cerebral artery territory, 
including the lateral cortex, hippocampus, striatum and thalamus. In situ hybridization 
histochemistry was performed using a TGF-Bl cONA probe comprising neariy the entire coding 

25 sequence of TGF-Bl, provided by Dr. R. Derynck. Hybridization histochemistry was performed 

essentially as described in McCabe, et aL, .1 Histnchem. Cvtochem.. 34:45-50 (1986) and bi Smith, 
et al., Ann. NeuroDhathoL. 64:319-332 (1984). 

After hybridization, the sections were washed 4 times in 2xSSC plus lOmM fi- 
mercaptoethanol at room temperature for 10 minutes each, 4 times in 2xSSC at room temperature 

30 for 10 minutes each, twice in 2xSSC at SO'C for 10 minutes each. 

Controls were performed using RNAase A (40//g/ml 0.5M NaCI / 20mM Tris, pH 7.5 / ImM 
EDTA at 37'C). RNAase pretreatment almost entirely depressed the signal Northern blots on each 
probe revealed the antidpated major band at 2.5kb. 

The resulting signal for TGF-Bl mRNA as measured by in situ hybridization showed an 

35 induction of the TGF-Bl mRNA restricted to the areas of neuronal damage. FoBowing mild 

asphyxia (15 minutes). Induction of TGF-Bl mRNA was observed in the figated brain in layer 3 of 
the cerebral cortex, the dentate gyrus, CAl and CA 2 regions of the pyramidal layer of the 
hippocampus. 



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Following severe asphyxia (90 minutes), TGF*61 mRNA was detectable by one hour post 
insult in the hippocampal dentate gyrus, CA 1 and CA 2 regions, and choroid plexus. By 5 hours it 
was detectable in the cortex and striatum on the ligated side. By 72 hours marked expression 
was observed throughout the whole cerebral and puriform cortex, striatum, thalamus and 
5 hippocampus of the ligated side but no expression was observed on the non-ligated side in which 
no neuronal death was observed (Figure 1). 

The specificity of the induction was demonstrated by predominately unilateral expression on 
the ligated side, lesser induction in animals subjected to a lesser insult and by negative controls 
using RNAase A. The probe was also used to hybridize a Northern blot of rat liver poly(A) RNA 
10 samples. The bands after hybridization to the TGF B1 probe are in agreement with the data 
reported in the literature. 

Immunohistochemistry was performed using anti h rabbit TGF-B1 polyclonal anti-serum. Ceils 
staining for TGF B1 could be identified in the damaged region of the ligated hemisphere. This 
staining was seen in cells with macrophage-like appearance. 
15 The data suggests that following an hypoxic ischendc msult, TGF*B1 is induced in 

macrophages, particularly in the area of damage. 

EXAMPLE 2 

The objective of this study was to assess the effect of administerina TGF-BI after a neural 

20 insult . 

Adult rats (250-350 grams) were used. The experiment involved treating the rats with TGF- 
BI after a neural insult. These rats had an hypoxic-ischenuc insult to one cerebral hemisphere 
induced in a standard manner. One carotid artery was ligated and the animal was subjected two 
hours later to a defined period of inhalational hypoxia. The degree, length of hypoxia, ambient 
25 temperature and humidity were defined to standardize the degree of damage. The conditions were 
inhaled oxygen (6%), 10 minutes of hypoxia at ambient temperature of 3V and 85% hunudity. The 
animals were maintained in an incubator for one hour then returned to their standard cages. They 
were sacrificed five days later for histological analysis using stains (acid fuchsin) specific for 
necrotic neurons. 

30 In such experiments typical neuronal death is restricted to the side of the side of arterial 

ligation and is primarily in the hippocampus, dentate gyrus and lateral cortex of the ligated 
hemisphere. 

Unilateral hypoxic-ischemic injury was induced in adult (300 4^1 Og) male Wistar rats. The 
rats underwent unilateral carotid ligation under light halothane anaesthesia. Following one hour 
35 recovery they were placed in an incubator at 31°C and 85±5% humidity for one hour before insult. 
They were subject to 10 ndnutes inhalational asphyxia (Fi02 6.0%) and maintained in the incubator 
for one hour after asphyxia. Two hours after the termination of the inhalational insult, a single 



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stereotaxically controlled lateral cerebrovsntricuiar Injection of either 0.05//g recombinant TGF-B1 or 
artificial cerebrospinal fluid (CSF) was given. 

Recombinant TGF-Bl or diluent was prepared and administered to waght-matched pairs as 
follows: Two hours after asphyxia the rats were given a light helothane anaesthetic, and a single 
5 ICV injection of either 20pl of CSF (n-6) or TD/A of CSF plus COSjug TGF-Bl |n-6) was given. 
Recombinant TGF B1 (Genentech, Inc., South San Francisco, Cafiforaia 94080 USA) was dissolved 
in the CSF dfluent at 2.5jug/ml. This solution was diluted 9 times with 0.15M PBS (phosphate 
buffered saline) givring a pH of 7.0. 

The animals were then maintained for 120 hours, anaesthetized and the brains fixed jn gtu 
10 with formaldehyde-acetic acid-methanol (1:1:8) for histological assessment. 

Surviving and dead neurons were discriminate with the use of an thionin/add fuschin staining 
technique. WJIiams, et al., PeOiS. 22:561-565 (1990); Brown, at al., J. Neurol Sd. , 16:59-84 
(1971). 

The degree of neural damage suffered was quantified by measuring the neuronal loss score. 
15 The neuronal loss scores are the average from the susceptflile regions of tiie hippocan^ius and 
cerebral cortex (100% equals total loss of neurons, 0% equals 0 loss). 

The percentage of dead neurons was estimated by two Independent observers, one of whom 
was blinded to the experiment The correlation between scores obtained by tha two observers 
was r-0.92 p,0.0001. The effect of treatment was evahiated with MANOVA followed by pair 
20 wise comparisons of each region using Fisher's least-agnificant-diffBrence procedure. 

The results are shown in figure 2. TGF-Bl therapy reduced the extent of nouronel death m 
the figated hemisphere compared to the CSF-treated conUols (p<0.01). A single central injection 
of TGF-Bl foDowing an a^hyxial insult in the adult rat was assoaated witfi a marked inq)rovanent 
m outcome as assessed histologicdiy (See Table 1). 
75 Table 1: Effect of TGF-Bl treatment on percent neuronal loss following hypoxic-ischemc inpiry (6 
groups; mean +. sem). 



Region 
(0.0!^g) 

Hippocanqial CA1-2 
Hippocan^al CAS 
Hippocampal CA4 
Dentate gyrus 
Pyriform cortex 
Lateral cortex 



% Naurand las qm treatment witb 
CSF (control) TGHJl 



30 



98.4+4 

100.0 

100.00 

97.2+3 

93.2+7 

98.0+2 



7.6+5 
4.0+4 



0.0 
0.0 
0.0 



35 



5.0+5 



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EXAMPLE 3 

ThB objective of this study was to confirm the observations of Example 2 and establish the 
most effective dosage range . 

The experiment was the same to that of Example 2 except that two further groups were 
5 treated with higher doses of TGF-B1. 

Hypoxic-ischemic insult was induced in rats as discussed for Example 2. Rats (n-6 for 
each treatment) were administered either CSF, CSF+0.05//g TGF-B1, CSF + O.Bjjq TGF-B1 or CSF 
4 5/jq TGF-B1 two hours after the inhalational insult. Rats (n-1) from each treatment were 
treated simultaneously. The same techniques for measuring the degree of insult at those discussed 
10 for Example 2 where employed. 

The results are shown in Figure 3. As can be seen, 5//g TGF-&1 had no significant effect 
on neuronal loss. On the other hand, O.Sjjg TGF-B1 reduced (p<0.05) neuronal loss in the 
cerebral cortex but 0.05//g TGF-B1 was significantly (p<0.05) more effective. Similar effects 
were seen in other neuronal areas. Further experiments have shown that dosages in the range of 
15 about 0.01//g to 0.05//g are most effective. 

DISCUSSION 

The results of the experiments described above were statistically highly significant. In 
Examples 2 and 3, where TGF-fil was given post-asphyxia, T6F-B1 therapy at doses less than 

20 0.5;/g/rat or lower markedly improved outcome compared to CSF treated controls. We therefore 
conclude that therapeutic elevation of T6F-B1 in the cerebral spinal fluid either directly or indirectly 
after an insult is advantageous to outcome. The results in Example 3 further show that the 
greatest efficacy is seen at low doses of TGF-B1. 

The present invention, therefore, recognizes the role of an administration of TGF-;91 and/or 

25 other compounds of similar effect into a patient prior to, simultaneous with, or following a CNS 
insult with the consequential result that CNS damage is m'mintized by preventing the otherwise 
consequential damage that otherwise would occur following the injury. The invention provides 
methods and pharmaceutical compositions for treating or for preventing neural damage. Neural 
damage may be associated with asphyxia, hypoxia, toxins, ischemia or trauma. Although it will be 

30 appreciated that the main application of the invention is to humans, the usefulness of the invention 
is not limited thereto and treatment of non-human animals (especially mammals) is also within the 
scope of the invention. 



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ID- 
CLAIMS 

What is daitned is: 

1. A method of treating central narvous system injury in a mammal, comprising administering 
to the central nervous system of said mammal an effective amount of TGF-^1 or a biologically 

5 acthre anaioguB of TGF-;ffl. 

2. A method of claim 1 vifheran the centtal nervous system injury is hypojdc 'mpiry. 

3. A method of daim 1 vnheran the central nervous system mpry is ischemic injury. 

4. A method of daim 1 wherein the central nervous system injury is traumatic injury. 

5. A method of daim 1 wherein the central nervous system injury affects nonxholinergic 
10 neuronal cells. 

6. A method of daim 1 wheron the central nervous system injury affects gHal cells. 

7. A method of daim 1 wherein the central nervous system injury is a consequence of 
Parldnson's disease. 

8. A method of daim 1 wherein the central nervous system injury is a consequence of multiple 
15 sderosis. 

9. A method of daim 1 wherein tiie central nervous system injury is a consequence of a 
demyelinating disorder. 

10. A method of daim 1 wherein the TGF-;?1 or hiologically active analogue of TGF-^1 Is 
administered in the period from the time of the central nervous system inpiry to 100 hours after 

20 the injury. 

n. A method of daim 1 wherein the TGF-^1 or bidogically active analogue of TGF-^1 is 
adninistered at least once in the period from the time of the central nervous system injury to 
about 8 hours subsequentiy. 

12. A metiiod of daim 1 wherein tiie TGF-jSI or biologicaUy active analogue of TGF-^1 is 

25 administered to tiie mammal in an amount from about 0.0001 to 100//g of TGF-^1 per lOOgm of 
body wnght of the mammal. 

13. A method of daim 1 wherem the biologically active analogue of TGF-^1 is selected from 
tiie group consisting of TGF-i82, m fiU, TGF-^, TGF-jS2,3, TGF-;?4, ami TGF-^. 

14. A method of daim 1 wherem the TGF-^I or biologically active analogue of IGF-1 is 

30 admmistered to the mammal through a surgically Inserted shunt into the cerebro ventride of the 
mammal. 

15. A method of daim 1 wherein tiie TGF-iffl or biologically active analogue of TGF-^1 is 
administered peripherally into tiie mammal for passage into the lateral ventride of the brain. 



wo 93/09802 



PCT/US92/09974 



1/3 



C 




wo 93/09802 



PCr/US92/09974 



2/3 



FIG.2 



Hippoc. CAl 



Hippoc. CAS 



Hippoc. CA4 

Dentate Gyrus 
Pyriform Cortex 

Lateral Cortex 




Vehicle 
0.05/2g 



r 
0 



■ 




— 1 1 1 1 r- 

20 40 ^ 60 80 1 00 



Neuronal loss score 

sem 



r.UDSTiTUTE SHEET 



wo 93/09802 PCT/US92/09974 

3/3 



FIG.3 



Cortical neuronal 
loss score 
100 -1 




SUBSTITUTE SHEET 



PATENT COOPERATION TREAT'^ 

PGT 

DECLARITION OF NON-ESTABLISHMENT OF INTERNATIONAL SEARCH REPORT 

(PCT Artide 17(2)(a) aod Rule 39) 



Applicant's or agent's file reference 

800P1 


IMPORTANT DECLARATION 


Date of mvi^mzidaylmomhfyear) 

1 a OZ 93 


International application No. 

PCT/ US 92/09974 


International filing 4zX6( day f month/year) 

20/11/92 


(Earliest) Priority daite(dayimonthlyear) 

zz/nm 


International Patent Classification (IPC) or both national classification and IPC 


IPCS: A 61 K 37/02 


Applicant 

GENENTECH, INC. et al. 



This International Searching Authority hereby declares, according to Article 17(2)(a), that no tnteniational search report wiQ 
be rsta Ji Ush ed on the international appUcaticn tar the reasons indicated tmiow 

1. [Y] The subject matter ot the international application relates to: 

a. I [ scientific theories, 

b. I [mathematical theroies 
c I I plant varieties. 

d. animal varieties. 

e. Q essentially biological processes for the production of plants and animals, other than microbiological processes 

and the products of such processes. 

f. sctiemes, rules or methods of doing business. 

g. I 1 schemes, rules or memcxts of performmg purely mental acts. 

h. I [schemes, rules or methods of playing games. 

i* \X} methods fdr treatment of me human body by surgery or therapy, 
j. \X\ methods for treatment of the animal body by surgery or therapy. 
Ic Q diagnostic methtxis practised on the human or antmal body. 
I. Q mere presentations of tnformatioa 

ni. 1^ computer programs fdr which this International Searching Authority is not equipped to search prior art. 

2. Q The failure of the following parts of ttie international application to comply with prescribed requirements prevents a 

meaningful search from being carried out 



I I the description 



fx] thedatms 



I I the drawing 



^' D failure of the nudeoOde and/or amino add sequence listing to comply with the prescribed requirements prevents a 
meaningful search from being earned out: 

it does not comply with the prescribed standard 
I I it is not in me presaibed machine readable form 
4. Further comments: 



Name and mailing address of the Internaiional Searching Authority 

European Patent OfHoe, P.B. 5818 Patentlaan2 
NL-2280 HV Rijswijk 
Tel. (+31-70) 340.2040, Tx. 31 651 epo nl. 
Fax: (+31-70> 340.3016 



Authorized ofTtoer 

BOHM e.h. 



Form PCr/ISA/203 (July 1992) 



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