CA2266659A1 - Pretreatment with growth factors to protect against cns damage - Google Patents

Abstract

Methods for protecting neural tissue from the effects of aging, trauma, toxic insult, neurological diseases or disorders comprise administering growth factors to the neural tissue of a mammal at a time prior to the onset of trauma or the manifestations of neurological disease or aging. The growth factors induce multipotent neural stem cells and neural stem cell progeny to proliferate and generate new neural cells which provide supportive and protective roles against the effects of aging, trauma, toxic insult, neurological diseases or disorders.

Description

CA 022666~9 l999-03-l7 PRETREATMENT WITH GROWTH FACTORS TO PROTECT AGAINST CNS
DAMAGE

Field of the Invention:
This invention relates to methods for protecting neural cells against injury and death, 5 as a result of trauma, neurodegenerative disease processes, toxic insult, malfunction~
or aging by providing factors to the central nervous system of a subject.

Back~rollnll of the Invention-Neurodegcne.~ /e ~licPqces such as Parkinson's disease, ~ h~im~r's disease, T~nntington's disease, multiple and amyotrophic lateral sclerosis, stroke, 10 schizophrenia, epilepsy and diabetic peripheral n~UlOpdlhy are ~licP~ces or disorders which affect millions of people. It is the loss of normal neuronal function which produces the behavioral and physical deficits which are characteristic of each of the different neurological disorders. In addition to chronic and acute neurodegtl~la~i~/e disorders, the aging process and physical trauma to the central nervous system both 15 result in the loss of neural cells accompqni~od by the associated behavioral and physical deficits. In recent years neurological disorders have become an important concern due to the expqn~lin~ elderly population which is at greà~ l risk for these disorders.

Recently, in vitro and in vivo studies have shown that ,q,.l,.,;,,icl,dljon of certain 20 agents prior to the oc~ e"ce of a neurological insult are capable of exerting a protective effect, resllhing in a decrease in neuronal loss. U.S. Patent No.
5,519,035, issued on May 21, 1996, provides a method of treating a patient at risk CA 022666~9 1999-03-17 for stroke with protein kinase C inhibitor in order to protect neuronal cells from death as a result of cerebral ischemia. Sufficient protein kinase C inhibitor isminictered on a daily basis to ensure that, should an i.cchPrnir insult occur, the levels of the agent would be high enough to counteract the neurotoxic effects of5 nitric oxide produced during the ischemic event.

Parkinson's disease, characterized by the loss of dopamine neurons in the nigro-striatal pathway, is a relatively common neurological disorder. The dop~ ic neurotoxin, l-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces parkinsonian sy~ o-lls when a~mini.ctered to humans and is frequently used to 1 0 induce parkinsonionism in animal models of Parkinson's disease. Tomac et al.(1995) (Nature 373 pp 33S-339) reported that glial cell line derived neurotrophic factor (GDNF) has a protective effect on dopdlllill~lgic neurons when the factor is injectP~ into mouse brain 24 hours before ~lminictration of MPTP. Similar results were reported for the neuloprotective effects of GDNF on neurons in the locus 1 5 coeruleus. Grafting of genPtir~lly engillee.ed fibroblasts expressing high levels of GDNF into the locus coeruleus, 24 hours before a~ ,c~.aLion of another do~u.lillc~;ic neurotoxin, 6-hydroxydol.~,lhle (6-OHDA), prevented more than 80% of the 6-OHDA-in~re~l degel~ ion of noradle"~ ic n~ulolls in that region (Arenas et al., Neurons 15: 1465-1473 [1995]). Basic fibroblast growth factor 20 (bFGF or FGF-2) has a protective role for nigrostriatal cells exposed to MPTP or methylpyridini--mion (MPP ) (Park & Mytilineou Brain Res. 599:83-97 [1992~);
Chadi et al. Exp. Brain Res. 97 :145-158 (1993); Otto and Unsicker J. Neurosci.
~es. 34:382-393 (1993). Results from in vitro and in vivo studies inrljc~t~cl that FGF-2, ~minictPred for a period of up to 4 days before exposure to the neurotoxins 25 and ~-lmini.ctered for several days after exposure to the toxins si~nifir~ntly decreased the number of dop~ in~ic neurons damaged by the toxins. Similar results were reported with EGF on cultured cells (Park & Mytilineou, supra). In addition, FGF-2 has been shown to have nGulolJlolective effects in the hippocampus. Koketsu etal. (Ann. Neurol. 35 :451~57 [1994]) reported that FGF-2~ "-i.,;.~L~,led by 30 continuous intracG,l,lo~ kicular (icv) infusion for 3 days before and 1 day after focal cerebral i.cchr.~ reduced the infarct size produced by the icchPn-ir rh~llPn~e.

They conclude that the protective effect observed was due to direct trophic action of FGF-2 on neulolls, via ne-l-ollal gene e~r~ssion that antagonizes "cell death"
programs, or due to trophic effects on glial cells and blood vessels. However, no evidence for glial cell proliferation was observed. Liu et al. Brain ~es. 626:335-5 338 (1993) reported that FGF-2, delivered icv for 2 days prior to, and 5 days after inLIdp~liLolleal injections of the seizure-inducing neurotoxin kainic acid did not affect the behavioral responses to the toxin but did prevent n~llronal cell loss in thehippocampus. They suggest the plol~-,live effect of the growth factor on the neurons is due to FGF-2 prevention of the rise in n~ulo"al intracellular c~lchlm1 0 levels which normally occurs in response to kainic acid ~rlminictratjon.

U.S. Patent 5,438,121, issued on August 1, 1995, relating to brain-derived neurotrophic factor (BDNF), disclosed that BDNF may be added to cell cultures 24hours prior to exposure to the neurotoxins MPP and 6-OHDA to reduce the amount of cellular damage caused by the llCUloto~ s. U.S. Patent 5,554,601, issued on 1 5 Se~ttl,l~el 10, 1996 dicclose(~ a method of plU~ illg cells in the central llel~OUS
system from cell death ~uu~lgh the stim~ tion of the production of ~ olro~hic growth factors in o~,~rc~L~ ll~i~ed animals. The method relies on the chronic a.l,.,i"i~iLl~tion of an estrogen compound having a unique structure which allows it to cross the blood brain barrier in order to st;m~ t~ the production of neuloL,uphic 20 growth factor proteins such as nerve growth factor (NGF) and BDNF. The effect of the esL,og~ compounds was believed to be due to their prote~ e actions against hypoglycemia and excitatory amino acids, and their stim~ tory effects on nt:ulollophic growth factor production in ovarectomi7~od anim~lc. It was noted ~at the protective effect was not due to a mitogenic effect of the steroid on neural tissue.

25 International application no. WO 95/13364 discloses the ~l,..;n;~l.ation of growth factors to neural tissue in vivo to induce a patient's stem cells to divide to replace cells that have been ll~m~ged as a result of disease.

,, S~ of thf Inve~tion Methods are provided for protecting m~mm~ n neural tissue from trauma or insult or from the manifestations of neurological disease or disorders or the aging process.
The method comprises ~minictering to a m~mm~l an effective amount of one or 5 more growth factors to induce multipotent neural stem cell proliferation whichresults in a ~loLecli~/e effect on the neural tissue. The growth factor treatmf nt can be completed while the neural tissue is still healthy to provided a prolonged protective effect which lasts weeks and even months after cessation of the tre~tmf nt Gro~ t~. fact~rs s~ 3 E _F, ~lp~;ircgulill, rlbrobiasl growtn factor, I.all~lo~ .g 10 growth factor alpha, and the like, and combinations thereof can be exogenously ~(lminictered to the m~mm~l, for example, to one or more CNS ventricles of the m~mm~l. Alternatively, the growth factors can be ~timini.ctered by genetically modifying cells of the m~mm~ n neural tissue to produce the growth factors.

The growth factors are a-imini.ctf. red at a time prior to the onset of the trauma or the 15 manifestations of the disease or aging process, in amounts sufficient to cause the neural cell population to be protected from progressive cell darnage leading to the death of the cells or their malfunction.

The invention is also di~ ed to the use of a growth factor, or a combination of growth factors in the m~m-f~rtnre of a m~ mrnt for the preventative tre~tmrnt of20 neurological or neurode,gf~ Li~re ~lic~o~ces or disorders? neurological traurna? and/or the aging of neural tissue.

Desc, ~ on of thf Fi.gl-re:
Figs. lA-lF depict photomicrographs of Nissl stained coronal sections (lO ~m, bregma -3.3 mm) of control (no pre~r.~t..~f~/~t, non-icrhfmir; lA, lB) or one week 25 post-icchf~mir (lC to 2F) rats that received either vehicle (lC and lD) or EGF (lE
and lF) i.c.v. infusion t months prior to an icrh.omir insult. CAl neulollv are present in the non-icchfmir controls (lA and lB) and in the EGF-~,cLlcaLed i~chf ~ir ~nim~lc (lE and lF), but are absent in the vehicle-pre~eated icrhf mic RECTIFIED SH~ET (RULE 91) ISA~EP

CA 022666~9 1999-03-17 anim~l~ (lC and lD), demonstrating that EGF p~cLle~ can protect against icch~miq.-inrlucerl cell death in the CA1 region of the hippocampus.

Detailed Descr~ption of th~- Invention The present invention provides methods for the protection of neural tissue from the 5 effects of aging, trauma, toxic insult, neurological ~i~e~q~es or disorders. Growth factors are q,-1mini~tered to the neural tissue, at a ti ne prior to the onset of trauma or the mal~ires~Lions of neurological disease or aging, to induce multipotent neural stem cells and neural stem cell progeny to proliferate and gel~,ate progeny cells that are capable of dir~ ting into neurons, astrocytes, and oligodendrocytes.
10 Astrocytes in particular have been shown to provide a supportive and protective role in the brain. These cells buffer the en~/holllllelll around neurons and secrete factors which enhance the survival and function of the neurons. Normally, ast..)cy turnover in the CNS is limited and large scale repl~c~ t of dysfunctional astrocytes has not been reported.

15 A "mulLipotelll neural stem cell" is an undirre.~L ~ d neural cell which is capable of extensive self renewal, i.e., is capable of replacing itself during cell division over an extended period of time, and is capable of generating the major cell types of the tissues in which it is located (i.e. neurons and glia - astrocytes and oligodendrocytes). The non-stem cell progeny of a neural stem cell are termed 20 progenitor cells. Methods of proliferating neural stem cells in vitro and in vivo have been previously described (see e.g. WO 93/0127~, WO 94/10292, and U.S. Ser.
No. 08/486,648).

The term "neural progenitor cell", as used herein, refers to an undirr~lc~ (l cell derived from a neural stem cell, and is not itself a stem cell. A distinguishing25 feature of a prog~niLol cell is that, unlike a stem cell, it has limited prolif~"~tive ability and thus does not exhibit self-mqi-~ re. It is co~ d to a particular path of dirr~ iation and will, under ap~ iat~ conditions, eventually dirf~,.ellLiate into either glia (astrocytes or oligodendlocyLes) or neurons, and is thus not multipotent.

CA 022666~9 1999-03-17 Wo 98/22127 PCT/CAg7/008~9 The term "precursor cells", as used herein, refers to the progeny of neural stemcells, and thus includes both progenitor cells and t~ ghtPr neural stem cells.

The majority of neural stem cells can be found in the tissue lining the CNS
ventricles (including the subependyma) of adult m~mm~l~. The term "ventricle"
5 refers to any cavity or passageway within the CNS through which cerebral spinal fluid flows and includes any collapsed portions of the ventricular system. Thus, the term not only encomp~c.ses the lateral, third, and fourth ventricles, but also encomp~cses the central canal, cerebral ~ e~lct, and other CNS cavities and collapsed CNS cavities.

10 One or more growth factors may be used to induce the proliferation, migration and/
or di~Çele~lLiaLion of the multipotent neural stem cells/and or their progeny in vivo.
As used herein, the term "growth factor" refers to a protein, peptide or other molecule having a growth, proliferative, di~re.~ iative, or tropic effect on neural stem cells and/or neural stem cell progeny. Growth factors which may be used for15 inducing proliferation include any factor that allows neural stem cells and ~cul~or cells to proliferate, inr~ ing any molecule which binds to a lece~L()l on the surface of the neural stem cell to exert a tropic, or growth-inducing effect on the cell, including any protein, amino acid, vitamin, carbohydrate, or other molecule or atom. Pl~r~llcd proliferation-inducing growth factors include EGF, amphiregulin,20 acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), L~al~ lg growth factor alpha (TGFa), proliferation-inducing ligands which bind to the EGF and FGF l~ce~tols, and combinations thereof. A
plefel,ed colllbinalion of proliferation-inducing growth factors is EGF or TGFa with FGF-1 or FGF-2. To detellllh,e whether a particular growth factor will have25 proliferative effects when ~lminictered in vivo, the effects of the growth factor on in vi~ro neural stem cell proliferation can be tested using mPthofic already known in the art and disclosed in WO 93/01275, WO 94/10292, and U.S. Ser. No. 08/486,648.
Growth factors that induce neural stem cell proliferation in vitro have been found to also have proliferative effects in vivo.

WO 98122127 PCT/CA97hf)0859 Experimental results in~ir~te that neural stem cell progeny that have been indllf ed to proliferate in vivo by ~flminictration of growth factors undergo dirr~len~iation into cell types that are beneficial to the treated area. A likely mf ch~ni.~m is that the neural stem cell progeny respond to extrinsic signals that influence them to 5 di~Ç~lwlLiate into needed cell types. Dirr lenliated progeny of neural stem cells include n~uluns, astrocytes (type I or type 2) and oligodendrocytes. The dirr~lcll~iative pathway of the neural stem cell progeny can be influenced by the addition of exogenous growth factors that increase the numbers of a particular cell type that are gen~ ed. Difrelelllialion-infl~Pnring growth factors that can be 10 exogenously added are disclosed in WO 94/10292 and U.S. Ser. No. 08/486,648.
Among the growth factors and other molecules that can be used to infl~nPnre the dirrelellLiation of precursor cells are FGF-1, FGF-2, ciliary n~-lrulruuhic factor (CNTF), NGF, BDNF, neulull~l.hin 3, n~ulol~ophil1 4, interleukins, lellk~mi~
inhibitory factor (LIF), cyclic af1enosinP monophosph~te, forskolin, high levels of 15 pot~csillm, amphiregulin, TGF-a, TGF-B, insulin-like growth &ctors, flP~...f !h~cone (glucocorticoid hormone), isobutyl 3-methylx~nthin~o~ sf~m~tost~tin, growth hormûne, retinoic acid, and PDGF, or ligands which bind to the l'eCe~101:~
for these growth factors.

The effects of various growth factors on dirr~lenliation can be tested in vitro on 20 cultures of multipotent neural stem cell progeny by using dual-label immllnc)cytorllf~mi~try various ~c:~llonal- and glial-specific antibodies, the effect of the exogenous growth factors on the dirr~.el.l;~tiQn of the cells can be ~le~ Pri.
Type I astrocytes, which are ~lirr~ tf~f~ glial cells, can be if1entifif~d by their imml-n--reactivity for GFAP but not A2B5. Type II astrocytes, which are 25 ~lirl~ d glial cells that display a stellate process-bearing morphology, can be identified using imml-nocytof ~P~n;. ~. y by their phenotype GFAP(+), A2BS(+) phenotype.

Growth factors can be af~ ,ed singly or in colllbil~ion to a patient. They can 30 also be ~ ,ed in a ~f ll.pol~l seql~enre (e.g. exposure to a first growth factor infl~PnrPs the expression of a second growth factor lecepLor, Ntqnron 4:189-201 CA 022666~9 1999-03-17 (1990). The growth factors may be prepared in a ph~rln~e~ltir~lly-~ccept~hle excipient. A~1minictration of the growth factors can be done by any method, including injection c~nn~ or injection to tne CNS, peripheral injection, timed-release apparati which can ~mini~t~r substances at the desired site, oral 5 a~1mini~tration, and the like. Growth factors can be ~r~mini~tered using methods in which the factors may either pass through or by-pass the blood-brain barrier.
Methods for allowing factors to pass through the blood-brain barrier include mi.~i"~i~it~g the size of the factor, or providing hydrophobic factors which may pass through more easily. For example, dimethyl sulfoxide (DMSO) or the like can be 10 ~llmini~tered to reversibly open the blood-brain barrier to allow the passage of intravenously (i.v.) h,llapeliLoneally (i.p.), or orally ~rimini~tered growth factor into the CNS. Doses of DMSO in mice would be from 2-30% DMSO in a volume of approximately 0.25 ml, ~minictered i.v., preferably 10-15%. An i,lllapeliLoneal Lion of DMSO and growth factor would require from 10-30% DMSO. It 15 would be possible to use Boradeption (Science 217: 166 (1982)) Another possibility would be to bind the growth factor to llal~rtllill to ~la~rcl it across the blood brain barrier.

It is ~lcsell~ly prcr~.lcd to ~ growth factors directly to one or more ventricles of the CNS. The fact that the majority of neural stem cells are located in 20 the tissues lining ventricles of mature brains offers several advantages for the mo-lific~tion and m~nip~ tion of these cells in vivo. Tre~tm~nt can be tailored accordingly so tnat stem cells ~ull~ullding ventricles near the desired region would be manipulated or mot1ifi~tl in vivo using the methods described herein. The ve.l~licular system is found in nearly all brain regions and thus allows easier access 25 to the desired areas. If one wants to modify the stem cells in vivo by exposing them to a composition colll~lisillg a growth factor or a viral vector, it is relatively easy to implant a device that a~imini~ters the composition to the ventricle and thus, to the neural stem cells. For example, a cannula ~tt~cl ~d to an osmotic pump may be used to deliver the composition. Alt~.llalively, the composition may be inject~(l directly 30 into the ventricles. The neural stem cell progeny can migrate into regions that may be subjected to possible damage as a result of injury, disease, or aging.

Wo 98/22127 PCT/CAg7/00859 g Furthermore, th~ ~lose proximity of the ventricles to many brain regions would allow for the diffilsion of a secreted neurological agent by the stem cells or their progeny to the app~ .iate region.

In addition, or as an alternative to exogenously ~rimini~tering growth factors to 5 precursor cells, the precursor cells can be ge~tir~lly modified in vivo by transfection of the cells with growth factor or hormone-e~ ssh~g vectors, so that the neural cells express various biological agents useful in the prevention of neurolo~ical disorders, trauma and the effects of aging. Methods for geneticallymodifying multipotent neural stem cells and their progeny are disclosed in published 10 international application no. WO 94/16718. In addition to genetic mo~iific~tion of the cells to express growth factors, the cells may be modified to express other types of neurological agents such as neu~ ilt~

Preferably, the genetic rno~ifir~tion is pelrulll,ed either by infection of the cells lining ventricular regions with recon1bil~nt retroviruses or transfection using 15 methods known in the art including CaPO4 llal~c~ion, DEAE-dextran transfection, polybrene transfection, by protoplast fusion, elec~ru~ola~ion, lipofection, and the like ~see Maniatis et al., Molecular Cloni~: A T ~horatory M~n~l (1989), 2nd Ed., Cold Spring Harbor, N.Y.]. Any method of genetic morlifir~tion, now known or later developed can be used. With direct DNA transfection, cells could be modified 20 by particle bombardment, receptor m~di~t~ delivery, and cationic liposomes.
When chimeric gene constructs are used, they generally will contain viral, for example retroviral long terminal repeat (LTR), simian virus 40 (SV40), cytomegalovirus (CMV); or ~.. ,.. ~li~n cell-specific promoters such as those for TH, DBH, phenylethanolamine N-methyltransferase, ChAT, GFAP, NSE, the NF
25 proteins (NF-L, NF-M, NF-H, and the like) that direct the e~lcsSiOn of the structural genes encoding the desired protein.

Any expression vector known in the art can be used to express the growth factor, as long as it has a promoter which is active in the cell, and applo~liate ~ inn and polyadenylation signals. These expression vectors include recombinant vaccinia .. ,.. . ... ~ .. .

CA 022666~9 lgg9-o3-l7 wo 98/22127 PcT/CA97/0~859 virus vectors including pSCll, or vectors derived various viruses such as from Simian Virus 40 (SV40, i.e. pSV2-dhfr, pSV2neo, pko-neo, pSV2gpt, pSVT7 and pBABY), from Rous Sarcoma Virus (RSV, i.e. pRSVneo), from mouse IllAllllllAly tumor virus (MMTV, i.e. pMSG), from adenovirus(pMT2), from herpes simplex 5 virus (HSV, i.e. pTK2 and pHyg), from bovine papillomavirus (BPV? i.e. pdBPV
and pBV-lMTHA), from Epstein-Barr Virus (EBV, i.e. p205 and pHEBo) or any other eukaryotic expression vector known in the art. If a retroviral construct is to be used to ge~tir~11y modify normally quiescent stem cells, then it is preferable to induce the proliferation of these cells using the methods described herein. For 10 example, an osmotic infusion pump could be used to deliver growth factors to the central canal several days prior to infection with the retrovirus. This assures that there will be actively dividing neural stem cells which are susceptible to infection with the retrovirus.

According to this invention, the deficits in movement, cognition, memory and other 15 behavioral S~ lOlllS which normally occur in ~ubjecL~ with neurological disorders caused by disease, trauma or aging or a combination of these factors are reduced as a con.~ecl~1enre of preventative ~ dLion of a growtn factor or growth factors capable of stimn1~ting the proliferation of multipotent neural stem cells and/or their progeny. The growth factor ~leLlcALIllent method achieves a prolonged protective20 effect, thereby e1iminAting the need for the exogenously a~mini.~tered growth factor to be present at the time of the trauma, disease, or disorder. The term "p.oLecli~re effect" means that the growth factor l-eA~ lr results in a ~ignifirAnt1y greaterllu llbel of normally functioning neural cells than would have otherwise been without the growth factor pre-llcA~ The growth factors may be ~ ,ed on an 25 ongoing basis or at regular intervals prior to the ~~I~Çe~L~Lions of aging orneurological disorders or disease and from one week to several weeks prior to ananticipated neurological trauma or insult, such as what might occur during brainsurgery. As used herein, the term "manifestation" refers to the outward sign of a neurological disease or disorder or the aging process. For example, clinical 30 memory loss is an oulw~.rd sign of ~17heimer's Disease, and can be an uuLward sign of the aging process. In contrast, certain biological processes that 1-1timAte1y lead to CA 022666~9 1999-03-17 W O 98/2~1t7 PCT/CA97/00859 -cell death, injury or dysfunction, such as the over- or under-expression of certain gene products, may be early events that begin prior to the onset of outward signs of the aging process, neurological f1ice~ces and/or disorders. Diagnosis of these biological processes or other precursor events of the aging process, neurological 5 disease and/or disorder followed by a suitable growth factor tre~tm~nt may lead to the prevention or reduction of the manifestations of the aging process and neurological ~licP~ces and disorders. For example, genetic screening can be used to diagnose patients who are predisposed to certain neurological disorders, such asHuntington's Disease, prior to the onset of manifestations of the disorder. Patients 10 found to be at risk for a neurological disease or disorder can be treated with growth factors to prevent or reduce the progression of the disease.

Growth factors are ~ d (or neural cells are geneli.,ally modified to express growth factors) prior to the onset of trauma or the manifestations of aging or disease processes at conce~ dlions that are ~..rric;~ to result in neural stem cell 15 proliferation. The protective effects that are provided by the growth factor tre~ nt are long-term and usually continue for at least one week after tre~tmPnthas ceased. The plo~clive benefits are often slTstAin~cl at least two weeks after cessation of ~lC~ , and in many cases, a protecli~e effect is sll~t~inPd for one, and up to two months, or more.

20 For the prevention or redn~tion of the m~nif~ost~tions of Hlmtington's Disease, Alzheimer's Disease, Parkinson's Disease, and other neurological disorders affecting primarily the forebrain, growth factors or other neurological agents would be delivered to the ventricles of the forebrain to affect in vivo proliferation of the stem cells. Alternatively, growth factors and other neurological agents can be easily 25 ~minictered to the lumbar cistern for circulation throughout the CNS. Animal models for various neurological dice~c~s, disorders and injury are used to assess the ,rolecli-/e effect of growth factor ~ alion to establish suitable dosages and m~thods of l~e~ .t Behavioral testing is pe~ ,led to compare the abilities of growth factor treated animals with non-treated control animals. Such behavioral 30 tests include learning and memory tests such as the Morris water maze and radial CA 022666~9 I ggg - 03 - l7 arm maze. The data obtained from accepted ln~ n animal models, particularly rodent, canine, and primate models are extrapolated to dcte~ e suitable protocols for growth factor treatment of huma~s. Generally, a~ l. ation of from about 100 to about 1000 ng growth factor per kilogram of body weight per hour over a period 5 of from about 1 to 10 days is sufficient to induce multipotent neural stem cell proliferation.

Example 2 below dPmn~ctrates that epidermal growth factor (EGF), a~lminictered icv at a physiologically effective dose, exer~s a cytoprotective effect on hippocarnpal neurons in vivo, preventing the normal neuronal degenerative lespollse following an 10 icchrmic insult. While not wishing to be bound by theory, this response appears to be due to the result of the mitogenic effect of the growth factors, resulting in the production of new neural cells, possibly as a result of the induction of proliferation, migration and/or dirf~lc,l~iation of ~uirsce~t neural stem and progenitor cells and leading to the production of new glial and/or lleulollal cells which secrete substances 15 or alter the envilol~llc.ll, providing a long term plote~;Live effect on the ne~lolls even after the exogenously ~dminict~red growth factors are no longer present. It is e~rrect~d that ~d~ aLion of large doses of tritiated Lhylli~dille or other anti-mitotic agents at the time of growth factor infusion would block the proliferation of the neural cells, and hence block the protective effect that is observed after i.sch~rnir 20 insult. This would in-iirare that the protective effect observed after growth factor treatm~nt results from the addition of new cells to the area.

The discovery that lle~ with growth factors provides a cytoplote-;Li~,re effect can be used advantageously to reduce the cll~nres of or lessen the severity of an expected CNS trauma. For example, a patient scll~d-lled to undergo brain surgery25 may be pleL~eaL~d with growth factors to prevent neurological trauma. Prior to surgery, growth factors would be aflminictered to brain regions that may be effected during the surgical procedure. Growth factors are ~mini.ctPred at least one weekprior to the e~rrect~ CNS trauma. Preferably, the growth factors would be c;d at least two, and more preferably, at least four or more weeks prior to 30 the e~rected trauma to provide time for the proliferation and dirrtle.lLiation of a , CA 022666~9 1999-03-17 Wo 98/22127 PCT/CA97/00859 beneficial ~lu~llbel of new neural stem cell progeny. If desired, the growth factor pretreatment could be completed several weeks in advance of surgery because the protective effect of growth factor plelle~r.-.~ nl is observed for prolonged periods after cessation of the tr~tm~nt Certain types of professional athletes, such as 5 boxers, are also at high risk for neurological trauma, and could be treated with growth factors to achieve a neLlul,loL~cLive effect. Additionally, patients who are at high risk for stroke can be treated with growth factors to reduce the amount of neurological damage should a stroke occur.

Example 3 below demonstrates that in very old animals, the ability to gen~dte an1 0 active con~ u~ively proliferating progenitor cell (CPC) population is irnpaired.
Neural precursor populations are shown to deciine with age, ~elhal)s as a result of a slowing stem cell cycle time. This in turn may lead to degenerative changes in brain morphology, increased susceptibility to i~f...ic and toxic insults, and i"cl~ased frequency of cognitive disorders that are often seen in aged anim~l~. As shown in 1 5 Example 3, ~ .dtion of growth factors such as EGF and FGF to the healthyCNS results in the ~l.,sence of more healthy, young cells in the aging brain. By the phrase "healthy CNS tissuen, it is meant that the stem cell cycle time and the neural tissue in general are nor nal for .~ lc of the same species and age group.
Preferably, at least two weeks after the growth factor tre~tm~ont, the treated ln~"~...;ll 20 has at least a 5% increase, and more preferably, a 10% increase, in the number of CPCs relative to the average CPC count for age-m~t~hlod 1ll~ of the same species. The increase in CPC count is snst~ined for extended time periods, preferably for at least one to two months, and more plef~l~bly, for at least four months after cessation of growth factor l.. ,.~.. It 25 F,Y~nPIe 1: ~n Vivo Pr~l;r~r~ n of Neural ~;it~m C~llc of ~ ~t~ral V~ ;
A replication hlco,.-pc~ retrovirus cont~inin~ the B-g~l~ctosi~ e gene ~as described in Walsh and Cepko, Science 241:1342, (1988)] was injected into the forebrain lateral ventricles of CD1 adult male mice (25-30 g from Charles River).
The injected retrovirus was harvested from the BAG cell line (ATCC CRL-9560) 30 according to the method of Walsh and Cepko (supra). Mice were an~cthPti7ocl using . .... ~

CA 022666~9 1999-03-17 65 mg/kg, i.p. sodium pentobarbital. Unilateral stereotactic injections of 0.2-1.0 ~1 of retrovirus were injected into the lateral ventricle using a 1 1ll H~milton syringe.
The coordinates for injection were AP +4.2 mm anterior to lambda, L + 0.7 mm, and DV -2.3 mm below dura, with the mouth bar at -2mm below the interaural line.
5 One day, or six days following the retrovirus injection, an infusion C~nn~
att~rhPd to a 0.5 ~I/hour ALZET osmotic mini-pumps filled with 3.3 - 330 llg/ml of EGF were surgically implanted into the lateral ventricles at the identi~l stereotactic coordinates as stated above. The infusion c~nn--l~ kits were obtained from ALZA.The infusion cann~ e were cut to 2.7 mm below the pedestal. The pumps were 10 secured to the mouse S'KUIl by use of acrylic cement and a S'KUll screw contralateral and caudal to the injection site. The osmotic mini-pump was situated subcutaneously under and behind the armpit of the left front paw and conn~octed to the infusion c~nn~ by the means of polyethylene tubing.

Six days following initiation of EGF infusion the animals were sacrificed with an 15 overdose of sodium ~ oba.L,ilal. Mice were ll~nG.ldrdially pelrused with 2%
buffered paraformaldehyde, and the brains were excised and post fixed overnight with 20% sucrose in 2% buffered paraformaldehyde. Coronal slices were prep~ d with -20 Celsius cryostat sectioning at 30 ~m. Slices were developed for B-gal histochemistry as per Morshead and Van der Kooy (supra).

20 Under these conditions, regardless of the day post retrovirus injection, inf~,lsion of EGF resulted in an e~rp~n~ion of the population of B-gal labeled cells from an average of 20 cells per brain up to an average of 150 cells per brain and the migration of these cells away from the lining of the lateral ventricles. Infusion of FGF-2 at 33 llg/ml resulted in an increase in the number of B-gal labeled cells, but 25 this increase was not accompani~o~l by any additional migration. Infusion of EGF
and FGF together resulted in an even greater e~pancion of the population of B-gal labeled cells from 20 cells per brain to an average of 350 cells per brain. The synergistic increase in B-galactosidase cell number when EGF and FGF are infusedtogether further reflects the direct association between the relatively qlliçscent stem " .. . . . .

CA 022666~9 lgg9-o3-l7 Wo 98/22127 PCT/CA97/00859 cell and the constitutively prolir~ ing progenitor cell.

This experiment can be modified to infuse growth factors in other CNS ventricles to achieve a similar expansion of cell nulllbel~ in other CNS regions.

~ple 2: (~rowth Factor Infusio~ Prior to Neurologjcal ln~--lt 5 Epidermal Growth Factor (EGF) (Chiron Corp.) was ~l~pal~,d in 1 mg/ml rat albumin (Calbiochem, Cat. No. 126722) in sterile physiological saline. The EGF
preparation was infused into the rostral lateral ventricle (Bregma +0.7 rnm) of adult male Wistar rats weighing 250- 350 g via a 30 gauge cannnl~ conn~ct~d to an osmotic lllilli~Ulllp (Alza, Model 2001) at a rate of 1 ,Lll/hr for 9 days. The EGF
10 was prepared at a concentration so as to provide each animal with 416 ng EGF per kilograrn of body weight every hour.

~ ;UI :~0- CPI~
Animals received BrdU (50 mg/kg) i.p. 3 tirnes per day for the last 3 days of growth factor in~u~iol~.

15 ~.cf~hPmi~ ~,eSi-~n~
The model used was a combination of rodent models used routinely for ~lansie.lt global i~cllPmi~ which combines bilateral common carotid artery occlusion with hemorrhagic hypotension (Smith et al. Act. Neurol. Sr~n~l 69:385-401 (1984);
Mudrick and Baimbridge, F~p'l RrAin Res. 86:233-247 (1991)). This model 20 produced lesions of the neurons in the CA1 region (100% of animals) and in the CA2 and CA3 regions (60% of animals) of the hip~oc~ s bilaterally. Two months after l,~ with EGF, the ~nim~l~ were A~ d with sodium pentobarbital (60 mgtkg i.p.) and treated with atropine (0.2 mg/kg i.p.) to reduce res~ toly secretions. A rectal l~nl~ Lurc: probe, conn~octPd to a le~ dL~lle 25 regulated heat pad, was used to m~int~in the animal body L~llp~,~tUre at 35~C. The comrnon carotid arteries and the femoral artery were isolated using blunt ~li.cs~ction and a 3-0 silk was looped around each vessel for later access. The femoral artery was then r~nnnl~t~d and heparin (150 units) was a~ ed to prevent clotting.

, . ... _.~

CA 022666~9 lggg-03-l7 wo 98/22127 PCT/CA97/00859 The baseline mean arterial pressulc was taken (~ 120/80 mmHg) via a tr~n~ cer and recorded on a chart recorder. The animal was hemorrhaged until mean arterialpressure fell to 3040 mmHg (removal of approximately 7-10 ml of blood). The common carotid arteries were quickly occl~ with atr~llm~tir arterial clamps for 5 20 mimltt~. Mean arterial pleS~u~e was m~int~in~d at 30~0 mmHg throughout the duration of the i.cch.orni~ by either further hemorrhage or reinfusion of blood. Blood flow was restored by removal of the arterial clamps, and reinfusion of shed b}ood.
Protamine sulfate (1.5 mg/animal) was a(l~-,i";~lered via the femoral c~nn~ to deactivate heparin to prevent post-surgical bleeding. Mean arterial pressure should 10 return to pre-icch~ornic levels or slightly higher. The animal was given S ml of lactated Ringers s.c. and allowed to recover under a warm lamp. The animal was single-housed and monitored closely for the next 48 hours.

Ti.c~ue 1~
Animals were allowed to survive 1 week after the i~rhPmi~ insult prior to tissue15 preparation. The ~nim~l~ were deeply ~nPsthrti7~d with 65 mg/kg sodium pentobarbital and transcardial}y perfused with ice cold saline (--350 ml) followed by ice cold 4% pal~foll.laldehyde (PFA) in 0.1M phnsph~te buffer at pH 7.4 (--500ml). Brains were removed and post fixed in 4 % PFA overnight at 4~C. The brains were then cryoprotected through serial overnight incubations in 10% to 30%
20 sucrose at 4~C and then inrl1k~trd for 2-24 hours in 2:1 30% sucrose:OCT. Thebrains were then frozen on dry ice and 10~m serial sections were cut on a cryostat.

~i~t-)lo~r~l ~,v~ tion of N~ I m.~l CPII nea.~h Tissue processed for morphology was ~lPf~ttPd in Histoclear (Diamed, Cat. No. HS-200), rehydrated through a tieScenrling series of alcohols, rinsed in ~ till~d water 25 and stained with Thionin (0.5%). Tissue was then dehydrated through a series of alcohols, cleared with ~i~toclP~r, and coverslipped with DPX (Aldrich, Cat. No.
31761-1). Thionin stains the Nissl su~ s~nre within the cytoplasm of nerve cells(Cajal, 1995). The ~hsenre of cells with the characteristic pyramidal cell molphology within the cell layer is indicative of a loss of hippoc~mpal pyramidal 30 neurons due to i~chPmi~-ind~lred cell death. A one week pOSt i.~ch~ survival time . . ~ , , CA 022666~9 l999-03-l7 Wo 98/22l27 PCT/CA97/00859 was chosen due to the unique characteristic of delayed neuronal death observed in CA1 pyramidal cells in response to transient global i~r~Pmi~ (~hi~eno et al. L
Neurosci. 11:2914-2919 (1991); Ordy etal. F.~'l Neurol. 119:128-139 (1993)) whereby select CA1 pyramidal cells appear unaffected by i.~chPmi~ for up to 3 days 5 post surgery. In addition7 rn~xim~l glial reaction has occurred and excitotoxic conditions have subsided by this time point.

Resllltc EGF pletlLO~ (icv infusion of EGF for a 9 day period) given 8 weeks prior to an ischemic insult produced complete protection in 3 of 7 animals from i.cch.olni~-1 0 int1uce~ neuronal cell death in the CA1 region. Nissl stain of EGF-treated anirnals revealed that the cells of the CAl, CA2 and CA3 displayed excellent morphology throughout the extent of the hippocampus (Figure 1). In addition, there was an increased number of glial cells observed within the hippocampus. BrdU l~hçiing was observed within the hippocampus.

1 5 F.x ~ lc 3: Growth F~~t~r A~ r~ n Prior to O~et of A~IV~ P~1 ~jr~
~s~ t of 1 ~ cells in rodents of ~ ;ous ages The adult subv~ lar zone (SVZ) consists of at least two populations of dividing cells that can be distinguished by differences in their cell cycle times: a slowly cycling stem cell population and a rapidly cycling, constitutively prolire.d~ g 20 proge.li~or cell (CPC) population. The purpose of this study was to ~uallLify the numbers and distribution of these cell types with age in the mouse and rat and the impact of growth factor infusion on cell u~ Cl~.

Mice of various ages (between 2 and 18 months) were injected with BrdU
(approximately 67 mg/kg) for 4 weeks (3-S times/day) and left un-li.ctl~rbed for a 25 further 8 weeks. This paradigm resulted in labeling of the slowly cycling stem cells of the subvel~ lar zone. Animals were perfused with 4% ~aldfo-lllaldehyde and the brains are removed and cryoprotected. Frozen sections were cut at 30 ~m.
BrdU was det~ctPd ;..~ ocytoc~lPmir~lly (rat anti-BrdU, Sera labs, followed by donkey anti-rat CY3 or FITC, Jackson Tmmlln~lesear~;h). l ~beled cells throughout ... . . . . .

CA 022666~9 lgg9-o3-l7 Wo 98/22127 PCT/CA97/008sg the brain were ql-~ntifi~d and changes in cell numbers or distribution with age were analyzed. In addition, the distribution of BrdU-imm-lnoreactive, post-mitotic stem cell progeny in the brain was ql-~ntifiPd and compared among ages.

The same tissue was double-labeled with antibodies to prolirelaLi.lg cell nuclear 5 antigen (PCNA), which is upregulated around the S phase of the cell cycle.
Prelimin~ry results in-lic~t~ that it marks the constitutively proliferating cell population. The number and distribution of PCNA-IR cells were ~ nti~l~d and compared among ages.

Results 10 The number of mitotically active stem cells (BrdU-IR) declined with age, particularly at stem cell "pockets" where there are higher col~ce.,lldlions of stem cells. At the rostral stem cell pocket (bregma 0.7), counts for the 7 month old group and the 13 month old animal were 65 % and 53 % of the 2 month values, respectively. Similarly, at the caudal stem cell pocket (bregma ~.3), counts for the 15 7 month and 13 month groups were 53% and 38% of the 2 month values, respectively.

The CPC population (PCNA-IR) also ~3eclinP~l with age. The ages of the animals at the time of sacrifice for CPC analysis were 5, 10, and 16 months. To avoid confusion when culllpaling data, the age at which the stem cell l~heling began (i.e.
20 2, 7 and 13 months) is used to identify the subset of animals referred to (rather than the age at sacrifice). At bregma 0.7, a previously identifled CPC peak, counts for the 7 month group were only 62% of the 2 month values, while the 13 month animalcontained only 30% of the 2 month counts. This data contradicts that of Kuhn et al.
J. Neuro Sci 16:2027-2033 (1996), who found no change in the llunlbel of BrdU-IR25 cells with age in a l~slli~;Led area of the rat lateral ventricle when analyzed 1 day after BrdU injection.

The number of BrdU-IR cells in the dentate gyrus of the hippocampus also ~ieclin~od with age.

CA 02266659 ls99-03-l7 Effect of growth factors on the aging 1~ oc~ss of roden~c EGF was infused into the lateral ventricles of two month old adult CD1 mice (n=3), using the procedure described in Exa nple 2 for a period of 6 days. Control mice(n=3) were infused with vehicle. The ~nim~l.c were sacrificed four months later.5 The number of PCNA-IR cells around the lateral ventricle was measured, using the procedures described above, in 10 ~m sections from the following bregmas: 2.7, 2.2, 1.7, 1.2, 0.7, 0.2, -0.3, -0.8. These bregma correspond to the peak cell distributions of CPCs and are the sites where ~ignifir~nt loss of PCNA-IR cells with age are seen. Two of the three animals that received EGF L ea~.l.ellt had significantly 10 higher PCNA counts (approx. 30% higher) than vehicle controls demonstrating that the EGF has a protective effect on the CPCs in aging animals.

In further studies, EGF-treated animals are allowed to age and are given a variety of memory and other behavioral tests and co~ Jaled with animals of the same age that have not received EGF L~ ;. Ill,ploved ~e~ re of EGF-treated ~nim~l~
15 compared to non-treated anim~l~ dell,ol~L ates that plelreal~llellL with growth factors can protect against or reduce age-related deçlinPs in cognitive func~ion.

F~ 4: Growth Factor A.l~ ';on Prior to OllcPt of N~ ulo~, Di.c~c~
r~ ..tn"ls nicP~ce M.~API
20 Animals receive EGF infusion Ll,,,t~ as des, ,ibed in Example 2 for 6 days (mice) or 9 days (rats). After various time points (1 wk, 2 wk, 1 mo., 2 mos., 4mos. and 8 mos.) after EGF l c~ nim~lc are hP~ionPd so as to model Palhinsoll's Disease. In rat models, lesions are made by knife-cuts of the dop~minPrgic fibers pro3ecting from the midbrain to the sl.iaLu.., or by one injection 25 of 8 ~g of 6-hydroxydop~ P directly into the SLliaLulll, nucleus ~c~l ..h~ or the ~b~ nigra. In mice, lesions are made by a ~ .ation of two injections at 16 hours interval, of 50 mg MPTP/Kg s~bc~ nPously in volume of 0.5 ml of 0.9%
saline solution. A subset of animals are allowed to survive one to eight days after lesioning and are then sacrificed. The numbers of surviving do~an.i~ gic cells are 30 assayed by tyrosine hydroxylase (TH) immlmncyt~ hPmi~try and are c~ arcd with WO 98/22127 PCTICA97/008~;9 control animals that are lesioned prior to sacrifice but which do not receive growth factor tre~tm~nt Another subset of ~nim~lc that are allowed to survive for prolonged periods and are subjected to behavioral testing and c~ll,pdled with lesioned controls ~nim~lc (no growth factor tre;ltrn~nt) or normal animals that are the 5 same age as the model ~nim~lc Al~hPimer's Dis~ce Model Female Wistar rats (180-200 g) are used for this model. The animals receive EGF
infusion ~ea~ as described in Example 2 for 9 days. After various time points (1 wk, 2 wk, 1 mo., 2 mos., 4 mos. and 8 mos.) after EGF L,Lat...~l-t, ~nim~l.c are 10 lesioned so as to model Alzheimer's Disease. A retractable wire knife (Scouten Knife) is used to partially lesion the fimbria-fornix pathway. Animals are killed at various time points after the lesion (from 1 day to 8 weeks), and the numbers ofsurviving cells are assayed by Nissel st~ining in the septum. Controls, lesioned and treated ~nim~lc are cc"l~a,- d. A subset of animals is allowed to survive for 15 prolonged periods for behavioral studies and co~ ,aled with control ~nim~lc.

~ ~r... 's Dic~e MnA~I
Following prell- d~ t with ~ n~ c~led growth factors âS described in Example I, the method described Levivier et al (1995) Neurosci. 69(1):43-50 is used to induce quinolinic acid lesions in striatal tissue. The numbers of neurons in the ~Lliatlllll are 20 compared between controls, lesioned and treated ~nim~lc.

.

Claims (15)

WHAT IS CLAIMED IS:

1. A method of preventing the decline of the constitutively proliferating neuralprogenitor cell population of mammalian neural tissue in an aging mammal comprising administering to a mammal an effective amount of one or more growth factors to induce multipotent neural stem cell proliferation and provide a protective effect on said constitutively proliferating neural progenitor cell population.

2. The method of claim 1 wherein said neural tissue is healthy.

3. The method of claim 1 wherein said one or more growth factors is selected from the group consisting of EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bPGF or FGF-2), transforming growth factor alpha (TGF.alpha.), proliferation-inducing ligands which bind to the EGF and FGF
receptors, and combinations thereof.

4. The method of claim 1 wherein said one or more growth factors is administeredto one or more ventricles of said mammal.

5. The method of claim 1 wherein said one or more growth factors is administeredby genetically modifying cells of said mammalian neural tissue to produce said one or more growth factors.

6. A method of protecting a mammalian neural tissue against neurological trauma or insult comprising administering to said mammal an effective amount of one ormore growth factors to induce multipotent neural stem cell proliferation and provide a protective effect on said neural tissue at a time prior to an anticipated neurological insult or trauma.

7. The method of claim 6 wherein administration of said growth factor to said mammal is completed at least one week prior to said neurological insult or trauma.

8. The method of claim 6 wherein said anticipated neurological insult or trauma is due to surgical procedures performed on said mammal's brain.

9. The method of claim 6 wherein said one or more growth factors is selected from the group consisting of EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGF.alpha.), proliferation-inducing ligands which bind to the EGF and FGF
receptors, and combinations thereof.

10. The method of claim 6 wherein said one or more growth factors is administered to one or more ventricles of said mammal.

11. A method of preventing or reducing the manifestations of a neurological disease or disorder in a mammal comprising administering to said mammal an effective amount of one or more growth factors to induce multipotent neural stem cell proliferation at a time prior to occurrence of said manifestations.

12. The method of claim 11 wherein prior to administration of said one or more growth factors to said mammal, said mammal is diagnosed with a precursor event to said manifestations of said neurological disease or disorder.

13. The method of claim 11 wherein said one or more growth factors is selected from the group consisting of EGF, amphiregulin, acidic fibroblast growth factor (aFGF or FGF-1), basic fibroblast growth factor (bFGF or FGF-2), transforming growth factor alpha (TGF.alpha.), proliferation-inducing ligands which bind to the EGF
and FGF receptors, and combinations thereof.

14. The method of claim 11 wherein said one or more growth factors is administered to one or more ventricles of said mammal.

15. The method of claim 11 wherein said one or more growth factors is administered by genetically modifying cells of said mammalian neural tissue to produce said one or more growth factors.