CA2176708A1

CA2176708A1 - Method of treating neurological disorders

Info

Publication number: CA2176708A1
Application number: CA002176708A
Authority: CA
Inventors: Christopher A. Maack; Debra Brown
Original assignee: Individual
Current assignee: Celtrix Pharmaceuticals Inc
Priority date: 1993-11-15
Filing date: 1994-11-15
Publication date: 1995-05-26
Also published as: JPH09509140A; AU693489B2; WO1995013823A1; EP0729361A1; EP0729361A4; AU1057095A

Abstract

This is a method for treating certain neurological disorders in subjects by administering a composition containing a complex of insulin-like growth factor (IGF) and insulin-like growth factor binding protein-3 (IGFBP-3).

Description

- 2~ 767~8 3 ` - 1 - PCT~US94~13177 MRT~OD OF 'T~R~TINt'7 NRTT~f~T -)G~ T, DT~RnET~q Field of the Tnvention This process relates generally to the f ield of 10 medical therapy and particularly to the treatment of neurological disorders by administering a th~r~rP~t i c composition cnnt~;nin~ a compleY of an insulin-like growth factor (IGF) and an insulin-like growth factor binding protein (IGFBP).

BackcTrmln~l ~rt Growth factors are polypeptides which sti lAte a wide variety of biological responses (eg. DNA
synthesis, cell division, expression of specific genes, 20 etc. ) in a deined population of target cells . A variety of growth factors have been i~l~nt~f~ including transforming growth factor-,B, (TGF-~), TGF-,~, TGF-~, growth factor (RGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-25 like growth factor-I (IGF-I), and IGF-II.
IGF- I and IGF- II are related in aTnino acid sequence and structure, with each polypeptide having a molecular weight of apprnY;~-tPly 7500 daltons. IGF-I
-1~t~c the major efect8 of growth hormone, and thus is 3 0 the primary mediator of growth af ter birth . IGF- I has also been implicated in the actions of various other growth factors, since treatment of cells with such growth factors leads to increased production of IGF-I. In contrast, IGF-II is believed to have a major role in 35 fetal growth. Both IGF-I and IGF-II ~ave in~ulin-like Wo 95/13823 ! 2 1 7 6 7 ~ 8 PCTIUS94/13177 activities (hence the name), and are mitogenic (st; l~tln~ cell division) for the cells in neural tissue, muscle, reproductive tissue, skeletal tissue and a wide variety of other tissues.
Unlike most growth factors, the IGFs are present in substantial quantity in the circ-ll; t;nn, but only a very small f raction of this IGF is f ree in the cir~ t;nn or in other body fluids. Most circlll~tlng IGF
is bound to an IGF-binding protein called IGFBP-3. IGF-I
may be measured in blood gerum to diagno8e ;Ihnnrr~l growth-related conditions, e.g., pituitary gigantism, a.:L , ly, dwarfism, vari~us growth hormone def iciencies, etc . Although IGF- I i8 produced in many tissues, most circl-l~tin~ IGF-I is believed to be synth~Ri 7ecl in the liver.
Almost all IGF circulates in a non-covalently associated ternary complex composed of IGF- I or - II, an IGF specif ic binding protein termed IGFBP - 3, and a larger protein termed the acid labile subunit (ALS). This ternary complex is composed of equimolar amounts of each of the three components. The ALS has no direct IGF
binding activity and appears to bind only a pref ormed IGF-I/IGFBP-3 complex. The ternary complex of IGF +
IGFBP-3 + ALS has a molecular weight of apprn~ t~ly 150, 000 daltons . This ternary complex i8 alleged to function in the cir~ tinn ~as a reservoir and a buffer f or IGF - I and IGF - II preventing rapid changes of f ree IGF . ~ See, Blum, W. F ., et al ., ~ Plasma IGFBP- 3 Levels as Clinical Indicators", In Modern Conceptg in Inclll ln-T.lkP
Growth Factors, (E. M. Spencer, ed., Elsevier, New York) 381-393, (l99l).
Nearly all of the~ IGF-I, IGF-II and IGFBP-3 in the cir~ t1on are in complexes, 80 very little free IGF
or IGFBP-3 is detectable. Moreover, a high level of free 35 IGF in plasma is 1ln~c1 r~hle. It would lead to serious ~VO 95/13823 i : - 2 l 7 6 7 ~ ~ PCT/US94~13177 hypoglycemia because IGF has insulin-like effects on circulating glucose levels. In contrast to the IGFs and IGFBP-3, there i9 a substantial pool of free ALS in plasma which assures that IGF/IGFBP-3 complex entering 5 the cir~ t;nn; 'ti~tPly formg ternary complex.
IGFBP-3 is the most ahllnA~nt IGF binding protein in the circulation, but at least five other distinct IGF binding proteins ( IGFBPs ) have been iAPnt;f;Pd in various tissues and body fluids. Although 10 these proteins bind IGFs, they each originate from separate genes and have distinct amino acid sequences.
Thus, the binding proteins are rlot merely analogs of a common precursor . Unlike IGFBP - 3, the other IGFBPs in the circulation are not s~tllr~tPd with IGFs. None of the 15 IGF binding proteins other than IGFBP-3 can form the 150 Kd cir~ t; n~ ternary complex.
IGF-I and IGFBP-3 may be purified from natural sources or produced by rer~-~h;n~nt means. For instance, IGF-I has been purified from huwan serum for a number of 20 years. See, ~;nAPrknPcht, E.W., et al., Proc Natl Acad Sci (YSA) 73, 2365-2369 (1976). Recombinant IGF-I
processes are shown in EPA 0,128,733, pllhl;~hP~l in December of 1984. IGFBP-3 may be purified from natural sources using a process such as that shown in Baxter et 25 al., "Growth ~ormone-Dependent Insulin-Like Growth Factors (IGF) Binding Protein from Human Plasma Differs f rom Other Eluman IGF Binding Proteins n ~ BiorhPm Bio~hy~
Res ~` 13~, 1256-1261 (1986). IGFBP-3 may be synthesized by recombinant organisms as discussed in 30 Sommer, A. S., et. al., In Mr~A~rn ~'fmc~eotg of Tne~ulin-J; kP Growth Factors (E . M. Spencer, ed., Elsevier, New York) 715-728 (1991). This rP~ ;n~nt IGFBP-3 binds IGF- I in a 1:1 molar ratio . The topical administration of the IGF-I/IGFBP-3 complex to rat and pig wounds was 35 significantly more effective than IGF-I alone. Sommer WO95/13823 ~ ~ ` 21 767~g PcrluS94/13177 et. al., ibid. Subcutaneous administration of the complex to hypophysf~rtr~n; 7~fl rats "substantially prevents the hypoglycemic effects of IGF-I" administered alone.
Sommer et. al., ibid.
U.S. Patent No. 5,093,317 issued on March 3, 1992 to Lewis et. al. discloses a method for using IGF-I
to enhance the survival of non-mitotic, rhr~l ;nF~rgic neuronal cells. The claims do not include the use of IGF
binding proteins in conjunction with IGF-I.
Patent Cooperation Treaty Publication No. WO
93/02695, published on February 18, 1993, and applied for by Genentech, Inc. and Auckland Univservices Ltd., discloses a method for treating injuries to or diseases of the central nervous system affecting glial or non-rh~l ;nC~rgic ~euronal cells with ;ntrAr~rebral infusions of IGF- I or IGF- I analogues .
Patent Corp~r~t; on Treaty Publication No. WO
92/19256, pllhl;~h.~l on November 12, 1992 and applied for by Kabi phAr~-r;~ A~3, discloses a method for ;nfll1r;ng nerve regeneration by treating subjects suffering from neuropathy or degenerative neural disorders with IGF- II
or IGF- II + IGF- I . The use of any IGF binding protein in these treatments is not disclosed.
European Patent Application EP 0 308 386 A1, 2~ published on March 22, 1989 and applied for by KabiVitrum A3, discloses a method for improving the regeneration of transected peripheral nerves by treating sub; ects with an effective amount of IGF-I. The use of any IGF binding protein in these treatments is not disclosed.
All of the important elements of the IGF system are f ound in the central and peripheral nervous systems of humans and other mammals, inrl~ ;n~ IGF-I and -II and the IGF binding proteins and receptors . IGF- I and IGF- II
have been implicated in the growth, survival and -W0 95/13823 i ; - ` 2 ~ 7 6 7 0 8 PcrtlrS94/13177 differPntiAtPrl function of several classes of neurons and glial cells.
The presence of IGF- I and IGF- II mRNA and protein in rat and human brain has been well ~o~ tP~, IGF-I and IGF-II mRNAs have rhArartpristic regional distributions in fetal and adult rat and human brain.
IGF-I mRNA is present at high levels in the olfactory bulb and cervical thoracic spinal cord and at ---' tP
levels in the m;r~hri~;n and cerebellum of adult rats.
IGF-I mR~A is also 5ynthP~i 7P~l by primary cultures of e~mbryonic astroglial and neuronal cells . IGF- II mRNA is both more Ahl~n~Ant in brain than IGF- I mRNA and is much more unif ormly distributed . This mRNA is somewhat elevated in the choroid plexus, cerebellum and medulla-pons, and somewhat reduced in midbrain and corpus striatum. In contrast to IGF-I, IGF-II mRNA is synthesized by cultured embryonic astroglial but not neuronal cells. Both mRNAs are highest at embryonic day 8-14 in rat brain, and decline from this peak to the adult level by the time of birth.
IGF- I and IGF- II have also been detected in cerebrospinal fluid ~CSF) and by; nh; ctochemistry in human, rat and cat brain. IGF-II; ~e:~ctivity in the brain is higher than that of IGF-I. Adult and fetal human brain contain both the normal form of IGF-I and a truncated form of IGF-I missing three r~-tPnm;nAl amino acids. IGF-I peptide is also secreted by cultured rat glioma cells . IGF- II immunoreactivity is highest in the anterior pituitary, dorsomedial hypothAl i - and 3 0 supraoptic nucleus of the brain . Larger than normal f orms of the IGF - II peptide have been extracted f rom human brain, and a large form of IGF-II is secreted by cultured PYpl Ant~ Of nPnnAti~l rat brain. The presence of IGF- I and IGF- II mRNA in the brain, and the secretion of 35 these peptides by cultured cells from the central nervous wo 95/13823 ` ' " 2 1 7 6 7 ~ 8 PCT~S94/131M ~
system suggests that at least some of the IGF found in the central nervous system i9 produced locally.
The expression of the IGF - I genes in neural tis6ue is under complex t 1 control. In vitro 5 studies indicate that basic fibroblast growth factor ~bFGF), which promotes survival of cultured neural tissue, sti lAtPC secretion of IGF-I from cultured fetal rat neuronal and glial cells. In contrast, ~ hAcnn~
and retinoic A, cid, which inhibit the growth of rat glioma 10 cells, reduce the ~Ar lAtion of IGF-I mR~7A and inhibit the secretion of IGF - I peptide by these cells .
Type I IGF receptors, which trAncflllr~ mitogenic and dif~er~nt;At;nn signals provided by the IGFs, are also present in the brain. ~owever, the ronr~rtrAt;nn 15 and distribution o~ this receptor varies during devPl ~ ~ . In the n~nnAt~l rat, brain Type I receptor levels are quite high (4-lO times higher than in the adult), and the receptor is especially Ahlln~9Ant in the superf icial cortical layers, nucleus ac~ 8 and 20 hippocampus. In the adult rat, Type I receptor levels are reduced and the receptor is more evenly distributed.
There is some receptor enric_ment in adult superficial and deep cortical layers, nl fArtnry bulb, endopiriform nucleus, basomedial nucleus of the amygdala, thalamic 25 nuclei and hippocampus. ~3rain Type I IGF receptor is present in two forms, a normal sized form and a somewhAt smaller form than that found in peripheral tissues.
Apparently this size difference is largely due to reduced glycosylation of the smaller of the brain species. Ag 30 with IGF-I, bFGF increases the synthesis of Type I IGF-receptors in cultured neuronal and glial cells.
Substantial quantities of the Type II IGF
receptor are also found in the brain, but the function of this receptor i~ obscure. In fetal and early postnatal 35 rats, the Type II IGF receptor is Ah-ln~Ant throughout the . = . 2 t 767~8 wo 9S/13823 PCT/US94113177 brain, whereas it is restricted to neurons in the forebrain (eg. hippocampus and dentate gyrus) in adult rats .
The third element of the IGF sygtem, the IGF
5 binding proteins, are also synth~ql ~ed by cells of the nervous system and are found in CSF and neural tissue.
IGFBP-2 mRNA is Ah11n~ nt in fetal rat brain stem, cerebral cortex, hypoth~ and choroid plexus and persists in adult brain. It is important to note that 10 IGF- II mRNA is also abundant in the choroid plexus, and that this region of the brain is important in generating CSF .
In neonatal and adult rat CSF, IGFBP-2 is the most ~hllntlAnt IGF binding protein, with substAnt;Ally 15 higher IGFBP-2 levels in n~nn~t~l CSF than in adult CSF.
~ower levels of IGFBP-3 are also present, as well as traces of lower molecular weight IGFBP species. Using immunocytochemistry with human fetal tissues, IGFBP-3 was localized to neuronal cell bodies in the upper region of 20 the cerebral cortex, while IGFBP-l and -2 were not detected in the cerebral cortex. IGFBP - 3 was not detectable in the meninges.
Cultured fetal, postnatal and adult rat glial and neuronal cells, gliomas and choroid plexus cells 25 synthesize and secrete IGFBP-2, IGFBP-3 and a 24 i~Da IGFBP. The glial cultures secreted approximately 5 times as much IGFBP- 2 as did the embryonic ~euronal cultures, and IGFBP- 2 was the only IGFBP secreted by choroid plexus cultures. In contrast, gliomas and 30 astrocytes synthesized prerlnmln~ntly IGFBP-3. The regulation of IGFBP synthesis has not been extensively studied, but it is known that bFGF treatment greatly increases IGFBP secretion by neuronal cultures and inhibits IGFBP secretion in glial cultures . IGF- I

Wo 95/13823 - 8 - PCrNS94/13177 stimulated IGFBP secretion in both cultures and in the rat neuroblastoma cell line Bl04.
As with many other tissues, IGF treatment of the various cell types of the nervous system leads to 5 both mitogenesis and the expression of tissue-specific differ~nt;Ate~l functions. These effects presumably act through the Type I IGF receptors . IGF- I and IGF- II are mitogenic ~ie. stimulate cell division) for oligodendrocyte precursor cells from cultured perinatal lO rat cerebrum explants, embryonic rat sympathetic neuroblasts, human neuroblastoma cells, newborn rat astroglial cells, and neonatal rat cerebral cortex as trocytes .
Furthermore, IGF- I has been shown to promote 15 the survival of various types of cultured nervous system cells. For example, IGF-I acts as a survival factor in cultured embryonic mouse neuroepithelial cells. bFGF, which is mitogenic f or these cells, induces endogenous production of IGF-I, which is reS~uired for the expression 20 of the mitogenic effect of bFGF. Similarly, autocrine production of IGF- I has been implicated as the mediator of at least part of the mitogenic effect of P~i~lPrr-l growth factor (EGF) on cultured newborn rat astroglial cells . IGF- I has also been shown to protect rat 25 hippocampal and septal neuronal cell cultures from hypoglycemia- induced damage by 5tAh; l; ~; n~ neuronal calcium homeostasis.
In cultures of undiffer~nt;At~-l neural cells, IGF-I promotes the differentiation of oligodendrocyte 30 precursor cells in explant cultures of perinatal rat cerebrum, catP~-hol Aml n~orgic precursor cells in cultured chick dorsal root ganglia, and cultured SH-SY5Y human neuroblastoma cells in synergy with the phorbol esters.
IGF- I also induces the synthesis of the rat brain glucose 35 transporter gene i primary rat neuronal and glial cell ~ W0 9~i113823 ' ~ 2 1 7 6 7 0 ~ p~us941l3l7~
cultures. Flnally, IGF-I, but not IGF-II, signi~icantly increases the potassium-evoked release of acetylcholine (a major neurotransmitter) from adult rat hippocampal tissue slices.
The importance of the IGFs in promoting the growth and function of the nervous system has been demonstrated in a number of in vivo studies. Several studies have shown that treatment with IGFs can stimulate neurite outgrowth and synapse f~ t1nn For example, both IGF-I and IGF-II subst~nt;~l~y stimulate rapid neurite outgrowth in embryonic chick spinal cord motor neurons in culture . Similarly, IGF- II administered daily to mouse gluteus muscle caused rapid, marked t~rmln~l and nodal neuronal sprouting of neurites . This ef f ect was detectable after as little as 3 days of treatment and produced 10 fold more neurite sprouts in IGF-II treated than control muscle after one week of treatment. IGF-II
treatment also caused a nearly 5-fold increase in the number of endplates that had f ormed neurite sprouts .
This data was interpreted to support the possibility that the IG~s could act as the diffusible factors that are thought to be released by damaged, partially denervated muscle and which lead to increased neurite sprouting by the viable cells that innervate the muscle.
Another set of experiments showed that transgenic mice expressing a human IGF- IA transgene developed subst~nt; ~ l l y larger brains than their control littermates. The brains of the transgenic mice expressing hIGF- I also r~mt~ 1 n-~-l substantially more myelin than did the brains of their control littermates.
These effects of IGF-I on brain dev~ occurred with only a modest increase in brain and serum IGF- I levels (1.5-2 fold) and with only a slight and insignificant increase in total body weight. These data demonstrate WO95/13823 ~ 1 7 6 7 0 8 PCT/US94/13177 the potent effect of IGF-I in stiTr''l~ting the development of brain tissue and myelination.
Dicclosl1re of the Invention In accordance with one orli of the present invention, there is provided a method for treating a subject for lIuntington's disease and Al 7hF.1 ' S disease, wherein the subject is administered a complex comprising an insulin-like growth factor (IGF) and insulin-like growth factor binding protein-3 (IGFBP-3) in an amount sufficient to alleviate said condition .
In accordance with another embodiment of the present invention, the IGF used in the complex is provided as IGF- I . In a further ' - ' ~, IGF and IGFBP are present in P~rl; ~l;lr amounts. In still another embodiment, both IGF and IGFBP - 3 are human proteins obtained f rom recombinant sources .
In accordance with another ~mhnrl; t of the present invention, the complex of IGF and IGFBP-3 is administered parenterally. In a further embodiment, the complex is administered by sl1hcut~n~oll~ inj ection .
In another embodiment, the subj ect to whom the complex is administered is a mammal.
In yet another ~ o~; , the method provides for treating a subject for exposure to neurotoxins, cerebrovascular hemorrhage, neuronal scission during surgery, meningitis or other infection of tissues of the nervous system. The method includes administration of the IGF/IGFBP-3 complex in an amoun~ sufficient to alleviate the condition.
In still other ' -';-- t,c,, the method provides a treatment for multiple sclerosis, amyotrophic lateral sclerosis or Charcot-Marie-Tooth disease, in which the 3~ subj ~ct is parenterally administered a complex of wo 95/13823 G~ ` 2 1 7 6 7 0 8 Pcrlus94ll3l77 IGF/IGFBP-3 in an amount sufficient to alleviate said condition .
The IGF/IGFBP complex can be administered using normal parenteral routes for the treatment of peripheral 5 nervous system disorders or f or the treatment of central nervous system disorders in which the blood brain barrier is c ~ qed ~eg. multiple sclerosis), thus allowing the passage of complex into the brain. Alternatively, in other conditions, such as Huntington's chorea and 10 Alzheimer' s disease, the IGF/IGFBP complex can be administered directly to the CNS by intrilr~Ani~l administration, such as by an implanted shunt into the ventricles of the brain.
While not wishing to be bound by any particular 15 theory, the Inventors propose that the administered complex of IGF and IGFBP- 3 results in the gradual release of free IGF in elevated levels. This graded, long lasting increase in bioavailable IGF stimulates the growth, survival and maturation of neuronal tissue cells 20 without causing the local or systemic side effects commonly Jbs~lv~d in treatments with free IGF (eg.
hypoglycemia, receptor down regulation, growth hormone suppression) .
25 ~ c For ~ n~ Out the Tnv~ntion Def initions:
As used herein, "Subjects" are defined as humans and l; ~n farm animals, sport animals and pets. Farm animals include, but are not limited to, 3 0 cows, hogs and sheep . Sport animals include, but are not limited to, dogs and horses. The category pets includes, but is not limited to, cats and dogs.
"Insulin-like growth factor (IGF) n comprises a family of factors, including, but not limited to, IGF-I
35 and IGF-II. IGF is a polypeptide having a molecular Wo 95/13823 -12 - PCTiUS94/13177 weight of about 7500 daltons. IGP may be obtained from natural sources or prepared by rec ` in~nt means.
~ Insulin-like growth factor binding proteins (IGFBPs) " comprises a family of binding proteins, 5 in~ 9;ng but not limited to IGFBP-l, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-5 and IGFBP-6. IGFBP may be obtained from natural sources or prepareq by recombinant means. At least one form of IGFBP (for example, IGFBP-3) complexes with IGF and with a third molecule known as ALS.
A "therapeutic composition" as used herein is defined as comprising IGF complexed with its binding protein IGFBP-3. The therapeutic composition also nnnt;iinc~ other gubgtanceg such as water, minerals and carriers such as proteins.
"Alleviation of the conditionn is said to occur when the sub; ect to whom the IGF/IGFBP- 3 complex is aqministered exhibits improved function of affected nervous tissue. For peripheral nervous system conditions, il.L~r uv~ ts:l include, but are not limited to, 20 improved coordination of ~ V , improved muscle function and strength, decreased pain, reduced nllmhn~o~s of extremities, and increased sensory function (eg.
touch). For central nervous system conditions, ~ lL~V. t~3 include, but are not limited to, improved 25 ability to reason, improved memory, improved speech, improved coordination or ~ ..L, reduced pain and improved sensory function (eg. sight, hearing).
Descril~tion of the Invention The method of the present invention contemplates treating neurological disorders by aqministering a complex of ~IGF and IGFBP-3.
Nearly all IGF- I or IGF- II complexes with IGFBP-3. IGF/IGFBP-3 nor~ally circulates in the form of 35 a complex in humans and other m~m~als. This complex wo s5/l38Z3 ~ ~ ~ 7 6 7 0 8 PCT/US94113177 associates with a third protein ~ALS), which is present in excess over the ~-on~-~ntr~tion of= IGF and IGFBP-3.
Theref ore, A~S i8 f ound both associated with the IGF/IGFBP-3 complex and in the free form. The resultant 5 ternary complex has a size of about 150 kd.
Conditions which are treated by the method of the present invention include Huntington' s disease, _l ~h-1m~r~8 digease, exposure to neurotoxins, ceLcl)Luvc-scular h _Lllc-ge, neuronal scission during 10 surgery, meningitis, other infection of the tissues of the nervous system, multiple sclerosis, amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease.
"Huntington' 8 disease~ is defined as an autosomal ~lnmin~nt disorder usually beginning in middle 15 age and characterized by choreiform ~ and progressive intellectual deter~ or~t; n . There are estimated to be about 25,000 cases in the United States.
It is diagnosed on CT scans by characteristic llboxcar ventricles" which result from atrophy of the caudate 20 nucleus. With that atrophy, there are decreases in levels of the neurotransmitters ~y-aminobutyric acid (GA;3A) and substance P (an ll-amino acid peptide), _nkArhiql ~ n~ and dynorphin in the stratum and its sites of projection. However, somatostatin and ne:uLu~e:~tide y may 25 be relatively increased in t~e caudate nucleus and putamen . ~l~n~r~ 1 1 y there is a pattern of cell death similar to that reproduced exper~ t-l 1 y by glutamate receptor agonists that act on the N-methyl-D-aspartate subclass of glutamate receptors. Current treatment is 30 palliative: suppressing choreic v~ - and agitated behavior with phenoth~-~in- or buLyLu~llenone neuroleptics or reserpine. Administration of IGF/IGFBP-3 helps this condition through its trophic efects on nerve growth and ~- 1 n t W095/13823 ~ ~ 767~3 PCTNS94113177 z~l 7hPi r~ S disease is a ~orm of progressive atrophy of the brain. It is the cl ^-t cause of ~ 1~ in the elderly and has a freguency of almost 20~
in those over 80 years old. The primary feature is death and 9; q~rpPArance of cells from the brain, resulting in extensive convrl lt;on~l atrophy. Acetylcholine-transmitting neurons are part;cl1l~rly affected. ~oss o~
peptidergic neurons in the cerebrum is associated with reduced somatostatin and corticotropin releasing factor rnnrPntrations . Somatostatin is also ~hnnrr~l 1 y low in the CSF. An early symptom is memory 10BS, followed by slow disintegration of judgment and affect. The clinician must rule out organic causes, such as drug overdoses, vitamin deficiencies, alcohol, ischemic conditions, etc. At present, management of patients with Alzheimer 8 disease is largely supportive, limiting the confusion and frustration with their environment.
Patients with ~~7hp;mprls disease may improve with IGF/IGFBP-3 complex administration, such as with an improved sense of well-being, affect and/or memory.
Another condition in which the IGF/IGFBP
complex promotes healing is e~ O:~u~ e to neurotoxins.
Polyneuropathy can result from exposure to the following envi~ ' toxins: acrylamide (herbicide, grout), arsenic (herbicide, insecticide~, b1 rkthnrn, carbon disulfide, ~irhthPria dimethylamino propionitrile, ~y-diketone hoY~rArhnn solvents, inorganic lead, or~norhn~phates and th~ lm (rat poison). Many drugs have neurologic adverse rP~rt;nnc~ See, for example, Tables 363-1-3, which list polyneuropathies associated with systemic disease, drugs or enviL, ~' toxins, and genetically determined conditions, respectively rrisnn s pr;nr;rles of Intprn~l MP~l;r1nP, 12th ed.
McGraw-}Iill, New Yorl~ City, 1991, pp. 2099-2103~. In `35 addition, animal toxins, such as the tetanus toxin and W09!;113823 ;~ ~ 767~8 rcTNsg4/l3l77 the toxins of various snakes and scorpions, damage the nervous system and interfere with respiration, heart rate, etc. For each of these toxins, the specific or underlying disorder must be treated, and vital functions 5 may need to be supported. In addition, the administration of the IGF/IGFBP-3 complex speeds healing and ~nc~llrAges the sprouting of new neurites.
Cerebrovascular h~m~rrh~ge is characterized by rupture of a cerebral blood vessel and bleeding into the 10 intr~rrAn;Al space, which compresses and may damage cerebral nerve and glial cells. Similarly, during surgery, nerves may be inadvertently or necessarily compressed or severed (scission). In both situations, the administration of IGF/IGFBP-3 will help nervous 15 tis6ue recover.
Meningitis and other inf ections of tissues of the nervous system have bacterial and viral origins.
Acute viral ~nc~rhAl ;tig ig an acute ;nfl: ti~-n of the brain due to virus or hypersensitivity caused by a virus 2 0 or other f oreign protein. If the spinal cord structures also are affected, the condition is called ~n~-~rhAl omyelitis . It is frequently called '~aseptic"
because no organisms are f ound. Cerebral edema is present, along with numerous small h Lllages which are 25 scattered throughout the brain, brainstem, cerebellum and sometimes the spinal cord. Viral invasion may cause nerve necrosis and/or inclusion bodies ~ DemyPl ' nAt; ng lesions sometimes are seen around veins. Therapy of the underlying inf ection is the primary concern . General 30 therapy includes antiviral and/or Antlh~ctsr~Al therapy, fluid therapy without uv~LllydLcltion, and if indicated steroid therapy to counteract the swelling associated with meningitis . In additio~, IGF/IGFBP- 3 administration helps restore nerve and glial cells. IGF/IGFBP can be 35 administered intracranially with other therapies.

:2 1 76708 Wo 9S/13823 ~ -~ PCT~S9~/13177 Meningitis also iB associated with a number of non-bacterial/viral conditions, such as funyal infections (especially with AIDS or ~ n~ s~ive therapy), T~3, dissemination of malignant cells as in leukemia, 5 metastatic carcinoma (~Apel-~Ally of lung and breast), gliomas, syphilis and sart ni~lns; R. Current therapy includes treatment of the underlying disorder, as well as steroids (such as prednisone) to reduce ~nfl; t;cn. In addition, administration of IGF/IGFBP-3 f.nhAn~'P~ recovery lO of injured nervous and glial tissue.
Multiple sclerosis has been characterized as "a slowly progressive CNS disease characterized by diss~m; nAt~l patches of demyelination in the brain and spinal cord, resulting in multiple and varied neurologic 15 sy~nptoms and signs, usually with remissions and ~ Af'~rhAti nn~ . " TH~ Mg~cK Ma~L, 15th ed., Merck ~ Co., Rahway, N.J., 1987, pp. 1414-17. There are plaques of demyelination, destroyed oligodendroglia and perivascular inflammation throughout the CNS. I,ater, nerves also may 20 be destroyed. The administration of IGF/IGFBP-3 will encourage the rerl Ar,~m~nt of neurites and glial cells .
In contrast, amyotrophic lateral sclerosis primarily affects motor neurons, producing muscular weakness and atrophy. Cramps are an early sign. I,ater 25 fasciculations, spasticity and hyperactive reflexes are observed . IGF/IGFBP- 3 administration will encourage the formation of new neurites.
Charcot-Marie-Tooth disease is an autosomally r~nminAnt disorder of the peripheral nervous system in 30 which weakness and atrophy, particularly of the peroneal and distal leg muscles, gradually develops over years.
Biopsy may show sesmental demyelination and remyelination. At present there is no specific treatment, aside from bracing weak muscles such as to W0 95/13823 ( r ~ ~; 2 1 7 6 7 0 3 PCT/US94/13177 limit foot drop. The administration 0~ IGF/IGFBP-3 ic supportive therapy, intended to enhance remyelination.
Systemic ~, n;~trAtion of IGF and IGFBP-3, either from natural or re- ~m~;nAnt sources, as a 5 preformed complex results in the forr-tion of the normal ternary complex with the excess AIS. This ty-pe of treatment produces a prolonged increase in the level of circulating IGF, which is gradually released from the ternary complex. This mode of administration avoids the lO detrimental side effects associated with administration of free IGF-I, namely, hypoglycemia, ~,u~l~Lc~ssion of growth hormone and AIS production, and release of endogenous IGF - II since administered exogenous f ree IGF- I
replaces endogenous IGF- II in normally circulating 15 IGF-II/IGF~P-3 complexes.
The f, lAt;on~ method of administration and dosage will depend upon the disorder to be treated and the medical history of the patient. These factors are readily A~t~;nod in the course of therapy. Suitable 20 patients with neurological disorders can be identified by medical history-, physical f;nrl;ngq and laboratory tests.
The medical history may reveal such facts as loss of coor~l;nAt;~n, muscle weakness, tremors, 11;77;nG~R, h.,A~. h~, loss of memory, impaired speech, cognitive 25 difficulties and the specific f~nrl;ng~ ~so~-iAted with the individual conditions 1; Cr~ d above. Patients may have physical findi~gs such as muscle weakness, impaired reflexes, impaired coordination, disorientation, memory loss, impaired language function, impaired sensory 30 function as well as specific findings associated with the individual conditions discussed above. Tnrl' rAtive diagnostic procedures include computerized t~ yLGyhy (CT) scans, magnetic resonance imagery (~I), electrc~nr~rhAl ography (EEG), c~L~Lot,~inal fluid (CSF) 35 analysis and the like.

W0 95ll3823 ` ~ ~ ~ 7 6 7 0 8 PCTIUS94113177 In accordance with the method of the present inverltion, the f~ lat;on comprises a complex of IGF and IGFBP - 3 . Pref erably, the IGF i9 IGF- I, although IGF - II
also is useful. Because IGF and IGFBP-3 n~tllr~1 ly S complex in a 1:1 molar ratio, a compo8ition Of f"I"; l~r amounts of IGF and IGFBP-3 is preferred. The product can be f ormulated with IGF: IGFBP - 3 molar ratios ranging f rom about 0.5 to 1.5. More preferably, the molar ratio is about 0.9 to 1.3; and most preferably, the product is 10 formulated with approxistely a 1 1 molar ratio.
In accordance with the method of the present invention, the IGF and IGFBP- 3 are human proteins obtained from natural or re~ ' ;n~nt sources. Most pref erably, IGF and IGFBP - 3 are human IGF - I and IGFBP- 3 15 made by re~ ' ;n~nt means and designated rhIGF-I and rhIGFBP - 3, respectively . rhIGFBP - 3 may be in glycosylated or non- glycosylated f orm . E . coli is a source of the non-glycosylated IGFBP-3. Glycosylated IGFBP-3 may be obtained from Chinese hamster ovary (CH0) 20 cells.
The method of the present invention provides for f~ l~t;n~ the complex in modes which are readily apparent to those skilled in the art. Preferably, the IGF and IGFBP- 3 are complexed prior to administration to 25 the treated subject. Preferably, the complex is formed by mixing approximately equimolar amounts of IGF- I and IGFBP-3 dissolved in physiologically c~ t;hle carriers such as normal saline solution or ~h~ph~te buffered saline solution. Most preferably, a concPntr~trfl 30 solution of rhIGF-I a~d a concentrated solution of rhIGFBP-3 are mixed together for a ~l1ff;ri~nt time to form an equimolar complex. ~
DPr~n~l; ng on the mode of administration, compositions of the complex may be in the form of solid, 35 semi-solid or liquid dosage preparations, such as for -WO 95113823 2 ~ 7 6 7 0 8 PCTIIJS94/131M

example, tablets, pills, powders, capsules, liquids, suqp~onqionq or the like. Physiologically t;hle carriers include intravenous solutions, such as normal saline, serum albumin, 5% dextrose, plasma preparations, 5 and other protein-r~ntA;n;n~ solutions. The preferred carrier for parenteral administration of the complex is a sterile, isotonic aqueous solution, such as normal saline or 596 dextrose. Alternatively, a solution of the complex may be placed. into an implant, such as an osmotic pump, 10 for the slow release of the complex over an ~Yt~n~lPd period of time. Alternatively, the complex may be provided in sustained release carrier formulations such as semi-p~ -hle polymer carriers in the form of suppositories or microcapsules. See, for instance, U.S
Patent No. 3,773,919 for Microcapsular Sustained Release Matrices Including Poly~Act;~q; Sidman et al., B i o~olYmers 2 2 ( 1 ): 5 4 7 - 5 5 6 ( 1 9 8 3 ) f or copolymers o f ~ -glutamic acid and y-ethyl-~-glutamate; ~anger et al., ~ Biom~-~l Res ~: 267-277 (1981) for 2 0 pol y ( 2 - lly dL U_'Ly ~:: thylme thacryl ate ) o r the 1 ike .
The mode of administration delivers the complex to the subject in a safe, physiologically effective manner. The complex may be given by intranasal, subcutaneous, intravenous, intramuscular, 25 lntrAr~ritoneal, ;ntr~ rAn;Al or other conV~nt;~-nZ~l routes of administration. Preferably, the complex is injected qllhrlltAnPollcly, intravenously or ;n qcularly. Most preferably, the complex is administered by subcutaneous injection. By gub~ ~t~n~ollq 30 injection, the complex appears not to be toxic or mitogenic at the inj ection site .
The dose of complex to be administered can be readily det~rm;n~ by those skilled in the art, based on the usual patient symptoms discussed above. Preferably, 35 when the complex is administered to humans daily, the ~0 95/13823 ! 7 0 8 PCr/US94/13177 dosage of complex is about 0 . Ol to lO mg of IGP-I or IGF-II/kg of body weight/day, complexed to an ~ r amount of IGFBP-3. More preferably, the daily dosage of the complex for humans is about 0.05 to 7.5 mg 5 IGF/kg/day, complexed to an equimolar amount of IGFBP-3.
Most preferably, the daily dosage of the complex for humans is about 0 . l to 5 mg IGF/kg/day, complexed to an equimolar amount of IGF3P - 3 . If daily dosages in excess of about 0 . 5 mg IGF/kg must be given, the dosage may be 10 divided and injected subcut~n~-) lRly at two or more sites.
If the IGF/IGFBP- 3 comFlex were administered to humans twice a week, each dose of complex is preferably about 0.05 to lO mg IGF/kg of body weight, complexed to an equimolar amount of IGFBP-3. More preferably, for 15 twice weekly administration, the dose of the complex is about O.l to 7.5 mg IGF/kg, complexed to an equimolar amount of IGFBP-3. Most preferably, for twice weekly administration, the dose of the complex i9 about 0 . 5 to 5 mg IGF/kg, complexed to an eguimolar amount of IGFBP-3.
20 There is no known upper limit of dosage; however, it is preferable that a slngle dose ~ot exceed lO mg IGF/kg o~
body weight, when the IGF is complexed to an equimolar amount of IGFBP-3. These doses of IGF/IGFBP-3 complex are not eYpected to cause signif icant hypoglycemia since 25 the IGF/IGFBP-3 slowly releases IGF to cellular insulin receptors .
Preferably, the patient is started with a relatively low dose of the complex, such as 0 . 05 mg IGF/kg of body weight/day. Physical oY~minAtions, 30 functional tests and diagnostic procedures such as those outl ;n~ above should be performed on the treated patients to determine; f there is il..~LVV~lL .
Preferably, the patient shows il..l)LVV~ -Cl in the structure and/or function of the pPr;rh~r;ll or central 35 ne ~ous tissue affected by the neurological disorder WO 9S/13823 ` 2 1 7 6 7 0 ~ PCT/~JS~4/~3177 f ollowing such treatment . If the patient improveg with the low dose, the low dose preferAbly should be ~ nt;
until acceptable clinical endpoints have been achieved.
If the patient does not respond to low dose 5 IGF/IGFBP-3 complex with sufficient clinical iLL~Luv~ t, the dose of complex should be increased gradually until such an outcome is achieved.
The invention has been disclosed by direct description. Following are examples showing the efficacy 10 of the IGF/IGFBP- 3 complex in stimulating processes critical to the growth, survival and functioning of neurological tissue. The P~ Pq are only examples and should not be taken in any way as limiting to the scope of the process.

R~l~qpr.R.c F le 1 This example is designed to demonstrate the 20 ability of the rhIGF-I/IGFBP-3 complex to 8t; lAtP the sprouting of neurites (nerve processes~ in cultured embryonic chick spinal cord motor neurons. The sprouting of neurites leads to the re-est~hl; ql ~ of innervation and consequently full function of partially denervated 25 neuromuscular junctions and disrupted central nervous system neural connections.
In this experiment, cultures of motor neuron cells are prepared from embryonic chick spinal cord tissue. Dissociated lumbar and hr~ hi~l spinal cord 30 cells are purified by differential Ficoll grAt9;Pnt centrifugation, and the motor neuron frarti~n is isolated. Primary cultures of these cells are plated in laminin coated tis6ue culture dish wells in enriched L15 medium c~nt~;nlng 20~ horse serum and 20 ~g/ml embryonic 35 chick hind limb muscle proteir3 extract. The majority of 2 ~ 767~3 Wo 95/13823 =- PCr/US94/13177 the cells in this culture are neurons with large multipolar cell bodies, and non-neuronal cells represent only a few percent of the total. Cells are plated in either the above medium alone, or in medium rnnt~;nin~ 1 5 to lO0 ng/ml rhIGF-I complexed to an equimolar amount of rhIGF~3P- 3 . The extent of neurite outgrowth in each set of cultures is assessed by light microscopy daily for up to 7 days. The e~fect of each treatment is determined by measuring the total length of the neurite tree f or each lO neuron at each time point.
F le 2 This example is designed to demonstrate the ability of the IGF-I/IGF~3P-3 complex to stimulate 15 regeneration of severed neurons. Tr~ tic or surgical injury to peripheral nerves is troublesome since the regeneration of damaged nerves is often slow and functionally incomplete. There is a clinical need for agents that can promote more rapid and completely 20 functional reg~n~or~tinn of such damaged nerves.
In this experiment, incisions are made in the hind legs of Sprague-Dawley rats, and the sciatlc nerve of each rat is transected at the mid- thigh region. The proximal end of the nerve is placed into one channel of a 25 silicone block cnnt~in~n~ three rh~nnGl ~ arranged in a Y-shape. A small, i , 1 ~n}~hl e osmotic pump is connected through a piece of tubing to one of the two " ;n~n~
channel6 of the block. The third channel is left open, and the block, tubing and pump sutured in place.
30 Finally, the incision is sutured closed.
In one set o~ anlmals, groups of rats have the osmotic pump filled with various concentrations rhIGF-I/IGFBP-3 complex (O.l-l mg/ml of rhIGF-I complexed to an equimolar amount of rhIGFi3P-3) in physlologlcal sallne 35 plus l~ rat serum albumln (RSA~. In ~ group of control . ~ - - 21 76708 wo 95~13823 PCTIUS94/13177 rats, the pump is f illed with physiological saline plus 1~ RSA only. The pumps are left in place to pump at a rate of approximately 0.5 ~l/hr for apprn~ t~ly 3-4 weeks to allow nerve reg~n-~r~ t ~ nn At the end of the 3-4 week treatment period, the animals are sacrificed and the silicone blocks recovered. The pumps are removed from the block and any tissue in the block is fixed by immersing the opened block in standard glutaraldehyde fixative. The fixed tissue i8 then stained with osmium tetroxide and dehydrated. Each channel in the block is cut into short lengths ~ ~d starting from the severed nerve stump .
Thin sections are cut f rom each short length of channel and are stained with methylene blue and azur II. ~ight microscopy is used to evaluate the presence of myelinated axons from the regpn~r~t~ nerve in each short length of each channel.
Rl~mnl e 3 This example is designed to demonstrate the ability of the rhIGF-I/IGE}3P-3 complex to stimulate the growth and myelination of nervous tissue in the central nervous system. A variety of illnesses result in the demyelination of central nervous system neurons (eg.
multiple sclerosis, acute diSsf~m; n ~
pn~ h;.l omyelitis) . Thig demyelination results in defective nerve tr~n~mi ~ion and loss of sensory and motor function. An agent that would stimulate myelin production in such cases would be a useful therapeutic.
In this experiment myelin production is assessed in fetal rat brain aggregate cultures. Whole fetal rat brain cells are dissociated into single cells, filtered, and then placed into aggregate culture medium (Almazan et. al., Dev. N~llrosci. 7, 45-54 (1935) ) 35 supplemented with 1096 fetal calf serum. The cells are ' ` 21 7670~
WOg5/13823 PCTiUS94/l3l77 grown in suspension for=two days, at which time either f resh medium alone or f resh medium plu8 0 . 01-1 ~g/ml rhIGF-I complexed to an equimolar amount of rhIGF~3P-3 is added. Myelin produced by the cultures is isolated and 5 quantitated following 10-30 days of culture. At each time point, the number and maturity of oligodendrocytes is also quantitated by measuring the activity of the oligodendrocyte marker 2'-3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in cell homogenates.
This invention has been detailed both by example and by direct description. It should be apparent that one having ordinary skill in this art would be able to surmise equivalents to the invention as described in 15 the claims which follow but which would be within the spirit of the description above. Those equivalents are -to be included within the scope of this invention.

Claims

-25-

1. A method for treating a subject for Huntington' s disease and Alzheimer's disease, said method comprising administering to said subject a complex comprising an insulin-like growth factor (IGF) and insulin-like growth fact or binding protein-3 (IGFBP-3), said complex being administered in an amount sufficient to alleviate said condition.

2. The method of claim 1 wherein the complex comprises equimolar amounts of IGF and IGFBP-3.

3. The method of claim 1 wherein the IGF is IGF-I.

4. The method of claim 3 wherein the IGF is recornbinant human IGF-I.

5. The method of claim 1 wherein the IGFBP-3 is recombinant human IGFBP-3.

6. The method of claim 1 wherein the IGF is IGF-II.

7. The method of claim 6 wherein the IGF-II is recombinant human IGF-II.

8. The method of claim 6 wherein the IGFBP-3 is recombinant human IGFBP-3.

9. The method of claim 1 wherein the administration is parenteral.

10. The method of claim 1 wherein the parenteral administration is performed by subcutaneous injection.

11. The method of claim 1 wherein the administration is intracranial.

12. The method of claim 1 wherein the sufficient amount is that amount of complex that results in improvements in the structure or function of the nervous system.

13. The method of claim 12 wherein the amount of complex administered is at least about 0.05 mg IGF/kg of body weight/day, said IGF being complexed to an equimolor amount of IGFBP.

14. The method of claim 1 wherein said subject is a mammal.

15. A method for treating a subject for exposure to neurotoxins, cerebrovascular hemorrhage, neuronal scission during surgery, meningitis or other infection of tissues of the nervous system, said method comprising administering to said subject a complex comprising an insulin-like growth factor (IGF) and insulin-like growth factor binding protein-3 (IGFBP-3), said complex being administered in an amount sufficient to alleviate said condition.

16. A method for treating a subject for multiple sclerosis, amyotrophic lateral sclerosis or Charcot-Marie-Tooth disease, said method comprising parenterally administering to said subject a complex comprising an insulin-like growth factor (IGF) and insulin-like growth factor binding protein-3 (IGFBP-3), said complex being administered in an amount sufficient to alleviate said condition.

17. The method of claim 16 wherein parenterally administering the IGF/IGFBP complex comprises subcutaneous injection.