Note: Descriptions are shown in the official language in which they were submitted.
- 2~ 767~8
WO 95/13823 ` - 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
--4 --
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
--5--
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
--7--
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
35
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
-10 -
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
-11-
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
-13-
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
-14-
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
-15-
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
-16 -
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
-17-
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
-18 -
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
-19-
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
-21-
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
-22 -
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
-23 -
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
-24 -
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.
