U.S. patent application number 15/486734 was filed with the patent office on 2017-08-03 for synthetic analogues of neural regeneration peptides.
The applicant listed for this patent is CURONZ HOLDINGS COMPANY LIMITED. Invention is credited to Margaret Anne Brimble, Paul William Richard Harris, Frank Sieg.
Application Number | 20170218023 15/486734 |
Document ID | / |
Family ID | 40567715 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218023 |
Kind Code |
A1 |
Harris; Paul William Richard ;
et al. |
August 3, 2017 |
SYNTHETIC ANALOGUES OF NEURAL REGENERATION PEPTIDES
Abstract
Embodiments of this invention include synthetic compounds (NRP
analogues) of peptides termed neural regeneration peptides (NRPs).
NRP analogues are made by substituting amino acids in the native
peptide sequence, modifying amino acids chemically, by replacing
amino acids with synthetic moieties, by stabilizing .beta.-turns,
acetylation of terminal glycine residues or by cyclization. NRP
analogues can be used to treat a variety of conditions involving
degeneration of neural cells, and includes treating disorders of
the nervous system, including peripheral neuropathy, multiple
sclerosis, diabetic peripheral neuropathy, neurotoxin-induced
neurodegeneration, and amyotrophic lateral sclerosis.
Inventors: |
Harris; Paul William Richard;
(Waitakere City, NZ) ; Brimble; Margaret Anne;
(Auckland, NZ) ; Sieg; Frank; (Wellsford,
NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CURONZ HOLDINGS COMPANY LIMITED |
Wellsford |
|
NZ |
|
|
Family ID: |
40567715 |
Appl. No.: |
15/486734 |
Filed: |
April 13, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14701991 |
May 1, 2015 |
9650418 |
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15486734 |
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13651884 |
Oct 15, 2012 |
9040485 |
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14701991 |
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12754937 |
Apr 6, 2010 |
8309684 |
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13651884 |
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PCT/US2008/011951 |
Oct 17, 2008 |
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12754937 |
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60999292 |
Oct 17, 2007 |
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60999503 |
Oct 18, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/2878 20130101;
G01N 2800/2892 20130101; A61P 21/00 20180101; G01N 2800/2835
20130101; A61P 25/28 20180101; C07K 7/08 20130101; A61K 38/00
20130101; A61P 9/10 20180101; G01N 2800/28 20130101; C07K 7/06
20130101; A61P 25/02 20180101; C07K 14/48 20130101; A61P 25/00
20180101; G01N 2800/285 20130101; A61P 43/00 20180101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Claims
1. A neural regeneration peptide (NRP) compound selected from the
group comprising the sequence selected from the group consisting of
SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,
SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.
2. The NRP compound of claim 1 where the NRP compound is selected
from the group consisting of SEQ ID NO:4 and SEQ ID NO:5.
3. The NRP compound of claim 1 where the NRP compound is SEQ ID
NO:6.
4. The NRP compound of claim 1 where the NRP compound is SEQ ID
NO:7.
5. The NRP compound of claim 1 where the NRP compound is and SEQ ID
NO:8.
6. The NRP compound of claim 1 where the NRP compound is selected
from the group consisting of SEQ ID NO:9, SEQ ID NO:10 and SEQ ID
NO:11.
7. A pharmaceutical composition comprising a NRP compound of claim
1 and a pharmaceutically acceptable excipient.
8. A method of treating a neurological disorder characterized by
loss of cells in an animal, comprising administering to said animal
a pharmaceutically effective amount of NRP compound of claim 1 or a
pharmaceutical composition as above.
9. The method of claim 8, wherein said NRP compound consists of the
sequence SEQ ID NO:4 or SEQ ID NO:5.
10. The method of claim 8, wherein said NRP compound consists of
the sequence SEQ ID NO:6.
11. The method of claim 8, wherein said NRP compound consists of
the sequence SEQ ID NO:7.
12. The method of claim 8, wherein said NRP compound consists of
the sequence SEQ ID NO:8.
13. The method of claim 8, wherein said NRP compound consists of
the sequence SEQ ID NO:9, SEQ ID NO:10 or SEQ ID NO:11.
14. The method of claim 8, wherein said disorder is of neural
cells.
15. The method of claim 8, wherein said neurological disorder is
amyotrophic lateral sclerosis, neurotoxin injury, oxidative injury,
multiple sclerosis, peripheral neuropathy, hypoxia/ischemia,
traumatic brain injury, or coronary artery bypass graft
surgery.
16. The method of claim 8, where said neurological disorder is
diabetic peripheral neuropathy.
17. A synthetic NRP compound, wherein said compound is produced
from a NRP by modifying a .beta.-turn, a peptide domain APGR, a
peptide domain RAGG, a replacement of a glycine residue with
asparagine, replacement of an L-amino acid by a D-amino acid, or
cyclization of said NRP.
18. A method of treating a neurological disorder characterized by
neurodegeneration, comprising administering to a patient in need
thereof, a synthetic NRP compound of claim 17.
Description
CLAIM OF PRIORITY
[0001] This PCT International application claims priority to U.S.
Provisional Application No. 60/999,292 entitled "Synthetic Analogs
of Neural Regeneration Peptides," filed Oct. 17, 2007, and to U.S.
Provisional Patent Application No. 60/999,503, entitled "Synthetic
Analogs of Neural Regeneration Peptides," filed Oct. 18, 2007. Both
of these provisional applications are incorporated herein fully by
reference as if individually so incorporated.
FIELD OF THE INVENTION
[0002] This invention relates to synthetic analogues of peptides
that have neural regeneration, migration, proliferation,
differentiation and/or axonal outgrowth properties. These peptides
are termed "Neural Regeneration Peptides" or "NRPs." In particular,
this invention relates to analogues of relatively small peptides
that have one or more biological properties of NRPs.
BACKGROUND
[0003] Neural regeneration peptides (NRPs) are a class of peptides
that have been shown to exhibit properties desirable for promoting
neural function in mammals. These functions include neural
survival, neural proliferation, neuronal outgrowth, neural
migration and neuronal differentiation. Several NRPs have been
previously described, and include those disclosed in U.S. patent
application Ser. Nos. 10/225,838 and 10/976,699, PCT/US02/026782,
PCT/US2004/036203, PCT/US2006017534 and PCT/US2006026994. Each of
the above patent applications is expressly incorporated herein
fully by reference as if individually so incorporated.
SUMMARY
[0004] NRPs described to date have desirable pharmacodynamic
properties and promote neural regeneration, migration,
proliferation, differentiation and/or axonal outgrowth. We have
recently discovered synthetic NRP analogues that also have improved
pharmacokinetic properties. There is a need in the art for
synthetic molecules or modified peptides that have desirable
pharmacodynamic properties similar to those of NRPs but also have
improved pharmacokinetic properties and/or are chemically
stable.
[0005] Certain aspects of this invention include novel synthetic
NRP analogue molecules that can be used to treat disorders of the
nervous system or other systems in which NPRs is effective. Another
aspect is to provide therapies for disorders of cellular
degeneration and death, including certain nervous system disorders.
In some aspects, synthetic analogues of NRPs can be used to treat
adverse effects of amyotrophic lateral sclerosis (ALS), multiple
sclerosis (MS), oxidative stress (e.g., Huntington's disease) or
peripheral neuropathy (PN). An additional aspect of this invention
is the production of NRP analogues with improved stability.
[0006] It should be understood that the terms "NRP compound,"
"analogue of NRP" "SEQ ID NO:" and other such terms, for
simplicity, are used to identify the molecules of the invention and
not to provide their complete characterization. Thus, an "analogue
of NRP" may be characterized herein as having a particular amino
acid sequence, a particular 2-dimensional representation of the
structure, but it is understood that the actual molecule claimed
has other features, including 3-dimensional structure, mobility
about certain bonds and other properties of the molecule as a
whole. It is the molecules themselves and their properties as a
whole that are the subjects of this invention.
[0007] It should also be understood that the designation of a
peptide as an "NRP" does not mean that it solely has neural
effects. Rather, the term NRP is intended to include peptides
having similar structural components as described in the above
patent applications, but may have effects on other cell types,
tissues, and/or organs. In certain embodiments, analogues of
relatively short NRPs are provided that can have increased
stability, due at least in part to decreased enzymatic degradation.
In other embodiments, NRP analogues are provided having modified
amino acids. In yet further embodiments, NRP analogues are provided
that have non-amino acid substituents replacing amino acids.
BRIEF DESCRIPTION OF THE FIGURES
[0008] This invention will be described with reference to specific
embodiments thereof. Other features and aspects of this invention
can be appreciated by reference to the Figures, in which:
[0009] FIG. 1 depicts a graph of neuroprotective effects of two
NRPs, SEQ ID NO: 5 of this invention and SEQ ID NO: 1 in cell
cultures exposed to the neurotoxin 3-NP.
[0010] FIG. 2 depicts a graph of results of studies of
neuroprotective effective of SEQ ID NO:1 in which the peptide was
stored at either -20.degree. C. or -4.degree. C.
[0011] FIG. 3 depicts a graph of results of studies of
neuroprotective effects of SEQ ID NO: 5 of this invention in which
the peptide was stored at either -20.degree. C. or -4.degree.
C.
[0012] FIG. 4 depicts a graph of results of an enlarged study of
neuroprotective effects of SEQ ID NO:5 of this invention and SEQ ID
NO:1 in cell cultures treated with the neurotoxin 3-NP, similar to
those shown in FIG. 1.
[0013] FIG. 5 depicts a graph showing significant long-term effects
of sequence REGRRDAPGRAGG (SEQ ID NO:12) of this invention to
decrease the severity of motoric impairment in animals with EAE,
when the synthetic NRP was administered at the peak of the disease.
Score 1 is the lowest score and implies a flaccid tail only, while
the higher scores imply weakness (score 2) or complete paralysis of
the hind legs (score 3). Kruskal-Wallis-test was used for
statistical analysis; ** p<0.01 versus treatment day 1 score
(data expressed as mean.+-.SEM).
[0014] FIG. 6 depicts a graph of results of beam walking scores for
rats with peripheral neuropathy induced by pyridoxine (800
mg/kg/day) and treated with either vehicle or SEQ ID NO:5 of this
invention at two different doses.
[0015] FIG. 7 depicts a graph of results of beam walking scores for
rats with peripheral neuropathy induced by pyridoxine (1200 mg/kg)
and treated with either vehicle or SEQ ID NO:5 of this
invention.
[0016] FIG. 8 depicts graphs of results of studies of longevity of
mice with a murine model of amyotrophic lateral sclerosis (ALS) and
the effects of two different doses of a synthetic NRP of this
invention, SEQ ID NO:5.
DETAILED DESCRIPTION
[0017] In some embodiments, NRP compounds are provided that have a
sequence of a native peptide. For example, one such NRP is an 11
amino acid long (11-mer) peptide having the following sequence.
TABLE-US-00001 NH.sub.2-G.sup.1RRAAPGRAGG.sup.11-NH.sub.2 SEQ ID
NO: 1
[0018] It should be appreciated that synthetic compounds or
analogues of NRPs can have either amidated C-termini or can have
C-terminal hydroxyl residues (OH). It should also be appreciated
that the terms "NRP compound," "NRP analogue" and similar terms
refer to compounds of this invention or to previously disclosed NRP
peptides or NRP proteins.
Synthetic Analogues of NRPs
[0019] Synthetic analogues of NPRs are provided that can have one
or more of the following types of modifications: (1) stabilization
of .beta.-turns, (2) replacement of glycine residues, (3)
replacement of the N-terminal glycine residue and/or (4)
cyclization.
1. Stabilization of .beta.-Turns
[0020] Chou and Fasman probabilities for 1-turn prediction reveal
that probable 1-turns in SEQ ID NO:1 can be found in the domains
APGR (SEQ ID NO:2) and RAGG (SEQ ID NO:3), as shown in bold
below:
TABLE-US-00002 SEQ ID NO: 1.
NH.sub.2-G.sup.1RRAAPGRAGG.sup.11-NH.sub.2 SEQ ID NO: 1
[0021] .beta.-turns can be stabilised by introducing steric
constraints such as alkylated amino acids. Readily available amino
acids that can be used include aminoisobutyric acid (Aib, .alpha.-H
on alanine replaced with methyl) can be used as a replacement for
either or both of alanine and glycine residues.
[0022] A. Modification of the APGR Domain
[0023] In sequence APGR (SEQ ID NO: 2), the alanine or glycine can
be replaced with aminoisobutyric acid (Aib). Substitution of the
alanine with Aib produces the following analogue:
TABLE-US-00003 NH.sub.2-G.sup.1RRA-Aib-PGRAGG.sup.11-NH.sub.2 SEQ
ID NO: 4
[0024] B. Modification of the RAGG Domain
[0025] In another probable .beta.-turn sequence, RAGG (SEQ ID NO:
3), the alanine can be replaced with aminoisobutyric acid (Aib) to
produce the analogue having the sequence:
TABLE-US-00004 NH.sub.2-G.sup.1RRAAPGR-Aib-GG.sup.11-NH.sub.2 SEQ
ID NO: 5
[0026] Experiments showed that SEQ ID NO:5 was: neuroprotective
(see FIGS. 1-4), stable under storage conditions (FIGS. 2 and 3),
more neuroprotective than the unsubstituted NRP (FIGS. 1 and 4),
peripheral neuropathy (FIGS. 6 and 7) and ALS (FIG. 8).
2. Replacement of Glycine Residues
[0027] Replacement of the internal glycine residue by an asparagine
(N) can induce .beta.-turns due to asparagine having higher
.beta.-turn propensity than glycine. Therefore the internal glycine
residue can be replaced with asparagine at amino acid position 10
producing a peptide having the following sequence:
TABLE-US-00005 NH.sub.2-G.sup.1RRAAPGRANG.sup.11-NH.sub.2 SEQ ID
NO: 6
[0028] This NRP analogue was found to be neuroprotective in in
vitro model of neural toxicity induced by 3-NP.
3. Replacement of the N-Terminal Glycine Residue
[0029] Truncation of the G.sup.1 at the N terminus can result in
loss of biological activity. Replacement of the G.sup.1 with an
acetyl group can restore biological activity. The resulting NRP
analogue is acetylated providing a peptide having the following
sequence:
TABLE-US-00006 AcNH-RRAAPGRAGG.sup.11-NH.sub.2 SEQ ID NO: 7
[0030] This NRP analogue was found to be neuroprotective in the
face of toxicity induced by 3-NP.
4. Replacement of L-Amino Acids with D-Amino Acids
[0031] The secondary structure of a peptide can be affected by the
presence of D-amino acids replacing one or more L-amino acids
(naturally occurring). Replacement of the third amino acid from the
N-terminus produces a compound having the following sequence:
TABLE-US-00007 NH.sub.2-GR(D-Arg)AAPGRAGG-NH.sub.2 SEQ ID NO: 8
[0032] This NRP analogue was found to be neuroprotective in the in
vitro model of neural toxicity induced by 3-NP.
5. Cyclization
[0033] Synthesis of a cyclic peptide mimetic of SEQ ID NO:1 can be
carried out. One method involves adding a cysteine residue to each
end of the sequence, and then oxidizing the resultant product to
produce a cyclic disulfide having the following sequence:
##STR00001##
[0034] Alternatively, both the N and C terminal glycine residues
can be replaced with a cysteine residue and oxidized similarly as
above, producing an analogue having the following sequence.
##STR00002##
[0035] Direct cyclization of the C terminal residue to the N
terminal residue can be accomplished by creating an amide bond to
produce a peptide having the following sequence.
##STR00003##
[0036] The use of circular dichroism can indicate secondary
structure and the use of computer simulation software for the
modeling of small peptides can also be carried out using
conventional methods. Both of these techniques can be used for
determining structural features of the NRP analogues of this
invention.
Synthesis of Synthetic Analogues of NRPs
[0037] Starting materials and reagents used in preparing these
compounds are either available from commercial suppliers such as
Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance,
Calif.), Sigma (St. Louis, Mo.), or are prepared by methods well
known to the person of ordinary skill in the art following
procedures described in such references as Fieser and Fieser's
Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons,
New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols.
1-5 and supplements, Elsevier Science Publishers, 1989; Organic
Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991;
March J; Advanced Organic Chemistry, 4.sup.th ed. John Wiley and
Sons, New York, N.Y., 1992; and Larock: Comprehensive Organic
Transformations, VCH Publishers, 1989. In most instances, amino
acids and their esters or amides, and protected amino acids, are
widely commercially available; and the preparation of modified
amino acids and their amides or esters are extensively described in
the chemical and biochemical literature and thus well-known to
persons of ordinary skill in the art. For example,
N-pyrrolidineacetic acid is described in Dega-Szafran Z and
Pryzbylak R. Synthesis, IR, and NMR studies of zwitterionic
.alpha.-(1-pyrrolidine)alkanocarboxylic acids and their N-methyl
derivatives. J. Mol. Struct.: 436-7, 107-121, 1997; and
N-piperidineacetic acid is described in Matsuda O, Ito S, and
Sekiya M. each article herein expressly incorporated herein fully
by reference.
[0038] Conveniently, synthetic production of the polypeptides of
the invention may be according to the solid-phase synthetic method
described by Goodman M. (ed.), "Synthesis of Peptides and
Peptidomimetics" in Methods of organic chemistry (Houben-Weyl)
(Workbench Edition, E22a,b,c,d,e; 2004; Georg Thieme Verlag,
Stuttgart, N.Y.), herein expressly incorporated fully by reference.
This technique is well understood and is a common method for
preparation of peptides. The general concept of this method depends
on attachment of the first amino acid of the chain to a solid
polymer by a covalent bond. Succeeding protected amino acids are
added, on at a time (stepwise strategy), or in blocks (segment
strategy), until the desired sequence is assembled. Finally, the
protected peptide is removed from the solid resin support and the
protecting groups are cleaved off. By this procedure, reagents and
by-products are removed by filtration, thus eliminating the
necessity of purifying intermediaries.
[0039] Amino acids may be attached to any suitable polymer as a
resin. The resin must contain a functional group to which the first
protected amino acid can be firmly linked by a covalent bond.
Various polymers are suitable for this purpose, such as cellulose,
polyvinyl alcohol, polymethylmethacrylate and polystyrene. Suitable
resins are commercially available and well known to those of skill
in the art. Appropriate protective groups usable in such synthesis
include tert-butyloxycarbonyl (BOC), benzyl (Bzl),
t-amyloxycarbonyl (Aoc), tosyl (Tos), o-bromo-phenylmethoxycarbonyl
(BrZ), 2,6-dichlorobenzyl (BzlCl.sub.2), and phenylmethoxycarbonyl
(Z or CBZ). Additional protective groups are identified in Goodman,
cited above, as well as in McOmie JFW: Protective Groups in Organic
Chemistry, Plenum Press, New York, 1973, both references expressly
incorporated fully herein by reference.
[0040] General procedures for preparing peptides of this invention
involve initially attaching a carboxyl-terminal protected amino
acid to the resin. After attachment the resin is filtered, washed
and the protecting group on the alpha-amino group of the
carboxyl-terminal amino acid is removed. The removal of this
protecting group must take place, of course, without breaking the
bond between that amino acid and the resin. The next amino, and if
necessary, side chain protected amino acid, is then coupled to the
free amino group of the amino acid on the resin. This coupling
takes place by the formation of an amide bond between the free
carboxyl group of the second amino acid and the amino group of the
first amino acid attached to the resin. This sequence of events is
repeated with successive amino acids until all amino acids are
attached to the resin. Finally, the protected peptide is cleaved
from the resin and the protecting groups removed to reveal the
desired peptide. The cleavage techniques used to separate the
peptide from the resin and to remove the protecting groups depend
upon the selection of resin and protecting groups and are known to
those familiar with the art of peptide synthesis.
[0041] Peptides may be cyclized by the formation of a disulfide
bond between two cysteine residues. Methods for the formation of
such bonds are well known and include such methods as those
described in G. A. Grant (Ed.) Synthetic Peptides A User's Guide
2.sup.nd Ed., Oxford University Press, 2002, W. C. Chan and P. D.
White (Eds.) Fmoc Solid Phase Synthesis A Practical Approach,
Oxford University Press, 2000 and references therein.
[0042] Alternative techniques for peptide synthesis are described
in Bodanszky et al, Peptide Synthesis, 2nd ed, John Wiley and Sons,
New York, 1976, expressly incorporated herein fully by reference.
For example, the peptides of the invention may also be synthesized
using standard solution peptide synthesis methodologies, involving
either stepwise or block coupling of amino acids or peptide
fragments using chemical or enzymatic methods of amide bond
formation (see, e.g. H. D. Jakubke in The Peptides, Analysis,
Synthesis, Biology, Academic Press, New York, 1987, p. 103-165; J.
D. Glass, ibid., pp. 167-184; and European Patent 0324659 A2,
describing enzymatic peptide synthesis methods.) These solution
synthesis methods are well known in the art.
[0043] Commercial peptide synthesizers, such as the Applied
Biosystems Model 430A, are available for the practice of these
methods.
Therapeutic Uses of NRP Analogues
[0044] NRP analogues of this invention can be used to treat
neurological disorders. NPRs have been unexpectedly effective in
treating neural degeneration associated with autoimmune disorders
of the brain, including EAE and multiple sclerosis (MS),
amyotrophic lateral sclerosis (ALS) and toxic injury to neural
cells.
[0045] Disorders and Conditions Treatable with NRP Analogues
[0046] Disorders and conditions in which NRP compounds of this
invention can be of benefit include the following.
[0047] Nervous system conditions treatable with NRP analogues
include infections of the central nervous system including
bacterial, fungal, spirochetal, parasitic and sarcoid including
pyrogenic infections, acute bacterial meningitis or
leptomeningitis.
[0048] Cerebrovascular diseases include stroke, ischemic stroke,
hypoxia/ischemia, atherosclerotic thrombosis, lacunes, embolism,
hypertensive haemorrhage, ruptured aneurysms, vascular
malformations, transient ischemic attacks, intracranial
haemorrhage, spontaneous subarachnoid haemorrhage, hypertensive
encephalopathy, inflammatory diseases of the brain arteries,
decreased perfusion caused by, for example, cardiac insufficiency
(possibly resulting from coronary bypass surgery) and other forms
of cerebrovascular disease.
[0049] Craniocerebral traumas include basal skull fractures and
cranial nerve injuries, carotid-cavernous fistula, pneumocephalus,
aerocele andrhinorrhea, cerebral contusion, traumatic intracerebral
haemorrhage, traumatic brain injury, penetrating traumatic brain
injury and acute brain swelling in children.
[0050] Demyelinating diseases include neuromyelitis optica, acute
disseminated encephalomyelitis, acute and subacute necrotizing
haemorrhagic encephalitis, diffuse cerebral sclerosis of Schilder
and multiple sclerosis in conjunction with peripheral neuropathy.
Degenerative diseases of the nervous system including syndrome of
one or more of progressive dementia, diffuse cerebral atrophy,
diffuse conical atrophy of the non-Alzheimer type, Lewy body
dementia, Pick's disease, fronto-temporal dementia, thalamic
degeneration, non-Huntingtonian types of Chorea and dementia,
cortico-spinal degeneration (Jakob), the
dementia-Parkinson-amyotrophic lateral sclerosis complex (Guamanina
and others) and amyotrophic lateral sclerosis (ALS).
[0051] Peripheral neuropathies are common and disabling conditions
characterised by damage to or loss of peripheral neurons. There are
more than 100 types of peripheral neuropathy, each with its own
characteristic set of symptoms, pattern of development, and
prognosis. Peripheral neuropathy may be either inherited or
acquired. Inherited forms of peripheral neuropathy can be caused by
genetic mutations. Some types of peripheral neuropathy and features
common to them are shown below in Table 1. Table 1 above shows
comparisons between pyridoxine-, streptozotin- and
chemotherapy-induced peripheral neuropathy and diabetic peripheral
neuropathy.
TABLE-US-00008 TABLE 1 Features Common to Peripheral Neuropathies
Chemotherapy- Pyridoxine Streptozotocin- Diabetic Induced (Vitamin
B6) Induced Diabetic Peripheral Peripheral Intoxication Neuropathy
Neuropathy Neuropathy Model/Condition Experimental Experimental
Clinical Condition Experimental Model & Model Model &
Clinical Clinical Condition Condition Principle Pathology
Reversible Peripheral nerve Progressive Peripheral nerve peripheral
nerve axonopathy, peripheral nerve neuronopathy, as axonopathy,
resulting in reduced axonopathy, well as damage to resulting in
autonomic and resulting in dorsal root ganglia, reduced sensory
sensory fiber reduced sensory resulting in reduced fiber conduction
conduction fiber conduction sensory fiber velocity, restricted
velocity, with velocity, with conduction to large diameter
degeneration being degeneration velocity, with and cells, without
fiber length being fiber length without demyelination. dependent.
dependent. demyelination. [1, 2, 3, 4, 5, 6] [11, 12, 13] [19, 20,
21] [22, 23] Proposed Saturation of Selective toxicity Wallerian
axonal (For platinum Mechanism of pyridoxal kinase to islet
.beta.-cells of degeneration compounds) Toxicity in the liver may
the pancreas resulting from Disturbance of inhibit neuronal leading
to a hyperglycemic metabolism and pyridoxal hyperglycemic
neurotoxicity, axonal transport in phosphate, diabetic condition,
Raised glucose peripheral sensory altering neurosnal with resulting
favors metabolism nerves following metabolism neurotoxicity, as in
through the polyol accumulation in the Impaired neuronal clinical
diabetes. pathway that DRG lead to metabolism leads [14, 15, 16]
results in axonopathy. to an production of [22, 24] impoverished
oxidative stress. energy support of [16, 19, 20] the large axons.
[2, 3] Peripheral Nerves Primarily large Primarily Primarily large
Primarily large Affected descending autonomic and descending
descending sensory sensory nerves, sensory nerves, sensory nerves,
nerves, including including peroneal including peroneal including
peroneal peroneal and sural and sural nerves and other nerves and
sural nerves nerves that descend that descend from descending from
that descend from from the sciatic. the sciatic. the sciatic. the
sciatic. [22, 23] [3, 6, 7] [11, 13] [19, 20] Functional Motoric
Predominantly Initially positive Sensory Outcomes impairment of
positive symptoms symptoms of pain neuronopathy, with hind-limbs
due to of hyperalgesia/ or paresthesia, diminished loss of sensory
allodynia. then progressing vibration feedback, [17, 18] to
negative perception, particularly from symptoms with paresthesia,
loss of the hind limbs. loss of sensory tendon reflex, pain [3, 6,
7, 8, 9, 10] feedback, and, later, ataxia particularly from
(motoric the feet, impairment). [19, 20] [22, 23]
[0052] Acquired peripheral neuropathy may result from: physical
injury (trauma) to a nerve, tumors, toxins (including
chemotherapy), autoimmune responses, nutritional deficiencies,
alcoholism, vascular and metabolic disorders (e.g. diabetic
neuropathy). The HIV-associated peripheral neuropathy is a common
side effect of drugs targeting the reverse transcriptase of the HIV
virus. The symptoms of peripheral neuropathy can vary from
temporary numbness, tingling, and pricking sensations, sensitivity
to touch or muscle weakness, to more extreme symptoms such as
burning pain, muscle wasting, paralysis, organ or gland
dysfunction.
[0053] The first report of a human sensory neuropathy induced by a
high dose of pyridoxine (vitamin B6) derives from Schaumberg et
al., New Eng. J. Medicine 309:445-448, 1983. Daily intakes were in
the 2000-6000 mg/day range over periods from 2 to 14 months. All
patients displayed a "stocking-glove" sensory loss with numbness in
hands, feet and an unstable gait.
[0054] Using rat models of peripheral neuropathy enables the
examination of neurological abnormalities induced by pyridoxine.
For instance, 1,200 or 800 mg/kg/day of pyridoxine administered to
rats for 5-10 days results in necrosis of sensory neurons,
especially affecting large diameter neurons in the sciatic nerve
and the dorsal root ganglion (Xu et al., Neurology 39:1077-1083,
1989).
[0055] Pyridoxine-induced peripheral neuropathy in animals is a
recognized system for studying effects of therapeutic agents. In
particular, this system is predictive of effects of such agents on
peripheral neuropathy in human beings.
[0056] Metabolic Disorders
[0057] Acquired metabolic disorders of the nervous system including
metabolic diseases presenting as a syndrome comprising one or more
of confusion, stupor or coma-ischemia-hypoxia, hypoglycaemia,
hyperglycemia, hypercapnia, hepatic failure and Reye syndrome,
metabolic diseases presenting as a progressive extrapyramidal
syndrome, metabolic diseases presenting as cerebellar ataxia,
hyperthermia, celiac-sprue disease, metabolic diseases causing
psychosis or dementia including Cushing disease and steroid
encephalopathy, thyroid psychosis and hypothyroidism and pancreatic
encephalopathy. An example of a metabolic disorder that can result
in neuropathy is pyridoxine excess described more fully below.
[0058] Diseases of the Nervous System Due to Nutritional
Deficiency, Alcohol and Alcoholism
[0059] Disorders of the nervous system due to drugs and other
chemical agents include opiates and synthetic analgesics, sedative
hypnotic drugs, stimulants, psychoactive drugs, bacterial toxins,
plant poisons, venomous bites and stings, heavy metals, industrial
toxins, anti-neoplastic and immunosuppressive agents, thalidomide,
aminoglycoside antibiotics (ototoxicity) and penicillin derivatives
(seizures), cardioprotective agents (beta-blockers, digitalis
derivatives and amiodarone).
[0060] As illustrated by the preceding list, compositions and
methods of the invention can find use in the treatment of human
neural injury and disease. Still more generally, the compositions
and methods of the invention find use in the treatment of human
patients suffering from neural damage as the result of acute brain
injury, including but not limited to diffuse axonal injury,
perinatal hypoxic-ischemic injury, traumatic brain injury, stroke,
ischemic infarction, embolism, and hypertensive haemorrhage;
exposure to CNS toxins, infections of the central nervous system,
such as, bacterial meningitis; metabolic diseases such as those
involving hypoxic-ischemic encephalopathy, peripheral neuropathy,
and glycogen storage diseases; or from chronic neural injury or
neurodegenerative disease, including but not limited to Multiple
Sclerosis, Lewy Body Dementia, Alzheimer's disease, Parkinson's
disease and Huntington's disease. Patient's suffering from such
diseases or injuries may benefit greatly by a treatment protocol
able to initiate neuronal proliferation and migration, as well as
neurite outgrowth.
[0061] Still more generally, the invention has application in the
induction of neuronal and neuroblast migration into areas of damage
following insult in the form of trauma, toxin exposure, asphyxia or
hypoxia-ischemia.
[0062] Administration of NRP Analogues
[0063] NRP analogues can be used via direct administration to the
patient. An NRP analogue can be administered as part of a
medicament or pharmaceutical preparation. This can involve
combining an NRP analogue with any pharmaceutically appropriate
carrier, adjuvant or excipient. Additionally an NRP analogue can be
used with other non-NRP neuroprotective, proliferative, or other
agent. The selection of the carrier, adjuvant or excipient can
depend upon the route of administration to be employed.
[0064] The administration route can vary widely to suit a
particular condition. An NRP analogue may be administered in
different ways: intraperitoneally, intravenously or
intracerebroventricularly. The peripheral application may be a
route of choice because then there is no direct interference with
the central nervous system.
[0065] Any peripheral route of administration known in the art can
be employed. These can include parenteral routes for example
injection into the peripheral circulation, subcutaneous,
intraorbital, ophthalmic, intraspinal, intracisternal, topical,
infusion (using eg. slow release devices or minipumps such as
osmotic pumps or skin patches), implant, aerosol, inhalation,
scarification, intraperitoneal, intracapsular, intramuscular,
intranasal, oral, buccal, pulmonary, rectal or vaginal. The
compositions can be formulated for parenteral administration to
humans or other mammals in therapeutically effective amounts (eg.
amounts which eliminate or reduce the patient's pathological
condition) to provide therapy for the neurological diseases
described above.
[0066] One route of administration includes subcutaneous injection
(e.g., dissolved in 0.9% sodium chloride) and oral administration
(e.g., in a capsule).
[0067] It will also be appreciated that it may on occasion be
desirable to directly administer NRP analogue to the CNS of the
patient by any appropriate route of administration. Examples
include administration by lateral cerebroventricular injection or
through a surgically inserted shunt into the lateral cerebral
ventricle of the brain of the patient, into the cerebrospinal fluid
or directly into an affected portion of a patient's brain.
[0068] Therapeutic Doses of NRP Analogues
[0069] In some embodiments of this invention, methods for treating
brain damage comprise administering one or more NRP analogues in a
dose range of from about 0.01 .mu.g/kg body weight to about 100
.mu.g/kg body weight. In other embodiments, a dose of 1 .mu.g/kg
body weight to about 10 .mu.g/kg body weight can be useful. In
further embodiments, a dose of an NRP can be in the range of about
0.01 .mu.g/kg body weight to about 0.1 mg/kg.
[0070] In other embodiments, the determination of an effective
amount of an NRP analogue to be administered is within the skill of
one of ordinary skill in the art, and will be routine to those
persons skilled in the art. In certain embodiments, the amount of
an NRP analogue to be used can be estimated by in vitro studies
using an assay system as described herein. The final amount of an
NRP analogue to be administered will be dependent upon the route of
administration, upon the NRP analogue used and the nature of the
neurological disorder or condition that is to be treated. A
suitable dose range may for example, be between about 0.01 .mu.g to
about 15 .mu.g per 1 kg of body weight or in other embodiments,
about 20 .mu.g/kg to about 30 .mu.g/kg body weight per day.
[0071] For inclusion in a medicament, NRP analogue can be directly
synthesized by conventional methods such as the stepwise solid
phase synthesis method of Merrifield et al., 1963 (J. Am. Chem.
Soc. 15:2149-2154) or Goodman M. (ed.), "Synthesis of Peptides and
Peptidomimetics" in Methods of organic chemistry (Houben-Weyl)
(Workbench Edition, E22a,b,c,d,e; 2004; Georg Thieme Verlag,
Stuttgart, N.Y.), expressly incorporated herein fully by reference.
Such methods of peptide synthesis are known in the art, and are
described, for example, in Fields and Colowick, 1997, Solid Phase
Peptide Synthesis (Methods in Enzymology, vol. 289), Academic
Press, San Diego, Calif., expressly incorporated herein fully by
reference. Alternatively synthesis can involve the use of
commercially available peptide synthesizers such as the Applied
Biosystems model 430A.
[0072] As a general proposition, the total pharmaceutically
effective amount of an NRP analogue administered parenterally per
dose will be in a range that can be measured by a dose response
curve. For example, an NRP analogue in the blood can be measured in
body fluids of the mammal to be treated to determine dosing.
Alternatively, one can administer increasing amounts of an NRP
compound to the patient and check the serum levels of the patient
for the NRP analogue. The amount of NRP analogue to be employed can
be calculated on a molar basis based on these serum levels of the
NRP analogue.
[0073] One method for determining appropriate dosing of the
compound entails measuring NRP levels in a biological fluid such as
a body or blood fluid. Measuring such levels can be done by any
means, including RIA and ELISA. After measuring NRP analogue
levels, the fluid is contacted with the compound using single or
multiple doses. After this contacting step, the NRP analogue levels
are re-measured in the fluid. If the fluid NRP analogue levels have
fallen by an amount sufficient to produce the desired efficacy for
which the molecule is to be administered, then the dose of the
molecule can be adjusted to produce maximal efficacy. This method
can be carried out in vitro or in vivo. This method can be carried
out in vivo, for example, after the fluid is extracted from a
mammal and the NRP analogue levels measured, the compound herein is
administered to the mammal using single or multiple doses (that is,
the contacting step is achieved by administration to a mammal) and
then the NRP analogue levels are re-measured from fluid extracted
from the mammal.
[0074] NRP analogues are suitably administered by a
sustained-release system. Suitable examples of sustained-release
compositions include semi-permeable polymer matrices in the form of
shaped articles, for example, films, or microcapsules.
Sustained-release matrices include polylactides (U.S. Pat. No.
3,773,919, EP 58,481), poly(2-hydroxyethyl methacrylate) (Langer et
al., 1981), ethylene vinyl acetate (Langer et al., supra), or
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also include a liposomally associated compound.
Liposomes containing the compound are prepared by methods known to
those of skill in the art, as exemplified by DE 3,218,121; Hwang et
al., 1980; EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641;
Japanese Pat. Appln. 83-118008, U.S. Pat. Nos. 4,485,045 and
4,544,545 and EP 102,324. In some embodiments, liposomes are of the
small (from or about 200 to 800 Angstroms) unilamellar type in
which the lipid content is greater than about 30 mol. percent
cholesterol, the selected proportion being adjusted for the most
efficacious therapy. All U.S. parents referred to herein, both
supra and infra, are hereby expressly incorporated by reference in
their entirety.
[0075] PEGylated peptides having a longer life than non-PEGylated
peptides can also be employed, based on, for example, the conjugate
technology described in WO 95/32003 published Nov. 30, 1995.
[0076] In some embodiments, the compound can be formulated
generally by mixing each at a desired degree of purity, in a unit
dosage injectable form (solution, suspension, or emulsion), with a
pharmaceutically, or parenterally, acceptable carrier, i.e., one
that is non-toxic to recipients at the dosages and concentrations
employed and is compatible with other ingredients of the
formulation. For example, the formulation preferably does not
include oxidizing agents and other compounds that are known to be
deleterious to polypeptides. It can be appreciated that the above
doses are not intended to be limiting. Other doses outside the
above ranges can be determined by those with skill in the art.
[0077] In some embodiments, formulations can be prepared by
contacting a compound uniformly and intimately with liquid carriers
or finely divided solid carriers or both. Then, if desired, the
product can be shaped into the desired formulation. In some
embodiments, the carrier is a parenteral carrier, alternatively, a
solution that is isotonic with the blood of the recipient. Examples
of such carrier vehicles include water, saline, Ringer's solution,
a buffered solution, and dextrose solution. Non-aqueous vehicles
such as fixed oils and ethyl oleate are also useful herein.
[0078] The carrier suitably contains minor amounts of additives
such as substances that enhance isotonicity and chemical stability.
Such materials are desirably non-toxic to recipients at the dosages
and concentrations employed, and include, by way of example only,
buffers such as phosphate, citrate, succinate, acetic acid, and
other organic acids or their salts; antioxidants such as ascorbic
acid; low molecular weight (less than about ten residues)
polypeptides, e.g., polyarginine or tripeptides; proteins, such as
serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; glycine; amino acids such as glutamic
acid, aspartic acid, histidine, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, trehalose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as mannitol or sorbitol;
counter-ions such as sodium; non-ionic surfactants such as
polysorbates, poloxamers, or polyethylene glycol (PEG); and/or
neutral salts, e.g., NaCl, KCl, MgCl.sub.2, CaCl.sub.2, and the
like. In certain embodiments, a peptide of this invention can be
stabilized using 0.5 M sucrose or 0.5 M trehalose. Using such
sugars can permit long-term storage of the peptides.
[0079] An NRP compound can be desirably formulated in such vehicles
at a pH of from about 6.5 to about 8. Alternatively, the pH can be
from about 4.5 to about 8. It will be understood that use of
certain of the foregoing excipients, carriers, or stabilizers will
result in the formation of salts of the compound. The final
preparation may be a stable liquid or lyophilized solid.
[0080] In other embodiments, adjuvants can be used. Typical
adjuvants which may be incorporated into tablets, capsules, and the
like are a binder such as acacia, corn starch, or gelatin; an
excipient such as microcrystalline cellulose; a disintegrating
agent like corn starch or alginic acid; a lubricant such as
magnesium stearate; a sweetening agent such as sucrose or lactose;
a flavoring agent such as peppermint, wintergreen, or cherry. When
the dosage form is a capsule, in addition to the above materials,
it may also contain a liquid carrier such as a fatty oil. Other
materials of various types may be used as coatings or as modifiers
of the physical form of the dosage unit. A syrup or elixir may
contain the active compound, a sweetener such as sucrose,
preservatives like propyl paraben, a coloring agent, and a
flavoring agent such as cherry. Sterile compositions for injection
can be formulated according to conventional pharmaceutical
practice. For example, dissolution or suspension of the active
compound in a vehicle such as water or naturally occurring
vegetable oil like sesame, peanut, or cottonseed oil or a synthetic
fatty vehicle like ethyl oleate or the like may be desired.
Buffers, preservatives, antioxidants, and the like can be
incorporated according to accepted pharmaceutical practice.
[0081] Desirably, an NRP analogue to be used for therapeutic
administration may be sterile. Sterility can be readily
accomplished by filtration through sterile filtration membranes
(e.g., membranes having pore size of about 0.2 micron). Therapeutic
compositions generally can be placed into a container having a
sterile access port, for example an intravenous solution bag or
vial having a stopper pierceable by a hypodermic injection
needle.
[0082] In other embodiments, an NRP analogue can be stored in unit
or multi-dose containers, for example, sealed ampules or vials, as
an aqueous solution or as a lyophilized formulation for
reconstitution. As an example of a lyophilized formulation, 10-mL
vials are filled with 5 ml of sterile-filtered 0.01% (w/v) aqueous
solution of compound, and the resulting mixture is lyophilized. The
infusion solution can be prepared by reconstituting lyophilized
compounds using bacteriostatic water or other suitable solvent.
[0083] In still further embodiments, a kit may contain a
predetermined amount of lyophilized NRP compound, a physiologically
compatible solution for preparation of a dosage form, a mixing
vial, a mixing device, and instructions for use. Such kits can be
manufactured and stored according to usual practices in the
industry.
[0084] An NRP compound-containing composition may be administered
by one or more of a variety of routes. By way of example,
intravenous, intraperitoneal, intracerebral, intraventricular,
inhalation, lavage, rectal, vaginal, transdermal, subcutaneous
administration can be used.
EXAMPLES
[0085] The following examples are presented to illustrate specific
embodiments of this invention. Persons of ordinary skill can
utilize the disclosures and teachings herein to produce other
embodiments and variations without undue experimentation. All such
embodiments and variations are considered to be part of this
invention.
Example 1: Effects of NRP Compounds on Survival and Proliferation
of Cerebellar Microexplants
[0086] NRP Compound Preparation
[0087] NRP compounds were provided by Auspep (Australia). The
peptides were synthesized using standard solid-phase synthesis. The
peptides were supplied with an amidated C-terminus, and were more
than 95% pure as analyzed by MALDI-MS spectrum analysis. The
peptides were stored lyophilized at -80.degree. C. under argon in
0.5M sucrose or 0.5M trehalose until usage. They were reconstituted
in PBS, alternatively in 100 .mu.g/ml human transferrin/PBS or in
other embodiments in 100 .mu.g/ml BSA/PBS, in 0.5M sucrose or 0.5M
trehalose.
[0088] Cell Culture Preparation
[0089] Laminated cerebellar cortices of the two hemispheres were
explanted from a P3, P4, P7 or P8 Wistar rat, cut into small pieces
in GBSS with 0.65% D(+)glucose solution, and triturated by a 0.4 mm
gauge needle and subsequently pressed through a 125 .mu.m pore size
sieve. The obtained microexplants were centrifuged (60.times.g) 2
times for a medium exchange into serum-free BSA-supplemented START
V-medium (Biochrom). Finally, the microexplants were reconstituted
in 500 .mu.l STARTV-medium. For culturing, 38 .mu.l of the cell
suspension was incubated for 1 hour on a poly-D-lysine-coated cover
slip in a 35 mm Petri dish under an atmosphere comprising 5%
CO.sub.2 in air and 100% humidity at 34.degree. C. Subsequently,
the injuring toxins (as described below), NRP analogues and 1 ml of
STARTV-medium were added, and the cultures were evaluated after 2-3
days of culture.
[0090] For immunohistochemistry and neuronal migration experiments,
cerebellar microexplants were fixed after 2-3 days in culture after
the following regime: microexplants were fixed by 2-minute, serial
treatment with 0.4%; 1.2%; 3% paraformaldehyde, respectively,
followed by a 5 min incubation in 4% paraformaldehyde/0.25%
glutaraldehyde in 0.1 M sodium phosphate (pH 7.4).
[0091] Effects of NRP Compounds on Toxin-Induced Neural Injury
[0092] Oxidative stress can result in neurodegeneration. This is
one possible mechanism for the symptoms observed in human disorder
with Huntington's disease. The oxidative stress-producing toxin,
3-nitropropionic acid (3-NP) has been previously shown to produce
effects in experimental animals that mimic those effects seen in
human beings with Huntington's disease. Thus, studies of
therapeutic drugs in experimental animals with 3-NP induced
neurotoxicity are predictive of effects of those drugs in treating
human beings with Huntington's disease or other disorders
characterized by oxidative stress.
[0093] General methods for toxicological and drug administration
experiments were designed such that 1/100 parts of toxin and
neuroprotective drug were administered simultaneously to the
freshly prepared cerebellar microexplants. Glutamate was prepared
as a 50 mM stock solution in MilliQ water while 50 mM
3-nitropropionic acid (3-NP) was pH-adjusted (pH 6.8-7.2) in MilliQ
water. The concentrations of the oxidative stress inducing toxin,
3-nitropropionic acid (3-NP), and the excitotoxin, glutamate, in
the assay were at concentrations of 0.5 mM each. Lyophilized NRP
peptides were reconstituted in PBS or 100 .mu.g/ml human
transferrin as a 10 .mu.M stock solution. Subsequently, serial
dilutions were made. Cerebellar microexplants were cultivated for
48-72 hours at 34.degree. C., 5% CO.sub.2 in air and 100% humidity
before they were fixed by increasing amounts of paraformaldehyde
(0.4%, 1.2%, 3% and 4%--each treatment 2-3 min).
[0094] Using the toxins described above, cerebellar explants were
exposed for 24 hours, at the beginning of culturing to dilutions of
NRP (survival assay) or NRP and 0.1 .mu.M BrdU (proliferation
assay). Subsequently, 80% of the medium was changed without
addition of new toxins and NRPs. The cerebellar cultures were fixed
as described above after 3 days in vitro. The detection of the
incorporated BrdU level is performed as described previously.
[0095] Data Reduction and Statistical Analysis
[0096] For statistical analysis of survival, four fields (each
field having an area of 0.65 mm.sup.2) of each fixed cerebellar
culture with the highest cell densities were chosen, and cells
displaying neurite outgrowth were counted (survival assay).
[0097] Results
[0098] Neuroprotection
[0099] NRP analogues promoted increased neuronal survival in
explants treated with 3-NP (see Examples 2, 3 and 4).
Example 2: Promotion of Neural Cell Survival by NRP Compounds
[0100] We studied effects of different concentrations of SEQ ID
NO:1, and SEQ ID NO:5 of this invention on cell cultures prepared
according to Example 1 above. SEQ ID NO:1 and SEQ ID NO:5 have the
same amino acid sequence, with the exception that the Alanine (A)
in position 9 of the amino acid sequence of SEQ ID NO:1 was
replaced by aminoisobutyric acid (Aib), thus producing a compound
having the sequence shown in SEQ ID NO:5. One can appreciate that
in addition to the change in linear structure of the peptide, Aib
replacement of Alanine can stabilize the beta-turn, and thus,
produce a peptide having different 3-dimensional structure and
different mobilities about certain bonds compared to the
un-substituted peptide.
[0101] We exposed cell cultures to vehicle alone (open column) the
neurotoxin 3-NP alone (shaded column) to 3-NP plus different
concentrations of SEQ ID NO:5 (hatched columns) or to 3-NP plus
different concentrations of a non-substituted NRP, SEQ ID NO: 1
(dark shaded column). We then counted the numbers of cells having
neurites as an indicator of neuronal cell survival.
[0102] FIG. 1 depicts a graph of results of these studies. 3-NP
alone produced profound loss of cells displaying neurites compared
to vehicle-treated controls, indicating that the compound is indeed
neurotoxic. The peptide having the sequence SEQ ID NO:1 at
concentrations of 10 fM or 1 pM significantly decreased the
neurotoxic effects of 3-NP (mean.+-.SEM; p<0.001; n=4).
Similarly, SEQ ID NO:5 decreased 3-NP-induced neurotoxicity in a
concentration dependent fashion, with a threshold of below about 1
fM and a maximal effect at a concentration of about 100 fM
(mean.+-.SEM; p<0.001; n=4 each).
[0103] From this study, we conclude that SEQ ID NO:5 is
neuroprotective. This result means that SEQ ID NO:5 can be a
valuable therapeutic NRP compound for treating neural degeneration
in humans suffering from neurological disorders. Further, because
oxidative stress is known to induce neurodegeneration similar to
the neurodegeneration observed in human beings with Huntington's
disease, synthetic NRP compounds of this invention can be used to
treat human beings with neurodegeneration caused by oxidative
stress.
Example 3: Stability and Neuroprotective Effects of Un-Substituted
NRP Compounds
[0104] To determine the stability of NRPs of this invention in
storage, we carried out a series of studies using SEQ ID NO:1. In
this study, we synthesized SEQ ID NO: 1 and then stored the peptide
at temperatures of either -20.degree. C. or -4.degree. C. for nine
(9) weeks. We then tested the NRP for efficacy in protecting
cerebellar neurons against the neurotoxic effect of 3-NP as
described above. Cerebellar explants were treated with vehicle
alone (open column), the neurotoxic agent 3-NP alone (light
stippled column), or 3-NP plus four concentrations of SEQ ID NO:1
that had been stored for 9 weeks at -20.degree. C., or at
-4.degree. C.
[0105] FIG. 2 depicts results of these studies. Cerebellar explants
treated with 3-NP alone (light stippled bar) showed fewer cells
exhibiting neurites compared to vehicle-treated control explants
(open bar). SEQ ID NO:1 that had been stored at -20.degree. C.
exhibited neuroprotective effects at all concentrations tested
(from 10.sup.-13 M; 100 fM to 10.sup.-10 M; 100 pM), with
statistically significant effects observed at concentrations of
10.sup.-11 M and 10.sup.-10 M.
[0106] In contrast, SEQ ID NO:1 stored at a temperature of
-4.degree. C. produced little decrease in neurotoxic effect of
3-NP. We conclude from this study that the unsubstituted NRP loses
activity over the 9-week period with storage at -4.degree. C., and
that storing SEQ ID NO:1 at -20.degree. C. can protect its
potency.
Example 4: Stability and Neuroprotective Effects of Substituted
NRPs
[0107] In this study, we determined the stability of a substituted
NRP, SEQ ID NO:5, in different storage conditions and at different
concentrations as with Example 3 above. We synthesized SEQ ID NO:5
and then stored the peptide for 9 weeks at a temperature of either
-20.degree. C. or -4.degree. C. We then tested the efficacy of SEQ
ID NO:5 in decreasing the neurotoxic effect of 3-NP as described in
Examples 2 and 3 above.
[0108] FIG. 3 depicts a graph of these studies. Cerebellar explants
were treated with vehicle alone (open column), 3-NP alone (light
stippled column) or 3-NP plus concentrations of SEQ ID NO:5 in four
concentrations ranging from 10.sup.-13 M to 10.sup.-10 M) that had
been stored at either -20.degree. C. or at -4.degree. C. When
stored at -20.degree. C., SEQ ID NO:5 produced neuroprotective
effects similar to those found for SEQ ID NO:1 that had been stored
at -20.degree. C., as shown in Example 3 and depicted in FIG.
2.
[0109] We surprisingly found that even when stored at -4.degree.
C., SEQ ID NO:5 retained its neuroprotective effect. In fact, the
degree of neuroprotection provided by SEQ ID NO:5 after storage at
-4.degree. C. was not statistically different from the degree of
neuroprotection provided after storage at -20.degree. C. This
result was completely unexpected based on studies of SEQ ID NO:1
shown above. The increased stability of the NPR having SEQ ID NO:5
means that this compound will be more suitable for storage and
transportation under commonly used conditions.
Example 5: Neuroprotective Effects of Un-Substituted and
Substituted NRPs
[0110] In a larger set of data obtained using the methods of
Example 1, we confirmed the results shown in FIG. 1. FIG. 4 depicts
a graph of results of studies of cerebellar microexplants treated
with the neurotoxin 3-NP, demonstrating neuroprotection by SEQ ID
NO:5 and SEQ ID NO:1 in a study of 6 in each group. We conclude
that SEQ ID NO:1 and SEQ ID NO:5 protect neurons from dying after
exposure to the neurotoxin 3-NP. We also conclude that SEQ ID NO:5
and SEQ ID NO:1 can be useful therapeutic agents in treating human
beings suffering from neurotoxicity.
Example 6: Therapeutic and Prophylactic Effects of NRP Analogues in
a Model of Multiple Sclerosis
[0111] To determine whether NRPs have an impact on chronic
inflammation in the CNS that leads to severe axonal damage and
subsequent lesions (such as in multiple sclerosis; MS), we tested
NRPs in a mouse model of experimental autoimmune encephalitis (EAE)
that mimics the severe progressive state of MS, using myelin
oligodendrocyte glycoprotein (MOG) as the immunogen.
[0112] Methods and Materials
[0113] Animals
[0114] Female mice, 6-8 weeks-old, strain C57Bl/6J weighing an
average of 24 gms each were used.
[0115] NRP Preparation
[0116] The peptide having the sequence:
NH.sub.2--REGRRDAPGRAGG-NH.sub.2 SEQ ID NO: 12 (also known as SEQ
ID NO: 30 disclosed in U.S. patent application Ser. No.
10/976,699), was supplied by Auspep (Australia). The peptide SEQ ID
NO:12 was supplied with an amidated C-terminus, and was more than
95% pure as determined by MALDI-MS spectroscopy.HPLC. The sequence
was confirmed by mass spectroscopy. The peptide was stored
lyophilized at a temperature of -80.degree. C. under argon gas
until use. The peptide was reconstituted in PBS on the day of
use.
[0117] Induction EAE
[0118] A 200 .mu.ul L of an emulsion containing 200 .mu.ug of the
encephalitogenic peptide MOG35-55
TABLE-US-00009 MEVGWYRSPFSRVVHLYRNGK SEQ ID NO: 13
was obtained from C S Bio Co. USA) in complete Freund adjuvant
(Difco, Detroit, USA) containing 800 .mu.ug Mycobacterium
tuberculosis (Difco, Detroit, USA) was injected subcutaneously into
one flank. Mice were immediately injected intraperitoneally with
400 ng pertussis toxin (List Biological Laboratories, USA) and
again 48 hours later.
[0119] Treatment
[0120] Therapeutic
[0121] At the peak of the disease (day 17 after MOG-immunization)
animals were treated with SEQ ID NO: 12 intraperitoneally (i.p.)
for 14 days with a daily dose of 0.1 .mu.g peptide/animal (4.16
.mu.g/kg).
[0122] Assessment of Neurological Impairment
[0123] Mice were monitored daily and neurological impairment was
scored on an arbitrary clinical score as follows: 0, no clinical
sign; 1, flaccid tail; 2, hind limb weakness; 3, hind limb
paralysis; 4, hind limb weakness and fore limb weakness; 5,
paraplegia; 6, death.
[0124] Results
[0125] Therapeutic Effects of NRPs on EAE in Mice
[0126] The outcome 37 days after the first NRP treatment is shown
in FIG. 5. There is a significant therapeutic effect of SEQ ID NO:
12 when peripherally administered. A similar drug effect has been
shown for the neuroregenerative compound EPO in a combination
therapy with methylprednisolone. The disadvantage of EPO is its
large size as it cannot easily be synthesized or administered. We
conclude that NRP has significant long-term potential to decrease
the severity of motoric impairment occurring in the EAE disease
model of MS when administered as therapeutic drug at the peak of
the disease. Score 1 is the lowest score and implies a flaccid tail
only while the higher scores imply weakness (score 2) or complete
paralysis of the hind legs (score 3). **p<0.01 versus treatment
day 1 score.
Example 7: Effects of NRP Analogues in Animals with Peripheral
Neuropathy
[0127] To determine if NRP analogues of this invention can be
useful therapeutic agents for treating peripheral neuropathy, we
carried out a series of studies in rats with peripheral neuropathy
induced by pyridoxine.
[0128] We demonstrated that
NH.sub.2-G.sup.1RRAAPGR-Aib-GG.sup.11-NH.sub.2 (SEQ ID NO:5) at
very low doses applied as a single bolus per day can attenuate
motor deficits in animals treated with toxic doses of
pyridoxine.
[0129] Materials and Methods
[0130] Male Sprague-Dawley rats were used and were weighed 278-349
g at the commencement of intoxication with pyridoxine chloride.
Before intoxication, rats were habituated to walk across a beam at
daily intervals for a week.
[0131] Experiment I:
[0132] Rats were administered 400 mg/kg pyridoxine chloride
dissolved in sterile distilled water adjusted to neutral pH
intraperitoneally (i.p) twice daily for 8 days and concurrently the
peptide having the sequence SEQ ID NO:5 was administered i.p. for
at total of 10 days. The rats were observed for a total of 29
days.
[0133] Experiment II:
[0134] Rats were administered a higher dose of pyridoxine (1200
mg/kg/day) over a shorter period of time. Pyridoxine chloride
dissolved in sterile distilled water adjusted to neutral pH was
administered i.p. to the rats at a dose of 600 mg/kg twice daily
for 4 days. Concurrently the peptide having the sequence SEQ ID
NO:5 was administered for 4 days. On day 5, rats were tested for
motor deficits.
[0135] Motor deficits after pyridoxine intoxication and effects of
SEQ ID NO:5 were analyzed using a precision beam walk. Seven
subsequent foot steps across the 1.5 m long beam were videotaped
and according to the positioning of the foot tarsus, these steps
were scored between 1 to 4 (1--hind leg tarsus above the beam
median; 2--tarsus was touching the upper half of beam medium;
3--tarsus was touching the lower half of beam median and 4--tarsus
below the beam median). The score results of all seven steps were
added together. A score of 30 was given if the animal was only able
to stand on the beam but was unable to walk. In the event of
inability to stand on the beam a score of 32 was awarded.
[0136] Treatment Groups
[0137] Experiment I:
[0138] The two concentrations tested for SEQ ID NO:5 were 40
ng/kg/day and 4 .mu.g/kg/day.
[0139] Experiment II:
[0140] Saline and SEQ ID NO:5 at a dose of 4 .mu.g/kg/day were
tested.
[0141] Statistics Analysis
[0142] Motor deficit data were assessed by two-way analysis of
variance and Bonferroni post-hoc test. Statistical significance was
concluded if p<0.05 between drug treatment cohorts and vehicle
treatment.
[0143] Results
[0144] Experiment I:
[0145] Four days after cessation of pyridoxine-intoxication both
SEQ ID NO:5-treated rat cohorts show attenuated motor deficits (low
dosage: p<0.05; high dosage: p<0.01) compared to the
vehicle-treated group. At day 16 after start of
pyridoxine-intoxication the high dose SEQ ID NO:5 group had still
significantly less motor deficits than the control group
(p<0.05). (FIG. 6).
[0146] Experiment II:
[0147] Four subsequent days of high-dosage pyridoxine-intoxication
lead to substantial motor deficits in the control group of rats. On
day 5 the SEQ ID NO:5-treated rat group (dosage: 4 .mu.g/kg/day)
showed highly significantly attenuation of motor deficits
(p<0.001) (FIG. 7).
[0148] Conclusions
[0149] We conclude that SEQ ID NO:5 showed highly significant and
clinically substantial attenuation of motor deficits
pyridoxine-induced peripheral neuropathy. We also conclude that at
a dose of 4 .mu.g/kg/day, SEQ ID NO:5 was well tolerated. The 4
.mu.g/kg/day concentration worked equally well in the more chronic
model of pyridoxine intoxication (800 mg/kg/day for 8 days) and the
acute pyridoxine intoxication induced by 1200 mg/kg/day
administered for 4 days.
[0150] We further conclude from these studies that SEQ ID NO:5 can
be effective treatment for peripheral neuropathies in human
beings.
Example 8: Effect of SEQ ID NO:5 on Animals with Amyotrophic
Lateral Sclerosis (ALS)
[0151] In this series of studies, we examined the effects of SEQ ID
NO:5 on mice having a genetic defect (SOD-1) that produces a motor
neuron disease similar to that in humans with ALS. Animals with
this disorder show a progressive loss of motor coordination over
time, and ultimately die prematurely of the disease. This animal
system is useful in studying effects of agents potentially useful
in treating ALS in human beings. Thus, results obtained are highly
predictive of results obtained in humans with ALS.
[0152] Methods
[0153] Mice were randomly allocated to receive either vehicle or
SEQ ID NO:5 from the point of disease onset onwards. Disease onset
in each treatment allocation group was not significantly different:
92-93 days.
[0154] SEQ ID NO:5 treatment (40 .mu.g/kg, given 1/day, i.p. (FIG.
8A) or 0.4 .mu.g/kg, given 1/day, i.p. (FIG. 8B)) was started at
the day of onset of the disease.
[0155] Results
[0156] In two studies, SEQ ID NO:5 treatment resulted in a
significant extension of lifetime in the mice suffering from the
ALS-like disorder (FIGS. 8A and 8B). A daily dose of 40 .mu.g/kg
i.p. SEQ ID NO:5 significantly promoted longevity following disease
onset. FIGS. 8A and 8B show Kaplan-Meier survival probability
curves for the SOD-1 mutant (ALS) transgenic mice.
[0157] The vehicle-treated animals began to die at day 120, and
were all dead by day 143 (Study 1; FIG. 8A; solid line) Similarly,
in Study 2 (FIG. 8B; solid line), vehicle-treated animals began to
die at day 120 asn were all dead at day 154 (FIG. 8B; solid
line).
[0158] In contrast, in Study 1 (FIG. 8A; dashed line) SEQ ID
NO:5-treated mice began dying later (at day 131) than
vehicle-treated animals and lived longer (up to 156 days)
Significantly, the two Kaplan-Meier curves did not overlap (FIG.
8A; 40 .mu.g/kg SEQ ID NO:5; p<0.01 compared to survival of
animals treated with vehicle alone). In Study 2 animals treated
with SEQ ID NO:5 (FIG. 8B; dashed line; 0.4 .mu.g/kg) began to die
later (at day 125) than vehicle-treated animals (FIG. 8B; solid
line) and in general, lived longer (up to 189 days: FIG. 8B; dashed
line; SEQ ID NO:5.
Conclusions
[0159] We conclude from these studies that SEQ ID NO:5 is an
effective agent that has unexpectedly improved stability compared
to its un-substituted counterpart. We also conclude that
stabilizing the beta-turn of an NRP can improve therapeutic
efficacy and can improve stability, both of which can improve
therapeutic utility of NRP analogues. The unexpected findings that
substituted NRP analogues have both improved potency and improved
stability indicates that the NRP analogues can be valuable
therapeutic alternatives for treating a variety of conditions
characterized by neurodegeneration.
[0160] Therefore, NRP analogues of this invention can be useful in
treating acute as well as chronic neurodegenerative disorders
including ALS, neurotoxicity, neurodegeneration associated with
oxidative stress, autoimmune disorders, traumatic brain injury and
other neurological diseases and conditions. Further, we conclude
that NRP analogues can have beneficial therapeutic effects in
situations where loss of neurological function is a symptom.
Example 9: NRP Analogue-Mediated Migration in Physiological
(Injury-Free) Conditions
[0161] An NRP analogue is tested for
migration-inducing/chemoattractive activity on mouse neural stem
cells in a haptotactic migration assay as described below.
[0162] Methods
[0163] Initial NRP Coating
[0164] Control wells of Transwell plates (Corning) with 12 .mu.m
pore size are coated in 1.5 ml of the BSA/PBS vehicle. Remaining
plates are coated using 0.1 ng/ml of NRP analogue (prepared in PBS
containing 10 ug/ml BSA).
[0165] Extracellular Matrix Coating
[0166] Laminin (7 .mu.g/ml) is used as extracellular matrix (ECM)
coating for mouse primary stem cells. The matrix is incubated at
37.degree. C.; 5% CO.sub.2 for 2 hrs at room temperature. The cells
are seeded onto the inserts (30,000 cells per well). Plates are
fixed at 1-2 days in vitro (DIV).
[0167] Coating of Inserts
[0168] A 5 ug/mL PDL/PLL mixture (in PBS) is used to coat inserts.
Subsequently the inserts are rinsed with MilliQ water.
[0169] Cell Fixation
[0170] Inserts are discarded and wells fixed in successive
dilutions of PFA (0.4, 1.2, 3 and 4%) for 3-5 min in each dilution.
The wells are rinsed and stored in successive dilutions of PFA
(0.4, 1.2, 3 and 4%) 3-5 min in each dilution. The wells are rinsed
and stored in PBS until counting. All cells that display neurite
outgrowth and traveled to the bottom chamber are counted as
migrating cells.
[0171] Results
[0172] More cells migrate in plates treated with NRP analogue than
migrated in plates without NRP analogue. NRP analogues can induce
neuronal cell migration, and that they each can be used to treat
neurodegeneration associated with neural injury or disease.
Example 10: NRP Analogue-Mediated Migration in Injury
Conditions
[0173] An NRP analogue is tested for
migration-inducing/chemoattractive activity on mouse neural stem
cells in a haptotactic migration assay in injury conditions, as
described below.
[0174] Methods
[0175] Production of a Monolayer of Astrocytes
[0176] P1 (postnatal day 1) Wistar or Sprague Dawley rats are
sacrificed by decapitation. Cortical heminspheres are removed and
collected into separate tubes containing 4 ml DMEM--1 cortex per
tube. The tissue is mechanically triturated. Cells are transferred
into medium using a sterile pipette and filtered through a 100 um
cell strainer into a 50 ml centrifuge tube. Each tube is stocked up
to 50 ml with DMEM. The tubes are centrifuged for 5 mins at
350.times.g at 22.degree. C. The cells are resuspended in 40 ml of
DMEM+10% FBS. The cells are then seeded into a 12-well plate+5 nM
ocadaic acid (to remove neurons by inducing apoptotic cell death)
and incubated at 37.degree. C./10% CO.sub.2 for 24 hrs in a Boyden
Chamber. The medium+FBS is replaced after 1 day with fresh DMEM+10%
FBS. The cell growth is monitored until confluency (14-18
days).
[0177] Pharmacological and Mechanical Injury
[0178] Induction of injuries to the astrocytic monolayer is
accomplished using the pharmacological agent transforming growth
factor .beta.1 (TGF.beta.1) and simultaneous mechanical scratching
of the monolayer in order to activate astrocytics. 10 ng/ml
TGF.beta.1 is administered to the astrocytic monolayer for 24 hrs.
Additionally, astrocytic cultures re mechanically injured by a
scalpel (one scratch throughout the bottom of the well).
[0179] Seeding of Pre-Labelled Stem Cells
[0180] Undifferentiated fluorescein diacetate-labelled embryonic
mouse neural stem cells (NSCs) are seeded into Poly-D-Lysine
(PDL--5 .mu.g/ml) coated inserts. The lower compartment of the
Boyden chamber receives 100 fM of an NRP analogue.
[0181] Cell Fixation
[0182] Inserts are discarded and wells fixed in successive
dilutions of PFA (0.4, 1.2, 3 and 4%) for 3-5 min in each dilution.
The wells are rinsed and stored in successive dilutions of PFA
(0.4, 1.2, 3 and 4%) 3-5 min in each dilution. The wells are rinsed
and stored in PBS until counting. All cells that display neurite
outgrowth and traveled to the bottom chamber are counted as
migrating cells.
[0183] Analysis
[0184] Migrated stem cell number of labelled cells re analysed
after 24 hrs by a fluorescence-based computerized imaging system
(Discovery-1).
[0185] Results
[0186] NRP analogues stimulate more stem cells to migrate than
vehicle-treated controls. We conclude that NRP analogue induces
neuronal stem cell migration, and that NRP analogues can be useful
to treat neurodegeneration associated with neural injury or
disease.
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[0211] All patents, patent applications and other publications are
incorporated herein fully by reference as if separately so
incorporated. The Sequence Listing appended to this application is
also incorporated herein fully by reference.
[0212] A person of ordinary skill in the art will not have to
undertake undue experimentation, taking account of that skill and
the knowledge available, and of this disclosure, in developing one
or more suitable synthetic compounds. All such compounds and
methods for their manufacture and use are considered to be part of
this invention.
[0213] Compounds and compositions of this invention find industrial
use in many aspects of commerce, including pharmaceutical
manufacturing, formulation, and sale. Methods of using compounds
and compositions of this invention find industrial use in medical
fields of neurology, and in particular, for treatment of
neurological disorders in animals and human beings.
Sequence CWU 1
1
13111PRTHomo sapiensMOD_RES(11)..(11)AMIDATION 1Gly Arg Arg Ala Ala
Pro Gly Arg Ala Gly Gly 1 5 10 24PRTHomo sapiens 2Ala Pro Gly Arg 1
34PRTHomo sapiens 3Arg Ala Gly Gly 1 411PRTArtificial
sequenceSynthetic peptide 4Gly Arg Arg Ala Xaa Pro Gly Arg Ala Gly
Gly 1 5 10 511PRTArtificial sequenceSynthetic peptide 5Gly Arg Arg
Ala Ala Pro Gly Arg Xaa Gly Gly 1 5 10 611PRTArtificial
sequenceSynthetic peptide 6Gly Arg Arg Ala Ala Pro Gly Arg Ala Asn
Gly 1 5 10 710PRTArtificial sequenceSynthetic peptide 7Arg Arg Ala
Ala Pro Gly Arg Ala Gly Gly 1 5 10 812PRTArtificial
sequenceSynthetic peptide 8Gly Arg Asp Arg Ala Ala Pro Gly Arg Ala
Gly Gly 1 5 10 913PRTArtificial sequenceSynthetic peptide 9Cys Gly
Arg Arg Ala Ala Pro Gly Arg Ala Gly Gly Cys 1 5 10
1011PRTArtificial sequenceSynthetic peptide 10Cys Arg Arg Ala Ala
Pro Gly Arg Ala Gly Cys 1 5 10 1111PRTArtificial sequenceSynthetic
peptide 11Gly Arg Arg Ala Ala Pro Gly Arg Ala Gly Gly 1 5 10
1213PRTArtificial sequenceSynthetic peptide 12Arg Glu Gly Arg Arg
Asp Ala Pro Gly Arg Ala Gly Gly 1 5 10 1321PRTMycobacterium
tuberculosis 13Met Glu Val Gly Trp Tyr Arg Ser Pro Phe Ser Arg Val
Val His Leu 1 5 10 15 Tyr Arg Gln Gly Lys 20
* * * * *