U.S. patent application number 17/496986 was filed with the patent office on 2022-01-20 for methods and compositions for treating aging-associated conditions.
The applicant listed for this patent is THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, The United States of America as represented by the Department of Veterans Affairs, The United States of America as represented by the Department of Veterans Affairs. Invention is credited to Joseph M. Castellano, Anton Wyss-Coray.
Application Number | 20220016224 17/496986 |
Document ID | / |
Family ID | |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220016224 |
Kind Code |
A1 |
Wyss-Coray; Anton ; et
al. |
January 20, 2022 |
METHODS AND COMPOSITIONS FOR TREATING AGING-ASSOCIATED
CONDITIONS
Abstract
Methods of treating an adult mammal for an aging-associated
condition are provided. Aspects of the methods include enhancing a
TIMP activity, e.g., a TIMP2 activity, in the mammal in a manner
sufficient to treat the adult mammal for the aging-associated
condition. Also provided are compositions for use in practicing
methods of the invention. A variety of aging-associated conditions
may be treated by practice of the methods, which conditions include
cognitive impairments.
Inventors: |
Wyss-Coray; Anton; (Palo
Alto, CA) ; Castellano; Joseph M.; (Tuckahoe,
NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY
The United States of America as represented by the Department of
Veterans Affairs |
Stanford
Washington |
CA
DC |
US
US |
|
|
Appl. No.: |
17/496986 |
Filed: |
October 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16842073 |
Apr 7, 2020 |
11141469 |
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17496986 |
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15736583 |
Dec 14, 2017 |
10617744 |
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PCT/US2016/036032 |
Jun 16, 2016 |
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16842073 |
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62175981 |
Jun 15, 2015 |
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International
Class: |
A61K 38/55 20060101
A61K038/55; A61P 25/28 20060101 A61P025/28 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with Government support under
contract AG045034 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method of treating an adult mammal for an aging-associated
condition, the method comprising: enhancing a TIMP activity in the
mammal in a manner sufficient to treat the adult mammal for the
aging-associated condition.
2. The method according to claim 1, wherein the TIMP activity is a
TIMP1, TIMP2, TIMP3 or TIMP4 activity.
3. The method according to claim 2, wherein the TIMP activity is a
TIMP2 activity.
4. The method according to claim 1, where the method comprises
enhancing a systemic TIMP activity.
5. The method according to claim 1, wherein the method comprises
increasing a systemic level of a TIMP active agent in the
mammal.
6. The method according to claim 5, wherein the systemic level of a
TIMP active agent is increased by administering a TIMP active agent
to the mammal.
7. The method according to claim 6, wherein the TIMP active agent
is a TIMP polypeptide or mimetic thereof.
8. The method according to claim 7, wherein the TIMP active agent
is a TIMP polypeptide.
9. The method according to claim 8, wherein the TIMP polypeptide
has a sequence that is at least 60% identical to any of SEQ ID NOS:
1 to 4.
10. The method according to claim 1, wherein the method comprises
enhancing expression of an endogenous TIMP coding sequence.
11. The method according to claim 1, wherein the mammal is a
primate.
12. The method according to claim 1, wherein the adult mammal is an
elderly mammal.
13. The method according to claim 11, wherein the primate is a
human.
14. The method according to claim 1, wherein the aging-associated
condition comprises a cognitive impairment or a cognitive decline
disease condition
15. The method according to claim 1, wherein the adult mammal
suffers from an aging associated disease condition.
16. The method according to claim 6, wherein the TIMP active agent
comprises a nucleic acid.
17. The method according to claim 6, wherein the TIMP active agent
comprises a modified TIMP polypeptide.
18. The method according to claim 6, wherein the TIMP active agent
comprises a pegylated TIMP polypeptide.
19. The method according to claim 6, wherein the TIMP active agent
comprises an TIMP polypeptide fused to an antibody or portion
thereof.
20. The method according to claim 19, wherein the antibody is an
IgG antibody.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 15/736,583, filed Dec. 14, 2017, now U.S. Pat. No.
10,617,744, issued Apr. 14, 2020, which is a U.S. 371 national
phase entry of International Patent Application No.
PCT/US2016/036032, filed Jun. 6, 2016, which claims priority to the
filing date of the U.S. Provisional Patent Application Ser. No.
62/175,981 filed Jun. 15, 2015, the disclosure of which
applications are incorporated herein by reference in their
entireties.
INTRODUCTION
[0003] Aging in an organism is accompanied by an accumulation of
changes over time. In the nervous system, aging is accompanied by
structural and neurophysiological changes that drive cognitive
decline and susceptibility to degenerative disorders in healthy
individuals. (Heeden & Gabrieli, "Insights into the ageing
mind: a view from cognitive neuroscience," Nat. Rev. Neurosci.
(2004) 5: 87-96; Raz et al., "Neuroanatomical correlates of
cognitive aging: evidence from structural magnetic resonance
imaging," Neuropsychology (1998) 12:95-114; Mattson & Magnus,
"Ageing and neuronal vulnerability," Nat. Rev. Neurosci. (2006) 7:
278-294; and Rapp & Heindel, "Memory systems in normal and
pathological aging," Curr. Opin. Neurol. (1994) 7:294-298).
Included in these changes are synapse loss and the loss of neuronal
function that results. Thus, although significant neuronal death is
typically not observed during the natural aging process, neurons in
the aging brain are vulnerable to sub-lethal age-related
alterations in structure, synaptic integrity, and molecular
processing at the synapse, all of which impair cognitive
function.
[0004] In addition to the normal synapse loss during natural aging,
synapse loss is an early pathological event common to many
neurodegenerative conditions, and is the best correlate to the
neuronal and cognitive impairment associated with these conditions.
Indeed, aging remains the single most dominant risk factor for
dementia-related neurodegenerative diseases such as Alzheimer's
disease (AD) (Bishop et al., "Neural mechanisms of ageing and
cognitive decline," Nature (2010) 464: 529-535 (2010); Heeden &
Gabrieli, "Insights into the ageing mind: a view from cognitive
neuroscience," Nat. Rev. Neurosci. (2004) 5:87-96; Mattson &
Magnus, "Ageing and neuronal vulnerability," Nat. Rev. Neurosci.
(2006) 7:278-294).
[0005] As the human lifespan increases, a greater fraction of the
population suffers from aging-associated cognitive impairments,
making it crucial to elucidate means by which to maintain cognitive
integrity by protecting against, or even counteracting, the effects
of aging (Hebert et al., "Alzheimer disease in the US population:
prevalence estimates using the 2000 census," Arch. Neurol. (2003)
60:1119-1122; Bishop et al., "Neural mechanisms of ageing and
cognitive decline," Nature (2010) 464:529-535).
[0006] Tissue inhibitor of metalloproteinase 2 (TIMP-2) is a member
of a group of specific inhibitors of matrix metalloproteinases. The
proteins these inhibitors regulate, matrix metalloproteinases
(MMPs), play a role in several physiological processes including
growth, wound healing, tissue repair, and cellular development and
homeostasis. Broad categories of MMPs consist of collagenases,
gelatinases, stromelysins, matrilysins, membrane-type MMPs
(MT-MMPs), and others. These enzymes must be precisely regulated as
the loss of control over MMP activity may result in arthritis,
cancer, atherosclerosis, aneurysms, nephritis, tissue ulcers,
fibrosis, and other tissue damage (Visse and Nagase, "Matrix
Metalloproteinases and Tissue Inhibitors of Metalloproteinases,"
Circulation Research (2003) 92: 827-39). Four TIMPs (1-4) have been
identified in vertebrates.
SUMMARY
[0007] Methods of treating an adult mammal for an aging-associated
condition are provided. Aspects of the methods include enhancing a
TIMP activity, e.g., a TIMP2 activity, in the mammal in a manner
sufficient to treat the adult mammal for the aging-associated
condition. Also provided are compositions for use in practicing
methods of the invention. A variety of aging-associated conditions
may be treated by practice of the methods, which conditions include
cognitive impairments.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIG. 1A shows the results on brain slices isolated from aged
wildtype mice that were treated with recombinant TIMP2 (i.p., 50
.mu.g/kg)(bottom) or a control (top). FIG. 1B provides
quantification of the maintenance phase of the PSA shown in FIG.
1A.
[0009] FIG. 2 shows the results of anti-TIMP2 antibody treatment on
object discrimination.
DETAILED DESCRIPTION
[0010] Methods of treating an adult mammal for an aging-associated
condition are provided. Aspects of the methods include enhancing
TIMP activity, e.g., a TIMP2 activity, in the mammal in a manner
sufficient to treat the adult mammal for the aging-associated
condition. Also provided are compositions for use in practicing
methods of the invention. A variety of aging-associated conditions
may be treated by practice of the methods, which conditions include
cognitive impairments.
[0011] Before the present methods and compositions are described,
it is to be understood that this invention is not limited to a
particular method or composition described, as such may, of course,
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting, since the scope of the present
invention will be limited only by the appended claims.
[0012] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limits of that range is also specifically disclosed. Each
smaller range between any stated value or intervening value in a
stated range and any other stated or intervening value in that
stated range is encompassed within the invention. The upper and
lower limits of these smaller ranges may independently be included
or excluded in the range, and each range where either, neither or
both limits are included in the smaller ranges is also encompassed
within the invention, subject to any specifically excluded limit in
the stated range. Where the stated range includes one or both of
the limits, ranges excluding either or both of those included
limits are also included in the invention.
[0013] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, some potential and preferred methods and materials are
now described. All publications mentioned herein are incorporated
herein by reference to disclose and describe the methods and/or
materials in connection with which the publications are cited. It
is understood that the present disclosure supersedes any disclosure
of an incorporated publication to the extent there is a
contradiction.
[0014] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
[0015] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and reference to "the peptide" includes reference to one or more
peptides and equivalents thereof, e.g., polypeptides, known to
those skilled in the art, and so forth.
[0016] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates which
may need to be independently confirmed.
Methods
[0017] As summarized above, aspects of the invention include
methods of treating an aging-associated condition in an adult
mammal. The aging-associated condition may manifest in a number of
different ways, e.g., as aging-associated cognitive impairment
and/or physiological impairment, e.g., in the form of damage to
central or peripheral organs of the body, such as but not limited
to: cell injury, tissue damage, organ dysfunction, aging-associated
lifespan shortening and carcinogenesis, where specific organs and
tissues of interest include, but are not limited to skin, neuron,
muscle, pancreas, brain, kidney, lung, stomach, intestine, spleen,
heart, adipose tissue, testes, ovary, uterus, liver and bone; in
the form of decreased neural plasticity, etc.
[0018] In some embodiments, the aging-associated condition is an
aging-associated impairment in cognitive ability in an individual,
i.e., an aging-associated cognitive impairment. By cognitive
ability, or "cognition", it is meant the mental processes that
include attention and concentration, learning complex tasks and
concepts, memory (acquiring, retaining, and retrieving new
information in the short and/or long term), information processing
(dealing with information gathered by the five senses),
visuospatial function (visual perception, depth perception, using
mental imagery, copying drawings, constructing objects or shapes),
producing and understanding language, verbal fluency
(word-finding), solving problems, making decisions, and executive
functions (planning and prioritizing). By "cognitive decline", it
is meant a progressive decrease in one or more of these abilities,
e.g., a decline in memory, language, thinking, judgment, etc. By
"an impairment in cognitive ability" and "cognitive impairment", it
is meant a reduction in cognitive ability relative to a healthy
individual, e.g., an age-matched healthy individual, or relative to
the ability of the individual at an earlier point in time, e.g., 2
weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5
years, or 10 years or more previously. Aging-associated cognitive
impairments include impairments in cognitive ability that are
typically associated with aging, including, for example, cognitive
impairment associated with the natural aging process, e.g., mild
cognitive impairment (M.C.I.); and cognitive impairment associated
with an aging-associated disorder, that is, a disorder that is seen
with increasing frequency with increasing senescence, e.g., a
neurodegenerative condition such as Alzheimer's disease,
Parkinson's disease, frontotemporal dementia, Huntington's disease,
amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,
muscular dystrophy, vascular dementia, and the like.
[0019] By "treatment" it is meant that at least an amelioration of
one or more symptoms associated with an aging-associated condition
afflicting the adult mammal is achieved, where amelioration is used
in a broad sense to refer to at least a reduction in the magnitude
of a parameter, e.g., a symptom associated with the impairment
being treated. As such, treatment also includes situations where a
pathological condition, or at least symptoms associated therewith,
are completely inhibited, e.g., prevented from happening, or
stopped, e.g., terminated, such that the adult mammal no longer
suffers from the impairment, or at least the symptoms that
characterize the impairment. In some instances, "treatment",
"treating" and the like refer to obtaining a desired pharmacologic
and/or physiologic effect. The effect may be prophylactic in terms
of completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of a partial or complete cure
for a disease and/or adverse effect attributable to the disease.
"Treatment" may be any treatment of a disease in a mammal, and
includes: (a) preventing the disease from occurring in a subject
which may be predisposed to the disease but has not yet been
diagnosed as having it; (b) inhibiting the disease, i.e., arresting
its development; or (c) relieving the disease, i.e., causing
regression of the disease. Treatment may result in a variety of
different physical manifestations, e.g., modulation in gene
expression, increased synaptic efficacy, increased neurogenesis,
rejuvenation of tissue or organs, etc. Treatment of ongoing
disease, where the treatment stabilizes or reduces the undesirable
clinical symptoms of the patient, occurs in some embodiments. Such
treatment may be performed prior to complete loss of function in
the affected tissues. The subject therapy may be administered prior
to the symptomatic state of the disease, during the symptomatic
stage of the disease, and in some cases after the symptomatic stage
of the disease.
[0020] In some instances where the aging-associated condition is
aging-associated cognitive decline, treatment by methods of the
present disclosure slows, or reduces, the progression of
aging-associated cognitive decline. In other words, cognitive
abilities in the individual decline more slowly, if at all,
following treatment by the disclosed methods than prior to or in
the absence of treatment by the disclosed methods. In some
instances, treatment by methods of the present disclosure
stabilizes the cognitive abilities of an individual. For example,
the progression of cognitive decline in an individual suffering
from aging-associated cognitive decline is halted following
treatment by the disclosed methods. As another example, cognitive
decline in an individual, e.g., an individual 40 years old or
older, that is projected to suffer from aging-associated cognitive
decline, is prevented following treatment by the disclosed methods.
In other words, no (further) cognitive impairment is observed. In
some instances, treatment by methods of the present disclosure
reduces, or reverses, cognitive impairment, e.g., as observed by
improving cognitive abilities in an individual suffering from
aging-associated cognitive decline. In other words, the cognitive
abilities of the individual suffering from aging-associated
cognitive decline following treatment by the disclosed methods are
better than they were prior to treatment by the disclosed methods,
i.e., they improve upon treatment. In some instances, treatment by
methods of the present disclosure abrogates cognitive impairment.
In other words, the cognitive abilities of the individual suffering
from aging-associated cognitive decline are restored, e.g., to
their level when the individual was about 40 years old or less,
following treatment by the disclosed methods, e.g., as evidenced by
improved cognitive abilities in an individual suffering from
aging-associated cognitive decline.
[0021] In some instances, treatment of an adult mammal in
accordance with the methods results in a change in a central organ,
e.g., a central nervous system organ, such as the brain, spinal
cord, etc., where the change may manifest in a number of different
ways, e.g., as described in greater detail below, including but not
limited to molecular, structural and/or functional, e.g., in the
form of enhanced synaptic plasticity. In some instances, treatment
of a subject in accordance with the methods results in a change in
a peripheral organ, such as liver, muscle, heart, blood, etc.,
where the change may manifest in a number of different ways, e.g.,
as described in greater detail below.
[0022] As summarized above, methods described herein are methods of
treating an aging-associated condition, e.g., as described above,
in an adult mammal. By adult mammal is meant a mammal that has
reached maturity, i.e., that is fully developed. As such, adult
mammals are not juvenile. Mammalian species that may be treated
with the present methods include canines and felines; equines;
bovines; ovines; etc., and primates, including humans. The subject
methods, compositions, and reagents may also be applied to animal
models, including small mammals, e.g., murine, lagomorpha, etc.,
for example, in experimental investigations. The discussion below
will focus on the application of the subject methods, compositions,
reagents, devices and kits to humans, but it will be understood by
the ordinarily skilled artisan that such descriptions can be
readily modified to other mammals of interest based on the
knowledge in the art.
[0023] The age of the adult mammal may vary, depending on the type
of mammal that is being treated. Where the adult mammal is a human,
the age of the human is generally 18 years or older. In some
instances, the adult mammal is an individual suffering from or at
risk of suffering from an aging-associated impairment, such as an
aging-associated cognitive impairment, where the adult mammal may
be one that has been determined, e.g., in the form of receiving a
diagnosis, to be suffering from or at risk of suffering from an
aging-associated impairment, such as an aging-associated cognitive
impairment. The phrase "an individual suffering from or at risk of
suffering from an aging-associated cognitive impairment" refers to
an individual that is about 50 years old or older, e.g., 60 years
old or older, 70 years old or older, 80 years old or older, and
sometimes no older than 100 years old, such as 90 years old, i.e.,
between the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70, 75,
80, 85 or about 90 years old. The individual may suffer from an
aging associated condition, e.g., cognitive impairment, associated
with the natural aging process, e.g., M.C.I. Alternatively, the
individual may be 50 years old or older, e.g., 60 years old or
older, 70 years old or older, 80 years old or older, 90 years old
or older, and sometimes no older than 100 years old, i.e., between
the ages of about 50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85,
90, 95 or about 100 years old, and has not yet begun to show
symptoms of an aging associated condition, e.g., cognitive
impairment. In yet other embodiments, the individual may be of any
age where the individual is suffering from a cognitive impairment
due to an aging-associated disease, e.g., Alzheimer's disease,
Parkinson's disease, frontotemporal dementia, Huntington's disease,
amyotrophic lateral sclerosis, multiple sclerosis, glaucoma,
muscular dystrophy, dementia, and the like. In some instances, the
individual is an individual of any age that has been diagnosed with
an aging-associated disease that is typically accompanied by
cognitive impairment, e.g., Alzheimer's disease, Parkinson's
disease, frontotemporal dementia, progressive supranuclear palsy,
Huntington's disease, amyotrophic lateral sclerosis, spinal
muscular atrophy, multiple sclerosis, multi-system atrophy,
glaucoma, ataxias, muscular dystrophy, dementia, and the like,
where the individual has not yet begun to show symptoms of
cognitive impairment.
[0024] As summarized above, aspects of the methods include
enhancing a TIMP activity, e.g., a systemic TIMP activity, in the
mammal in a manner sufficient to treat the adult mammal for the
aging-associated condition. By enhancing a TIMP activity is meant
increasing one or more target TIMP activities in the subject. In
some instances, the TIMP activity that is enhanced is a systemic
TIMP activity, by which is meant a TIMP activity in the circulatory
system of the mammal. The magnitude of the increase may vary, where
in some instances the magnitude of the increase is 2-fold or
greater, such as 5-fold or greater, including 10-fold or greater,
e.g., 15-fold or greater, 20-fold or greater, 25-fold or greater
(as compared to a suitable control). The TIMP activity that is
increased by practice of the methods is a TIMP mediated process
that is beneficial in treating an aging associated condition. In
other words, the TIMP activity that is enhanced is one that results
in treatment, e.g., as described above, of the subject for the
aging associated condition.
[0025] The target TIMP activity that is enhanced may vary. In some
instances, the target TIMP activity is a TIMP2 activity, i.e., an
activity exhibited by a TIMP2 protein. As such, by TIMP2 activity
is meant an activity of interest of a TIMP2 protein, i.e., an
activity that results in treatment of an aging-associated
condition, e.g., as described above. Of interest are mammalian
TIMP2 proteins, such as but not limited to: primate, e.g., human,
canine, feline, equine, bovine, ovine, murine, lagomorpha, etc. The
sequence of human TIMP2 is:
TABLE-US-00001 (SEQ. ID NO: 01) 10 20 30 40 MGAAARTLPL ALGLLLLATL
LRPADACSCS PVHPQQAFCN 50 60 70 80 ADVVIRAKAV SEKEVDSGND IYGNPIKRIQ
YEIKQIKMFK 90 100 110 120 GPEKDIEFIY TAPSSAVCGV SLDVGGKKEY
LIAGKAEGDG 130 140 150 160 KMHITLCDFI VPWDTLSTTQ KKSLNHRYQM
GCECKITRCP 170 180 190 200 MIPCYISSPD ECLWMDWVTE KNINGHQAKF
FACIKRSDGS 210 220 CAWYRGAAPP KQEFLDIEDP
[0026] In some instances, the target TIMP activity is a TIMP1
activity, i.e., an activity exhibited by a TIMP1 protein. As such,
by TIMP1 activity is meant an activity of interest of a TIMP1
protein, i.e., an activity that results in treatment of an
aging-associated condition, e.g., as described above. Of interest
are mammalian TIMP1 proteins, such as but not limited to: primate,
e.g., human, canine, feline, equine, bovine, ovine, murine,
lagomorpha, etc. The sequence of human TIMP1 is:
TABLE-US-00002 (SEQ ID NO: 02) 10 20 30 40 MAPFEPLASG ILLLLWLIAP
SRACTCVPPH PQTAFCNSDL 50 60 70 80 VIRAKFVGTP EVNQTTLYQR YEIKMTKNYK
GFQALGDAAD 90 100 110 120 IRFVYTPAME SVCGYFHRSH NRSEEFLIAG
KLQDGLLHIT 130 140 150 160 TCSFVAPWNS LSLAQRRGFT KTYTVGCEEC
TVFPCLSIPC KLQSGTHCLW TDQLLQGSEK GFQSRHLACL PREPGLCTWQ SLRSQIA
[0027] In some instances, the target TIMP activity is a TIMP3
activity, i.e., an activity exhibited by a TIMP3 protein. As such,
by TIMP3 activity is meant an activity of interest of a TIMP3
protein, i.e., an activity that results in treatment of an
aging-associated condition, e.g., as described above. Of interest
are mammalian TIMP3 proteins, such as but not limited to: primate,
e.g., human, canine, feline, equine, bovine, ovine, murine,
lagomorpha, etc. The sequence of human TIMP3 is:
TABLE-US-00003 (SEQ ID NO: 03) 10 20 30 40 MTPWLGLIVL LGSWSLGDWG
AEACTCSPSH PQDAFCNSDI 50 60 70 80 VIRAKVVGKK LVKEGPFGTL VYTIKQMKMY
RGFTKMPHVQ 90 100 110 120 YIHTEASESL CGLKLEVNKY QYLLTGRVYD
GKKYTGLCNF 130 140 150 160 VERWDQLTLS QRKGLNYRYH LGCNCKIKSC
YYLPCFVTSK 170 180 190 200 NECLWTDMLS NFGYPGYQSK HYACIRQKGG
YCSWYRGWAP 210 PDKSIINATD P
[0028] In some instances, the target TIMP activity is a TIMP4
activity, i.e., an activity exhibited by a TIMP4 protein. As such,
by TIMP2 activity is meant an activity of interest of a TIMP4
protein, i.e., an activity that results in treatment of an
aging-associated condition, e.g., as described above. Of interest
are mammalian TIMP4 proteins, such as but not limited to: primate,
e.g., human, canine, feline, equine, bovine, ovine, murine,
lagomorpha, etc. The sequence of human TIMP4 is:
TABLE-US-00004 (SEQ ID NO: 04) 10 20 30 40 MPGSPRPAPS WVLLLRLLAL
LPPPGLGEAC SCAPAHPQQH 50 60 70 80 ICHSALNIRA KISSEKVVPA SADPADTEKM
LRYEIKQIKM 90 100 110 120 FKGFEKVKDV QYIYTPFDSS LCGVKLEANS
QKQYLLTGQV 130 140 150 160 LSDGKVFIHL CNYIEPWEDL SLVQRESLNH
HYHLNCGCQI 170 180 190 200 TTCYTVPCTI SAPNECLWTD WLLERKTYGY
QAQHYVCMKH 210 220 VDGTCSWYPG HLPMRKFFVD IVQP
[0029] The target TIMP activity or activities of interest may be
enhanced using any convenient protocol. In some instances, the
target TIMP activity is enhanced by increasing a systemic level of
a TIMP active agent in the mammal. By systemic level is meant the
level (e.g., concentration or amount) of the TIMP active agent in
the circulatory system of the mammal. The magnitude of the increase
may vary, where in some instances the magnitude of the increase is
2-fold or greater, such as 5-fold or greater, including 10-fold or
greater, e.g., 15-fold or greater, 20-fold or greater, 25-fold or
greater (as compared to a suitable control).
[0030] In these embodiments, the systemic level of the TIMP active
agent of interest may be increased using any convenient protocol.
In some instances, the systemic level is increased by administering
a TIMP active agent to the subject. In such instances, the TIMP
active agent may vary. TIMP active agents that may be employed in
these embodiments of the invention include TIMP polypeptides and
nucleic acids encoding the same.
[0031] TIMP polypeptides are polypeptides that, upon administration
to a subject, exhibit the desired TIMP aging associated condition
treatment activity, e.g., as described above. The term
"polypeptide" as used herein refers to full-length proteins as well
as portions or fragments thereof which exhibit the desired TIMP
activity. Also included in this term are variations of the
naturally occurring proteins, where such variations are homologous
or substantially similar to the naturally occurring protein, as
described in greater detail below, be the naturally occurring
protein the human protein, mouse protein, or protein from some
other species which naturally expresses a TIMP protein. In the
following description, the term TIMP is used to refer not only to
the human form of a TIMP protein, but also to homologs thereof
expressed in non-human species.
[0032] TIMP polypeptides of interest may vary in terms of amino
acid sequence length and molecular weight. In some instances, the
TIMP polypeptides range in length from 175 to 350, such as from 200
to 250 and including from about 200 to 225 amino acid residues, and
have a projected molecular weight based solely on the number of
amino acid residues in the protein and assuming an average
molecular weight of 110 Daltons that ranges from 19 to 39 kDa, such
as 22 to 28 kDa, including 22 to 25 kDa, where the actual molecular
weight may vary depending on the amount of glycosylation of the
protein and the apparent molecular weight may be considerably less
because of SDS binding on gels. TIMP polypeptides as described
herein may be obtained from naturally sources, e.g., via
purification techniques, chemically synthesized or produced using
recombinant protocols, as desired.
[0033] In some instances, the TIMP polypeptide that is administered
to the subject is a human TIMP2 protein, where the human TIMP2
protein has an amino acid sequence that comprises a region
substantially the same as or identical to the sequence appearing as
SEQ ID NO:01. By substantially the same as is meant a protein
having a region with a sequence that is 60% or greater, such as 75%
or greater, such as 90% or greater and including 98% or greater
sequence identity with the sequence of SED ID NO:01, as determined
by BLAST using default settings. In some instances, the TIMP
polypeptide that is administered to the subject is a human TIMP1
protein, where the human TIMP1 protein has an amino acid sequence
that comprises a region substantially the same as or identical to
the sequence appearing as SEQ ID NO:02. By substantially the same
as is meant a protein having a region with a sequence that is 60%
or greater, such as 75% or greater, such as 90% or greater and
including 98% or greater sequence identity with the sequence of SED
ID NO:02, as determined by BLAST using default settings. In some
instances, the TIMP polypeptide that is administered to the subject
is a human TIMP3 protein, where the human TIMP3 protein has an
amino acid sequence that comprises a region substantially the same
as or identical to the sequence appearing as SEQ ID NO:03. By
substantially the same as is meant a protein having a region with a
sequence that is 60% or greater, such as 75% or greater, such as
90% or greater and including 98% or greater sequence identity with
the sequence of SED ID NO:03, as determined by BLAST using default
settings. In some instances, the TIMP polypeptide that is
administered to the subject is a human TIMP4 protein, where the
human TIMP4 protein has an amino acid sequence that comprises a
region substantially the same as or identical to the sequence
appearing as SEQ ID NO:04. By substantially the same as is meant a
protein having a region with a sequence that is 60% or greater,
such as 75% or greater, such as 90% or greater and including 98% or
greater sequence identity with the sequence of SED ID NO:04, as
determined by BLAST using default settings.
[0034] In addition to the specific TIMP proteins described above,
homologs or proteins (or fragments thereof) from other species,
e.g., other animal species, may also be employed in embodiments of
the methods, where such homologs or proteins may be from a variety
of different types of species, including animals, such as mammals,
e.g., rodents, such as mice, rats; domestic animals, e.g., horse,
cow, dog, cat; etc. By homolog is meant a protein having 35% or
more, such as 40% and more and including 60% or more amino acid
sequence identity to the specific TIMP proteins as identified in
SEQ ID NOS: 01 to 04, where sequence identity is determined using
BLAST at default settings.
[0035] In addition to the naturally occurring TIMP proteins, e.g.,
as described above, TIMP polypeptides that vary from the naturally
occurring TIMP proteins may also be employed in practicing methods
of the invention. Different variations may be present, including
but not limited to substitution, insertion and/or deletion
mutations, as well as other types of non-amino acid sequence
variations, e.g., as illustrated below. TIMP polypeptides that may
be employed include proteins having an amino acid sequence encoded
by an open reading frame (ORF) of a TIMP gene, including the full
length TIMP protein and fragments thereof, such as biologically
active fragments and/or fragments corresponding to functional
domains; and including fusions of the subject polypeptides to other
proteins or parts thereof. Fragments of interest may vary in
length, and in some instances are 10 aa or longer, such as 50 aa or
longer, and including 100 aa or longer, and in some instances do
not exceed 150 aa in length, where a given fragment will have a
stretch of amino acids that is substantially the same as or
identical to a subsequence found in any of SEQ ID NOS:1 to 4; where
the subsequence may vary in length and in some instances is 10 aa
or longer, such as 15 aa or longer, up to 50 aa or even longer.
[0036] In some instances, TIMP polypeptides employed in methods of
invention include or more modifications. Modifications that may be
present may vary, and include but are not limited to: amide bond
substitutions, amino acid substitutions, including of cysteine
residues/analogues, cyclization, pegylation, etc. Examples of
modifications that may be found in TIMP polypeptides employed in
methods of the invention are now reviewed in greater detail.
[0037] In some cases, TIMP polypeptides include one or more
linkages other than peptide bonds, e.g., at least two adjacent
amino acids are joined via a linkage other than an amide bond. For
example, in order to reduce or eliminate undesired proteolysis or
other means of degradation, and/or to increase serum stability,
and/or to restrict or increase conformational flexibility, one or
more amide bonds within the backbone of a TIMP polypeptide can be
substituted. In another example, one or more amide linkages
(--CO--NH--) in a TIMP polypeptide can be replaced with a linkage
which is an isostere of an amide linkage, such as --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2CH.sub.2--, --CH.dbd.CH-- (cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2-- or --CH.sub.2SO--. One or more
amide linkages in a TIMP polypeptide can also be replaced by, for
example, a reduced isostere pseudopeptide bond.
[0038] One or more amino acid substitutions can be made in a TIMP
polypeptide. The following are non-limiting examples: a)
substitution of alkyl-substituted hydrophobic amino acids,
including alanine, leucine, isoleucine, valine, norleucine,
(S)-2-aminobutyric acid, (5)-cyclohexylalanine or other simple
alpha-amino acids substituted by an aliphatic side chain from
C.sub.1-C.sub.10 carbons including branched, cyclic and straight
chain alkyl, alkenyl or alkynyl substitutions; b) substitution of
aromatic-substituted hydrophobic amino acids, including
phenylalanine, tryptophan, tyrosine, sulfotyrosine,
biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,
2-benzothienylalanine, 3-benzothienylalanine, histidine, including
amino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,
bromo, or iodo) or alkoxy (from C.sub.1-C.sub.4)-substituted forms
of the above-listed aromatic amino acids, illustrative examples of
which are: 2-, 3- or 4-aminophenylalanine, 2-, 3- or
4-chlorophenylalanine, 2-, 3- or 4-methylphenylalanine, 2-, 3- or
4-methoxyphenylalanine, 5-amino-, 5-chloro-, 5-methyl- or
5-methoxytryptophan, 2'-, 3'-, or 4'-amino-, 2'-, 3'-, or
4'-chloro-, 2, 3, or 4-biphenylalanine, 2'-, 3'-, or 4'-methyl-,
2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine; c)
substitution of amino acids containing basic side chains, including
arginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,
homoarginine, including alkyl, alkenyl, or aryl-substituted (from
C.sub.1-C.sub.10 branched, linear, or cyclic) derivatives of the
previous amino acids, whether the substituent is on the heteroatoms
(such as the alpha nitrogen, or the distal nitrogen or nitrogens,
or on the alpha carbon, in the pro-R position for example.
Compounds that serve as illustrative examples include:
N-epsilon-isopropyl-lysine, 3-(4-tetrahydropyridyl)-glycine,
3-(4-tetrahydropyridyl)-alanine, N,N-gamma,
gamma'-diethyl-homoarginine. Included also are compounds such as
alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,
alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl
group occupies the pro-R position of the alpha-carbon. Also
included are the amides formed from alkyl, aromatic, heteroaromatic
(where the heteroaromatic group has one or more nitrogens, oxygens
or sulfur atoms singly or in combination), carboxylic acids or any
of the many well-known activated derivatives such as acid
chlorides, active esters, active azolides and related derivatives,
and lysine, ornithine, or 2,3-diaminopropionic acid; d)
substitution of acidic amino acids, including aspartic acid,
glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl,
and heteroaryl sulfonamides of 2,4-diaminopriopionic acid,
ornithine or lysine and tetrazole-substituted alkyl amino acids; e)
substitution of side chain amide residues, including asparagine,
glutamine, and alkyl or aromatic substituted derivatives of
asparagine or glutamine; and f) substitution of hydroxyl-containing
amino acids, including serine, threonine, homoserine,
2,3-diaminopropionic acid, and alkyl or aromatic substituted
derivatives of serine or threonine.
[0039] In some cases, a TIMP polypeptide includes one or more
naturally occurring non-genetically encoded L-amino acids,
synthetic L-amino acids, or D-enantiomers of an amino acid. For
example, a TIMP polypeptide can include only D-amino acids. For
example, a TIMP polypeptide can include one or more of the
following residues: hydroxyproline, .beta.-alanine, o-aminobenzoic
acid, m-aminobenzoic acid, p-aminobenzoic acid,
m-aminomethylbenzoic acid, 2,3-diaminopropionic acid,
a-aminoisobutyric acid, N-methylglycine (sarcosine), ornithine,
citrulline, t-butylalanine, t-butylglycine, N-methylisoleucine,
phenylglycine, cyclohexylalanine, norleucine, naphthylalanine,
pyridylalanine 3-benzothienyl alanine, 4-chlorophenylalanine,
2-fluorophenylalanine, 3-fluorophenylalanine,
4-fluorophenylalanine, penicillamine,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid,
.beta.-2-thienylalanine, methionine sulfoxide, homoarginine,
N-acetyl lysine, 2,4-diamino butyric acid, rho-aminophenylalanine,
N-methylvaline, homocysteine, homoserine, E-amino hexanoic acid,
.omega.-aminohexanoic acid, .omega.-aminoheptanoic acid,
.omega.-aminooctanoic acid, .omega.-aminodecanoic acid,
.omega.-aminotetradecanoic acid, cyclohexylalanine,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, .delta.-amino valeric acid,
and 2,3-diaminobutyric acid.
[0040] A cysteine residue or a cysteine analog can be introduced
into a TIMP polypeptide to provide for linkage to another peptide
via a disulfide linkage or to provide for cyclization of the TIMP
polypeptide. a TIMP polypeptide can be cyclized. One or more
cysteines or cysteine analogs can be introduced into a TIMP
polypeptide, where the introduced cysteine or cysteine analog can
form a disulfide bond with a second introduced cysteine or cysteine
analog. Other means of cyclization include introduction of an oxime
linker or a lanthionine linker; see, e.g., U.S. Pat. No. 8,044,175.
Any combination of amino acids (or non-amino acid moieties) that
can form a cyclizing bond can be used and/or introduced. A
cyclizing bond can be generated with any combination of amino acids
(or with an amino acid and --(CH2).sub.n-CO-- or
--(CH2).sub.n-C.sub.6H.sub.4--CO--) with functional groups which
allow for the introduction of a bridge. Some examples are
disulfides, disulfide mimetics such as the --(CH2).sub.n-- carba
bridge, thioacetal, thioether bridges (cystathionine or
lanthionine) and bridges containing esters and ethers. In these
examples, n can be any integer, but is frequently less than
ten.
[0041] Other modifications include, for example, an N-alkyl (or
aryl) substitution (.psi.[CONR]), or backbone crosslinking to
construct lactams and other cyclic structures. Other derivatives
include C-terminal hydroxymethyl derivatives, o-modified
derivatives (e.g., C-terminal hydroxymethyl benzyl ether),
N-terminally modified derivatives including substituted amides such
as alkylamides and hydrazides.
[0042] Modifications may be present that provide for improvements
in one or more physical properties of the TIMP polypeptide.
Improvements of physical properties include, for example,
modulating immunogenicity; methods of increasing water solubility,
bioavailability, serum half-life, and/or therapeutic half-life;
and/or modulating biological activity. Examples of such
modifications include, but are not limited to: pegylation,
glycosylation (N- and O-linked); polysialylation; albumin fusion
molecules comprising serum albumin (e.g., human serum albumin
(HSA), cyno serum albumin, or bovine serum albumin (BSA)); albumin
binding through, for example a conjugated fatty acid chain
(acylation); and Fc-fusion proteins.
[0043] Pegylation:
[0044] The clinical effectiveness of protein therapeutics may be
limited by short plasma half-life and susceptibility to protease
degradation. Studies of various therapeutic proteins (e.g.,
filgrastim) have shown that such difficulties may be overcome by
various modifications, including conjugating or linking the
polypeptide sequence to any of a variety of nonproteinaceous
polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes. This is frequently effected by a linking moiety
covalently bound to both the protein and the nonproteinaceous
polymer, e.g., a PEG. Such PEG-conjugated biomolecules have been
shown to possess clinically useful properties, including better
physical and thermal stability, protection against susceptibility
to enzymatic degradation, increased solubility, longer in vivo
circulating half-life and decreased clearance, reduced
immunogenicity and antigenicity, and reduced toxicity. In addition
to the beneficial effects of pegylation on pharmacokinetic
parameters, pegylation itself may enhance activity. PEGs suitable
for conjugation to a polypeptide sequence are generally soluble in
water at room temperature, and have the general formula
R(O--CH.sub.2--CH.sub.2).sub.nO--R, where R is hydrogen or a
protective group such as an alkyl or an alkanol group, and where n
is an integer from 1 to 1000. When R is a protective group, it
generally has from 1 to 8 carbons. The PEG conjugated to the
polypeptide sequence can be linear or branched. Branched PEG
derivatives, "star-PEGs" and multi-armed PEGs are contemplated by
the present disclosure. A molecular weight of the PEG used in the
present disclosure is not restricted to any particular range, and
examples are set forth elsewhere herein; by way of example, certain
embodiments have molecular weights between 5 kDa and 20 kDa, while
other embodiments have molecular weights between 4 kDa and 10 kDa.
Pegylated TIMP polypeptides may be conjugates wherein the PEGs have
different n values, and thus the various different PEGs are present
in specific ratios. For example, some compositions comprise a
mixture of conjugates where n=1, 2, 3 and 4. In some compositions,
the percentage of conjugates where n=1 is 18-25%, the percentage of
conjugates where n=2 is 50-66%, the percentage of conjugates where
n=3 is 12-16%, and the percentage of conjugates where n=4 is up to
5%. Such compositions can be produced by any convenient reaction
conditions and purification. Pegylation most frequently occurs at
the alpha amino group at the N-terminus of the polypeptide, the
epsilon amino group on the side chain of lysine residues, and the
imidazole group on the side chain of histidine residues. Since most
recombinant polypeptides possess a single alpha and a number of
epsilon amino and imidazole groups, numerous positional isomers can
be generated depending on the linker chemistry. General pegylation
strategies, such as those known in the art, can be applied herein.
PEG may be bound to a polypeptide of the present disclosure via a
terminal reactive group (a "spacer") which mediates a bond between
the free amino or carboxyl groups of one or more of the polypeptide
sequences and polyethylene glycol. The PEG having the spacer which
may be bound to the free amino group includes
N-hydroxysuccinylimide polyethylene glycol which may be prepared by
activating succinic acid ester of polyethylene glycol with
N-hydroxysuccinylimide. Another activated polyethylene glycol which
may be bound to a free amino group is
2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which may
be prepared by reacting polyethylene glycol monomethyl ether with
cyanuric chloride. The activated polyethylene glycol which is bound
to the free carboxyl group includes polyoxyethylenediamine.
Conjugation of one or more of the polypeptide sequences to PEG
having a spacer may be carried out by various conventional methods.
For example, the conjugation reaction can be carried out in
solution at a pH of from 5 to 10, at temperature from 4.degree. C.
to room temperature, for 30 minutes to 20 hours, utilizing a molar
ratio of reagent to protein of from 4:1 to 30:1. Reaction
conditions may be selected to direct the reaction towards producing
predominantly a desired degree of substitution. In general, low
temperature, low pH (e.g., pH=5), and short reaction time tend to
decrease the number of PEGs attached, whereas high temperature,
neutral to high pH (e.g., pH.gtoreq.7), and longer reaction time
tend to increase the number of PEGs attached. Various means known
in the art may be used to terminate the reaction. In some
embodiments the reaction is terminated by acidifying the reaction
mixture and freezing at, e.g., -20.degree. C. Pegylation of various
molecules is discussed in, for example, U.S. Pat. Nos. 5,252,714;
5,643,575; 5,919,455; 5,932,462; and 5,985,263. The present
disclosure also contemplates the use of PEG mimetics. Recombinant
PEG mimetics have been developed that retain the attributes of PEG
(e.g., enhanced serum half-life) while conferring several
additional advantageous properties. By way of example, simple
polypeptide chains (comprising, for example, Ala, Glu, Gly, Pro,
Ser and Thr) capable of forming an extended conformation similar to
PEG can be produced recombinantly already fused to the peptide or
protein drug of interest. This obviates the need for an additional
conjugation step during the manufacturing process. Moreover,
established molecular biology techniques enable control of the side
chain composition of the polypeptide chains, allowing optimization
of immunogenicity and manufacturing properties.
[0045] Glycosylation:
[0046] For purposes of the present disclosure, "glycosylation" is
meant to broadly refer to the enzymatic process that attaches
glycans to proteins, lipids or other organic molecules. The use of
the term "glycosylation" in conjunction with the present disclosure
is generally intended to mean adding or deleting one or more
carbohydrate moieties (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that may or may not be present in the native sequence. In
addition, the phrase includes qualitative changes in the
glycosylation of the native proteins involving a change in the
nature and proportions of the various carbohydrate moieties
present. Glycosylation can dramatically affect the physical
properties (e.g., solubility) of polypeptides such as TIMP
polypeptides and can also be important in protein stability,
secretion, and subcellular localization. Glycosylated polypeptides
may also exhibit enhanced stability or may improve one or more
pharmacokinetic properties, such as half-life. In addition,
solubility improvements can, for example, enable the generation of
formulations more suitable for pharmaceutical administration than
formulations comprising the non-glycosylated polypeptide. Addition
of glycosylation sites can be accomplished by altering the amino
acid sequence. The alteration to the polypeptide may be made, for
example, by the addition of, or substitution by, one or more serine
or threonine residues (for O-linked glycosylation sites) or
asparagine residues (for N-linked glycosylation sites). The
structures of N-linked and O-linked oligosaccharides and the sugar
residues found in each type may be different. One type of sugar
that is commonly found on both is N-acetylneuraminic acid
(hereafter referred to as sialic acid). Sialic acid is usually the
terminal residue of both N-linked and O-linked oligosaccharides
and, by virtue of its negative charge, may confer acidic properties
to the glycoprotein. A particular embodiment of the present
disclosure comprises the generation and use of N-glycosylation
variants. The polypeptide sequences of the present disclosure may
optionally be altered through changes at the nucleic acid level,
particularly by mutating the nucleic acid encoding the polypeptide
at preselected bases such that codons are generated that will
translate into the desired amino acids. Another means of increasing
the number of carbohydrate moieties on the polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide.
Removal of carbohydrates may be accomplished chemically or
enzymatically, or by substitution of codons encoding amino acid
residues that are glycosylated. Chemical deglycosylation techniques
are known, and enzymatic cleavage of carbohydrate moieties on
polypeptides can be achieved by the use of a variety of endo- and
exo-glycosidases. Dihydrofolate reductase (DHFR)--deficient Chinese
Hamster Ovary (CHO) cells are a commonly used host cell for the
production of recombinant glycoproteins. These cells do not express
the enzyme beta-galactoside alpha-2,6-sialyltransferase and
therefore do not add sialic acid in the alpha-2,6 linkage to
N-linked oligosaccharides of glycoproteins produced in these
cells.
[0047] In some embodiments, the polypeptides are non-naturally
glycosylated. By non-naturally glycosylated is meant that the
polypeptide has a glycosylation pattern, if present, which is not
the same as the glycosylation pattern found in the corresponding
naturally occurring protein. For example, a human TIMP2 employed in
methods of the invention of this particular embodiment is
characterized by having a glycosylation pattern, if glycosylated at
all, that differs from that of naturally occurring human TIMP2.
Thus, the non-naturally glycosylated TIMP polypeptides of this
embodiment include non-glycosylated TIMP polypeptides, i.e.
proteins having no covalently bound glycosyl groups.
[0048] Polysialylation:
[0049] The present disclosure also contemplates the use of
polysialylation, the conjugation of polypeptides to the naturally
occurring, biodegradable .alpha.-(2.fwdarw.8) linked polysialic
acid ("PSA") in order to improve the polypeptides' stability and in
vivo pharmacokinetics. PSA is a biodegradable, non-toxic natural
polymer that is highly hydrophilic, giving it a high apparent
molecular weight in the blood which increases its serum half-life.
In addition, polysialylation of a range of peptide and protein
therapeutics has led to markedly reduced proteolysis, retention of
in vivo activity, and reduction in immunogenicity and antigenicity
(see, e.g., G. Gregoriadis et al., Int. J. Pharmaceutics
300(1-2):125-30). As with modifications with other conjugates
(e.g., PEG), various techniques for site-specific polysialylation
are available (see, e.g., T. Lindhout et al., (2011) PNAS
108(18)7397-7402).
[0050] Albumin Fusion:
[0051] Additional suitable components and molecules for conjugation
include albumins such as human serum albumin (HSA), cyno serum
albumin, and bovine serum albumin (BSA). Mature HSA, a 585 amino
acid polypeptide (.about.67 kDa) having a serum half-life of
.about.20 days, is primarily responsible for the maintenance of
colloidal osmotic blood pressure, blood pH, and transport and
distribution of numerous endogenous and exogenous ligands. The
protein has three structurally homologous domains (domains I, II
and III), is almost entirely in the alpha-helical conformation, and
is highly stabilized by 17 disulphide bridges. The three primary
drug binding regions of albumin are located on each of the three
domains within sub-domains IB, IIA and IIIA. Albumin synthesis
takes place in the liver, which produces the short-lived, primary
product preproalbumin. Thus, the full-length HSA has a signal
peptide of 18 amino acids (MKWVTFISLLFLFSSAYS; SEQ ID NO:5)
followed by a pro-domain of 6 amino acids (RGVFRR; SEQ ID NO:6);
this 24 amino acid residue peptide may be referred to as the
pre-pro domain. HSA can be expressed and secreted using its
endogenous signal peptide as a pre-pro-domain. Alternatively, HSA
can be expressed and secreted using a IgK signal peptide fused to a
mature construct. Preproalbumin is rapidly co-translationally
cleaved in the endoplasmic reticulum lumen at its amino terminus to
produce the stable, 609-amino acid precursor polypeptide,
proalbumin. Proalbumin then passes to the Golgi apparatus, where it
is converted to the 585 amino acid mature albumin by a
furin-dependent amino-terminal cleavage. The primary amino acid
sequences, structure, and function of albumins are highly conserved
across species, as are the processes of albumin synthesis and
secretion. Albumin serum proteins comparable to HSA are found in,
for example, cynomolgus monkeys, cows, dogs, rabbits and rats. Of
the non-human species, bovine serum albumin (BSA) is the most
structurally similar to HSA (see, e.g., Kosa et al., November 2007
J Pharm Sci. 96(11):3117-24). The present disclosure contemplates
the use of albumin from non-human species, including, but not
limited to, those set forth above, in, for example, the drug
development process. According to the present disclosure, albumin
may be conjugated to a drug molecule (e.g., a polypeptide described
herein) at the carboxyl terminus, the amino terminus, both the
carboxyl and amino termini, and internally (see, e.g., U.S. Pat.
Nos. 5,876,969 and 7,056,701). In the HSA-TIMP conjugates
contemplated by the present disclosure, various forms of albumin
may be used, such as albumin secretion pre-sequences and variants
thereof, fragments and variants thereof, and HSA variants. Such
forms generally possess one or more desired albumin activities. In
additional embodiments, the present disclosure involves fusion
proteins comprising a polypeptide drug molecule fused directly or
indirectly to albumin, an albumin fragment, and albumin variant,
etc., wherein the fusion protein has a higher plasma stability than
the unfused drug molecule and/or the fusion protein retains the
therapeutic activity of the unfused drug molecule. In some
embodiments, the indirect fusion is effected by a linker, such as a
peptide linker or modified version thereof. Intracellular cleavage
may be carried out enzymatically by, for example, furin or caspase.
Cells express a low level of these endogenous enzymes, which are
capable of cleaving a portion of the fusion molecules
intracellularly; thus, some of the polypeptides are secreted from
the cell without being conjugated to HSA, while some of the
polypeptides are secreted in the form of fusion molecules that
comprise HSA. Embodiments of the present disclosure contemplate the
use of various furin fusion constructs. For example, constructs may
be designed that comprise the sequence RGRR, RKRKKR, RKKR, or
RRRKKR. The present disclosure also contemplates extra-cellular
cleavage (i.e., ex-vivo cleavage) whereby the fusion molecules are
secreted from the cell, subjected to purification, and then
cleaved. It is understood that the excision may dissociate the
entire HSA-linker complex from the mature TIMP polypeptide, or less
that the entire HSA-linker complex. As alluded to above, fusion of
albumin to one or more polypeptides of the present disclosure can,
for example, be achieved by genetic manipulation, such that the
nucleic acid coding for HSA, or a fragment thereof, is joined to
the nucleic acid coding for the one or more polypeptide sequences.
Thereafter, a suitable host can be transformed or transfected with
the fused nucleotide sequences in the form of, for example, a
suitable plasmid, so as to express a fusion polypeptide. The
expression may be effected in vitro from, for example, prokaryotic
or eukaryotic cells, or in vivo from, for example, a transgenic
organism. In some embodiments of the present disclosure, the
expression of the fusion protein is performed in mammalian cell
lines, for example, CHO cell lines. Transformation is used broadly
herein to refer to the genetic alteration of a cell resulting from
the direct uptake through the cell membrane, incorporation and
expression of exogenous genetic material (exogenous nucleic acid).
Transformation occurs naturally in some species of bacteria, but it
can also be effected by artificial means in other cells.
Furthermore, albumin itself may be modified to extend its
circulating half-life. Fusion of the modified albumin to a TIMP
polypeptide can be attained by the genetic manipulation techniques
described above or by chemical conjugation; the resulting fusion
molecule has a half-life that exceeds that of fusions with
non-modified albumin. TIMP2-albumin fusion proteins of interest
include those described in U.S. Pat. No. 7,163,805, the disclosure
of which is herein incorporated by reference.
[0052] Several albumin--binding strategies have been developed as
alternatives to direct fusion, including albumin binding through a
conjugated fatty acid chain (acylation). Because serum albumin is a
transport protein for fatty acids, these natural ligands with
albumin--binding activity have been used for half-life extension of
small protein therapeutics. For example, insulin determir
(LEVEMIR), an approved product for diabetes, comprises a myristyl
chain conjugated to a genetically-modified insulin, resulting in a
long-acting insulin analog. The present disclosure also
contemplates fusion proteins which comprise an albumin binding
domain (ABD) polypeptide sequence and the sequence of one or more
of the polypeptides described herein. Any ABD polypeptide sequence
described in the literature can be a component of the fusion
proteins. The components of the fusion proteins can be optionally
covalently bonded through a linker, such as those linkers described
herein. In some of the embodiments of the present disclosure, the
fusion proteins comprise the ABD polypeptide sequence as an
N-terminal moiety and the polypeptides described herein as a
C-terminal moiety. The present disclosure also contemplates fusion
proteins comprising a fragment of an albumin binding polypeptide,
which fragment substantially retains albumin binding; or a multimer
of albumin binding polypeptides or their fragments comprising at
least two albumin binding polypeptides or their fragments as
monomer units.
[0053] Conjugation with Other Molecules:
[0054] Additional suitable components and molecules for conjugation
include, for example, thyroglobulin; tetanus toxoid; Diphtheria
toxoid; polyamino acids such as poly(D-lysine:D-glutamic acid); VP6
polypeptides of rotaviruses; influenza virus hemaglutinin,
influenza virus nucleoprotein; Keyhole Limpet Hemocyanin (KLH); and
hepatitis B virus core protein and surface antigen; or any
combination of the foregoing. Thus, the present disclosure
contemplates conjugation of one or more additional components or
molecules at the N- and/or C-terminus of a polypeptide sequence,
such as another polypeptide (e.g., a polypeptide having an amino
acid sequence heterologous to the subject polypeptide), or a
carrier molecule. Thus, an exemplary polypeptide sequence can be
provided as a conjugate with another component or molecule. A
conjugate modification may result in a polypeptide sequence that
retains activity with an additional or complementary function or
activity derived from the second molecule. For example, a
polypeptide sequence may be conjugated to a molecule, e.g., to
facilitate solubility, storage, in vivo or shelf half-life or
stability, reduction in immunogenicity, delayed or controlled
release in vivo, etc. Other functions or activities include a
conjugate that reduces toxicity relative to an unconjugated
polypeptide sequence, a conjugate that targets a type of cell or
organ more efficiently than an unconjugated polypeptide sequence,
or a drug to further counter the causes or effects associated with
a disease, disorder or condition as set forth herein (e.g.,
cancer). A TIMP polypeptide may also be conjugated to large, slowly
metabolized macromolecules such as proteins; polysaccharides, such
as sepharose, agarose, cellulose, or cellulose beads; polymeric
amino acids such as polyglutamic acid, or polylysine; amino acid
copolymers; inactivated virus particles; inactivated bacterial
toxins such as toxoid from diphtheria, tetanus, cholera, or
leukotoxin molecules; inactivated bacteria; and dendritic cells.
Such conjugated forms, if desired, can be used to produce
antibodies against a polypeptide of the present disclosure.
Additional candidate components and molecules for conjugation
include those suitable for isolation or purification. Particular
non-limiting examples include binding molecules, such as biotin
(biotin-avidin specific binding pair), an antibody, a receptor, a
ligand, a lectin, or molecules that comprise a solid support,
including, for example, plastic or polystyrene beads, plates or
beads, magnetic beads, test strips, and membranes. Purification
methods such as cation exchange chromatography may be used to
separate conjugates by charge difference, which effectively
separates conjugates into their various molecular weights. For
example, the cation exchange column can be loaded and then washed
with .about.20 mM sodium acetate, pH .about.4, and then eluted with
a linear (0 M to 0.5 M) NaCl gradient buffered at a pH from about 3
to 5.5, e.g., at pH .about.4.5. The content of the fractions
obtained by cation exchange chromatography may be identified by
molecular weight using conventional methods, for example, mass
spectroscopy, SDS-PAGE, or other known methods for separating
molecular entities by molecular weight.
[0055] Fc-Fusion Molecules:
[0056] In certain embodiments, the amino- or carboxyl-terminus of a
polypeptide sequence of the present disclosure can be fused with an
immunoglobulin Fc region (e.g., human Fc) to form a fusion
conjugate (or fusion molecule). Fc fusion conjugates have been
shown to increase the systemic half-life of biopharmaceuticals, and
thus the biopharmaceutical product may require less frequent
administration. Fc binds to the neonatal Fc receptor (FcRn) in
endothelial cells that line the blood vessels, and, upon binding,
the Fc fusion molecule is protected from degradation and
re-released into the circulation, keeping the molecule in
circulation longer. This Fc binding is believed to be the mechanism
by which endogenous IgG retains its long plasma half-life. More
recent Fc-fusion technology links a single copy of a
biopharmaceutical to the Fc region of an antibody to optimize the
pharmacokinetic and pharmacodynamic properties of the
biopharmaceutical as compared to traditional Fc-fusion
conjugates.
[0057] Other Modifications:
[0058] The present disclosure contemplates the use of other
modifications, currently known or developed in the future, of TIMP
polypeptides to improve one or more properties. One such method for
prolonging the circulation half-life, increasing the stability,
reducing the clearance, or altering the immunogenicity or
allergenicity of a polypeptide of the present disclosure involves
modification of the polypeptide sequences by hesylation, which
utilizes hydroxyethyl starch derivatives linked to other molecules
in order to modify the polypeptide sequences' characteristics.
[0059] Linkers:
[0060] Linkers and their use have been described above. Any of the
foregoing components and molecules used to modify the polypeptide
sequences of the present disclosure may optionally be conjugated
via a linker. Suitable linkers include "flexible linkers" which are
generally of sufficient length to permit some movement between the
modified polypeptide sequences and the linked components and
molecules. The linker molecules are generally about 6-50 atoms
long. The linker molecules may also be, for example, aryl
acetylene, ethylene glycol oligomers containing 2-10 monomer units,
diamines, diacids, amino acids, or combinations thereof. Suitable
linkers can be readily selected and can be of any suitable length,
such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,
10-20, 20-30, 30-50 or more than 50 amino acids. Exemplary flexible
linkers include glycine polymers (G).sub.n, glycine-serine polymers
(for example, (GS).sub.n, GSGGS.sub.n, GGGS.sub.n,
(G.sub.mS.sub.o).sub.n, (G.sub.mS.sub.oG.sub.m).sub.n,
(G.sub.mS.sub.oG.sub.mS.sub.oG.sub.m).sub.n, (GSGGS.sub.m).sub.n,
(GSGS.sub.mG).sub.n and (GGGS.sub.m).sub.n, and combinations
thereof, where m, and o are each independently selected from an
integer of at least one), glycine-alanine polymers, alanine-serine
polymers, and other flexible linkers. Glycine and glycine-serine
polymers are relatively unstructured, and therefore may serve as a
neutral tether between components. Exemplary flexible linkers
include, but are not limited to GGSG, GGSGG, GSGSG, GSGGG, GGGSG,
and GSSSG.
[0061] In some instances, systemic TIMP polypeptide levels is
increased by administering a nucleic acid coding sequence to the
subject under conditions sufficient for the coding sequence to be
expressed in the subject. Depending on the desired TIMP
polypeptide, the nucleic acid coding sequence may vary. Nucleic
acids of interest include those encoding the TIMP polypeptides
provided above. Specific nucleic acids of interest include, but are
not limited to: Human TIMP2 (NCBI Reference Sequence: NM_003255.4);
Human TIMP1 (NCBI Reference Sequence: NM_003254.2); Human TIMP3
(NCBI Reference Sequence: NM_000362.4) and Human TIMP4 (NCBI
Reference Sequence: NM_003256.3).
[0062] By nucleic acid composition is meant a composition
comprising a sequence of DNA having an open reading frame that
encodes a TIMP polypeptide of interest, i.e., a TIMP coding
sequence, and is capable, under appropriate conditions, of being
expressed as a TIMP polypeptide. Also encompassed in this term are
nucleic acids that are homologous, substantially similar or
identical to the specific nucleic acids described above. In
addition to the above described specific nucleic acid compositions,
also of interest are homologues of the above sequences. In certain
embodiments, sequence similarity between homologues is 20% or
higher, such as 25% or higher, and including 30%, 35%, 40%, 50%,
60%, 70% or higher, including 75%, 80%, 85%, 90% and 95% or higher.
Sequence similarity is calculated based on a reference sequence,
which may be a subset of a larger sequence, such as a conserved
motif, coding region, flanking region, etc. A reference sequence
may be 18 nt long or longer, such as 30 nt long, and may extend to
the complete sequence that is being compared. Algorithms for
sequence analysis are known in the art, such as BLAST, described in
Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using default
settings, i.e. parameters w=4 and T=17). Of particular interest in
certain embodiments are nucleic acids of substantially the same
length as specific human TIMP1 to TIMP4 nucleic acids mentioned
above, where by substantially the same length is meant that any
difference in length does not exceed about 20 number %, usually
does not exceed about 10 number and more usually does not exceed
about 5 number %; and have sequence identity to any of these
sequences of at 90% or greater, such as 95% or greater and
including 99% or greater over the entire length of the nucleic
acid. In some embodiments, the nucleic acids have a sequence that
is substantially similar or identical to the above specific
sequences. By substantially similar is meant that sequence identity
is 60% or greater, such as 75% or greater and including 80, 85, 90,
or even 95% or greater. Nucleic acids of interest also include
nucleic acids that encode the proteins encoded by the above
described nucleic acids, but differ in sequence from the above
described nucleic acids due to the degeneracy of the genetic
code.
[0063] Nucleic acids as described herein may be present in a
vector. Various vectors (e.g., viral vectors, bacterial vectors, or
vectors capable of replication in eukaryotic and prokaryotic hosts)
can be used in accordance with the present invention. Numerous
vectors which can replicate in eukaryotic and prokaryotic hosts are
known in the art and are commercially available. In some instances,
such vectors used in accordance with the invention are composed of
a bacterial origin of replication and a eukaryotic promoter
operably linked to a DNA of interest.
[0064] Viral vectors used in accordance with the invention may be
composed of a viral particle derived from a naturally-occurring
virus which has been genetically altered to render the virus
replication-defective and to express a recombinant gene of interest
in accordance with the invention. Once the virus delivers its
genetic material to a cell, it does not generate additional
infectious virus but does introduce exogenous recombinant genes
into the cell, preferably into the genome of the cell. Numerous
viral vectors are well known in the art, including, for example,
retrovirus, adenovirus, adeno-associated virus, herpes simplex
virus (HSV), cytomegalovirus (CMV), vaccinia and poliovirus
vectors.
[0065] The DNA of interest may be administered using a non-viral
vector, for example, as a DNA- or RNA-liposome complex formulation.
Such complexes comprise a mixture of lipids which bind to genetic
material (DNA or RNA), providing a hydrophobic coat which allows
the genetic material to be delivered into cells. Liposomes which
can be used in accordance with the invention include DOPE (dioleyl
phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-.beta.-ol
3-urethanyl)-N',N'-dimethylethylene diamine). When the DNA of
interest is introduced using a liposome, in some instances one
first determines in vitro the optimal values for the DNA: lipid
ratios and the absolute concentrations of DNA and lipid as a
function of cell death and transformation efficiency for the
particular type of cell to be transformed. These values can then be
used in or extrapolated for use in in vivo transformation. The in
vitro determinations of these values can be readily carried out
using techniques which are well known in the art.
[0066] Other non-viral vectors may also be used in accordance with
the present invention. These include chemical formulations of DNA
or RNA coupled to a carrier molecule (e.g., an antibody or a
receptor ligand) which facilitates delivery to host cells for the
purpose of altering the biological properties of the host cells. By
the term "chemical formulations" is meant modifications of nucleic
acids to allow coupling of the nucleic acid compounds to a carrier
molecule such as a protein or lipid, or derivative thereof.
Exemplary protein carrier molecules include antibodies specific to
the cells of a targeted secretory gland or receptor ligands, i.e.,
molecules capable of interacting with receptors associated with a
cell of a targeted secretory gland.
[0067] DNA constructs may include a promoter to facilitate
expression of the DNA of interest within a target cell, such as a
strong, eukaryotic promoter. Exemplary eukaryotic promoters include
promoters from cytomegalovirus (CMV), mouse mammary tumor virus
(MMTV), Rous sarcoma virus (RSV), and adenovirus. More
specifically, exemplary promoters include the promoter from the
immediate early gene of human CMV (Boshart et al., Cell 41:521-530,
1985) and the promoter from the long terminal repeat (LTR) of RSV
(Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777-6781, 1982).
[0068] Instead of administration of a TIMP polypeptide, e.g., as
described above, the level of systemic TIMP active agent in the
subject may be enhanced by stimulating endogenous production and/or
release of a TIMP polypeptide in vivo.
[0069] Also of interest are potentiators of TIMP activity. By TIMP
potentiator is meant an agent or combination of agents that work to
increase the desirable TIMP activity of endogenous TIMP
polypeptides present in the subject being treated. The magnitude of
the increase may vary, where in some instances the magnitude of the
increase is 2-fold or greater, such as 5-fold or greater, including
10-fold or greater, e.g., 15-fold or greater, 20-fold or greater,
25-fold or greater (as compared to a suitable control). TIMP
potentiators of interest may work through a variety of different
mechanisms, e.g., by enhancing the binding interaction between a
TIMP polypeptide and a desired target; by increasing the
bioavailability of the endogenous pool, e.g., by sequestering
undesirable competitive binding targets, etc.
[0070] In yet other embodiments, the agent is a small molecule
agent that exhibits the desired TIMP activity. Naturally occurring
or synthetic small molecule compounds of interest include numerous
chemical classes, such as organic molecules, e.g., small organic
compounds having a molecular weight of more than 50 and less than
about 2,500 Daltons. Candidate agents comprise functional groups
for structural interaction with proteins, particularly hydrogen
bonding, and typically include at least an amine, carbonyl,
hydroxyl or carboxyl group, preferably at least two of the
functional chemical groups. The candidate agents may include
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among
biomolecules including peptides, saccharides, fatty acids,
steroids, purines, pyrimidines, derivatives, structural analogs or
combinations thereof. Such molecules may be identified, among other
ways, by employing the screening protocols described below.
[0071] In practicing methods of the invention, the active agent(s)
may be administered to the adult mammal using any convenient
administration protocol capable of resulting in the desired
activity. Thus, the agent can be incorporated into a variety of
formulations, e.g., pharmaceutically acceptable vehicles, for
therapeutic administration. More particularly, the agents of the
present invention can be formulated into pharmaceutical
compositions by combination with appropriate, pharmaceutically
acceptable carriers or diluents, and may be formulated into
preparations in solid, semi-solid, liquid or gaseous forms, such as
tablets, capsules, powders, granules, ointments (e.g., skin
creams), solutions, suppositories, injections, inhalants and
aerosols. As such, administration of the agents can be achieved in
various ways, including oral, buccal, rectal, parenteral,
intraperitoneal, intradermal, transdermal, intracheal, etc.,
administration.
[0072] In pharmaceutical dosage forms, the agents may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0073] For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders,
granules or capsules, for example, with conventional additives,
such as lactose, mannitol, corn starch or potato starch; with
binders, such as crystalline cellulose, cellulose derivatives,
acacia, corn starch or gelatins; with disintegrators, such as corn
starch, potato starch or sodium carboxymethylcellulose; with
lubricants, such as talc or magnesium stearate; and if desired,
with diluents, buffering agents, moistening agents, preservatives
and flavoring agents.
[0074] The agents can be formulated into preparations for injection
by dissolving, suspending or emulsifying them in an aqueous or
nonaqueous solvent, such as vegetable or other similar oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives.
[0075] The agents can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0076] Furthermore, the agents can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0077] Unit dosage forms for oral or rectal administration such as
syrups, elixirs, and suspensions may be provided wherein each
dosage unit, for example, teaspoonful, tablespoonful, tablet or
suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier.
[0078] The term "unit dosage form," as used herein, refers to
physically discrete units suitable as unitary dosages for human and
animal subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular compound employed and the effect
to be achieved, and the pharmacodynamics associated with each
compound in the host.
[0079] The pharmaceutically acceptable excipients, such as
vehicles, adjuvants, carriers or diluents, are readily available to
the public. Moreover, pharmaceutically acceptable auxiliary
substances, such as pH adjusting and buffering agents, tonicity
adjusting agents, stabilizers, wetting agents and the like, are
readily available to the public.
[0080] Where the agent is a polypeptide, polynucleotide, analog or
mimetic thereof, it may be introduced into tissues or host cells by
any number of routes, including viral infection, microinjection, or
fusion of vesicles. Jet injection may also be used for
intramuscular administration, as described by Furth et al., Anal
Biochem. (1992) 205:365-368. The DNA may be coated onto gold
microparticles, and delivered intradermally by a particle
bombardment device, or "gene gun" as described in the literature
(see, for example, Tang et al., Nature (1992) 356:152-154), where
gold microprojectiles are coated with the DNA, then bombarded into
skin cells. For nucleic acid therapeutic agents, a number of
different delivery vehicles find use, including viral and non-viral
vector systems, as are known in the art.
[0081] Those of skill in the art will readily appreciate that dose
levels can vary as a function of the specific compound, the nature
of the delivery vehicle, and the like. Preferred dosages for a
given compound are readily determinable by those of skill in the
art by a variety of means.
[0082] In those embodiments where an effective amount of an active
agent is administered to the adult mammal, the amount or dosage is
effective when administered for a suitable period of time, such as
one week or longer, including two weeks or longer, such as 3 weeks
or longer, 4 weeks or longer, 8 weeks or longer, etc., so as to
evidence a reduction in the impairment, e.g., cognition decline
and/or cognitive improvement in the adult mammal. For example, an
effective dose is the dose that, when administered for a suitable
period of time, such as at least about one week, and maybe about
two weeks, or more, up to a period of about 3 weeks, 4 weeks, 8
weeks, or longer, will slow e.g., by about 20% or more, e.g., by
30% or more, by 40% or more, or by 50% or more, in some instances
by 60% or more, by 70% or more, by 80% or more, or by 90% or more,
e.g., will halt, cognitive decline in a patient suffering from
natural aging or an aging-associated disorder. In some instances,
an effective amount or dose of active agent will not only slow or
halt the progression of the disease condition but will also induce
the reversal of the condition, i.e., will cause an improvement in
cognitive ability. For example, in some instances, an effective
amount is the amount that when administered for a suitable period
of time, usually at least about one week, and maybe about two
weeks, or more, up to a period of about 3 weeks, 4 weeks, 8 weeks,
or longer will improve the cognitive abilities of an individual
suffering from an aging-associated cognitive impairment by, for
example 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, in some instances
6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more relative to
cognition prior to administration of the blood product.
[0083] Where desired, effectiveness of treatment may be assessed
using any convenient protocol. Cognition tests and IQ test for
measuring cognitive ability, e.g., attention and concentration, the
ability to learn complex tasks and concepts, memory, information
processing, visuospatial function, the ability to produce and
understanding language, the ability to solve problems and make
decisions, and the ability to perform executive functions, are well
known in the art, any of which may be used to measure the cognitive
ability of the individual before and/or during and after treatment
with the subject blood product, e.g., to confirm that an effective
amount has been administered. These include, for example, the
General Practitioner Assessment of Cognition (GPCOG) test, the
Memory Impairment Screen, the Mini Mental State Examination (MMSE),
the California Verbal Learning Test, Second Edition, Short Form,
for memory, the Delis-Kaplan Executive Functioning System test, the
Alzheimer's Disease Assessment Scale (ADAS-Cog), the
Psychogeriatric Assessment Scale (PAS) and the like. Progression of
functional brain improvements may be detected by brain imaging
techniques, such as Magnetic Resonance Imaging (MRI) or Positron
Emission Tomography (PET) and the like. A wide range of additional
functional assessments may be applied to monitor activities of
daily living, executive functions, mobility, etc. In some
embodiments, the method comprises the step of measuring cognitive
ability, and detecting a decreased rate of cognitive decline, a
stabilization of cognitive ability, and/or an increase in cognitive
ability after administration of the blood product as compared to
the cognitive ability of the individual before the blood product
was administered. Such measurements may be made a week or more
after administration of the blood product, e.g., 1 week, 2 weeks, 3
weeks, or more, for instance, 4 weeks, 6 weeks, or 8 weeks or more,
e.g., 3 months, 4 months, 5 months, or 6 months or more.
[0084] Biochemically, by an "effective amount" or "effective dose"
of active agent is meant an amount of active agent that will
inhibit, antagonize, decrease, reduce, or suppress by about 20% or
more, e.g., by 30% or more, by 40% or more, or by 50% or more, in
some instances by 60% or more, by 70% or more, by 80% or more, or
by 90% or more, in some cases by about 100%, i.e., to negligible
amounts, and in some instances reverse, the reduction in synaptic
plasticity and loss of synapses that occurs during the natural
aging process or during the progression of an aging-associated
disorder. In other words, cells present in adult mammals treated in
accordance with methods of the invention will become more
responsive to cues, e.g., activity cues, which promote the
formation and maintenance of synapses.
[0085] Performance of methods of the invention, e.g., as described
above, may manifest as improvements in observed synaptic
plasticity, both in vitro and in vivo as an induction of long term
potentiation. For example, the induction of LTP in neural circuits
may be observed in awake individuals, e.g., by performing
non-invasive stimulation techniques on awake individuals to induce
LTP-like long-lasting changes in localized neural activity (Cooke
SF, Bliss TV (2006) Plasticity in the human central nervous system.
Brain. 129(Pt 7):1659-73); mapping plasticity and increased neural
circuit activity in individuals, e.g., by using positron emission
tomography, functional magnetic resonance imaging, and/or
transcranial magnetic stimulation (Cramer and Bastings, "Mapping
clinically relevant plasticity after stroke," Neuropharmacology
(2000)39:842-51); and by detecting neural plasticity following
learning, i.e., improvements in memory, e.g., by assaying
retrieval-related brain activity (Buchmann et al., "Prion protein
M129V polymorphism affects retrieval-related brain activity,"
Neuropsychologia. (2008) 46:2389-402) or, e.g., by imaging brain
tissue by functional magnetic resonance imaging (fMRI) following
repetition priming with familiar and unfamiliar objects (Soldan et
al., "Global familiarity of visual stimuli affects
repetition-related neural plasticity but not repetition priming,"
Neuroimage. (2008) 39:515-26; Soldan et al., "Aging does not affect
brain patterns of repetition effects associated with perceptual
priming of novel objects," J. Cogn. Neurosci. (2008) 20:1762-76).
In some embodiments, the method includes the step of measuring
synaptic plasticity, and detecting a decreased rate of loss of
synaptic plasticity, a stabilization of synaptic plasticity, and/or
an increase in synaptic plasticity after administration of the
blood product as compared to the synaptic plasticity of the
individual before the blood product was administered. Such
measurements may be made a week or more after administration of the
blood product, e.g., 1 week, 2 weeks, 3 weeks, or more, for
instance, 4 weeks, 6 weeks, or 8 weeks or more, e.g., 3 months, 4
months, 5 months, or 6 months or more.
[0086] In some instances, the methods result in a change in
expression levels of one or more genes in one or more tissues of
the host, e.g., as compared to a suitable control (such as
described in the Experimental section, below). The change in
expression level of a given gene may be 0.5 fold or greater, such
as 1.0 fold or greater, including 1.5 fold or greater. The tissue
may vary, and in some instances is nervous system tissue, e.g.,
central nervous system tissue, including brain tissue, e.g.,
hippocampal tissue. In some instances, the modulation of
hippocampal gene expression is manifested as enhanced hippocampal
plasticity, e.g., as compared to a suitable control. In some
instances, the one or more genes whose expression is modulated,
e.g., enhanced, is a gene encoding a product that is a member of a
plasticity related signaling pathway (i.e., a synaptic plasticity
regulation gene), e.g., Tlr4, Gria1, Kcnj10, Kdr, Ncam, Sdfr1,
Egr1, Fos proteins, e.g., c-Fos, Drd1a, Stxbp1, Mef2c, Cntn2, Junb,
Bdnf and CamK2a, etc. In some instances, the modulation of
hippocampal gene expression is manifested as enhanced hippocampal
plasticity, e.g., as compared to a suitable control. In some
instances, the one or more genes whose expression is modulated,
e.g., enhanced, is a gene encoding a product that is a member of
network related to synaptic plasticity and learning and memory,
such as but not limited to: RELN, NTRK3, EPHA4, etc.
[0087] In some instances, treatment results in an enhancement in
the levels of one or more proteins in one or more tissues of the
host, e.g., as compared to a suitable control (such as described in
the Experimental section, below). The change in protein level of a
given protein may be 0.5 fold or greater, such as 1.0 fold or
greater, including 1.5 fold or greater, where in some instances the
level may approach that of a healthy wild-type level, e.g., within
50% or less, such as 25% or less, including 10% or less, e.g., 5%
or less of the healthy wild-type level. The tissue may vary, and in
some instances is nervous system tissue, e.g., central nervous
system tissue, including brain tissue, e.g., hippocampal
tissue.
[0088] In some instances, the methods result in one or more
structural changes in one or more tissues. The tissue may vary, and
in some instances is nervous system tissue, e.g., central nervous
system tissue, including brain tissue, e.g., hippocampal tissue.
Structure changes of interest include an increase in dendritic
spine density of mature neurons in the dentate gyrus (DG) of the
hippocampus, e.g., as compared to a suitable control. In some
instances, the modulation of hippocampal structure is manifested as
enhanced synapse formation or function, e.g., as compared to a
suitable control. In some instances, the methods may result in an
enhancement of long term potentiation, e.g., as compared to a
suitable control.
[0089] In some instances, practice of the methods, e.g., as
described above, results in an increase in neurogenesis in the
adult mammal. The increase may be identified in a number of
different ways, e.g., as described below in the Experimental
section. In some instances, the increase in neurogenesis manifests
as an increase the amount of Dcx-positive immature neurons, e.g.,
where the increase may be 1.5-fold or greater. In some instances,
the increase in neurogenesis manifests as an increase in the number
of BrdU/NeuN positive cells, where the increase may be 1.5-fold or
greater.
[0090] In some instances, the methods result in enhancement in
learning and memory, e.g., as compared to a suitable control.
Enhancement in learning and memory may be evaluated in a number of
different ways, e.g., the contextual fear conditioning, Barnes
maze, and/or radial arm water maze (RAWM) paradigms described in
the experimental section, below. When measured by contextual fear
conditioning, treatment results in some instances in increased
freezing in contextual, but not cued, memory testing. When measured
by Barnes maze, treatment results in some instances in enhanced
learning and memory for escape hole location during the testing
phase of the task on any day of the task. When measured by RAWM,
treatment results in some instances in enhanced learning and memory
for platform location during the testing phase of the task. In some
instances, treatment is manifested as enhanced cognitive
improvement in hippocampal-dependent learning and memory, e.g., as
compared to a suitable control.
[0091] In some embodiments, the methods may be performed in
conjunction with an active agent having activity suitable to treat
aging-associated cognitive impairment. For example, a number of
active agents have been shown to have some efficacy in treating the
cognitive symptoms of Alzheimer's disease (e.g., memory loss,
confusion, and problems with thinking and reasoning), e.g.,
cholinesterase inhibitors (e.g., Donepezil, Rivastigmine,
Galantamine, Tacrine), Memantine, and Vitamin E. As another
example, a number of agents have been shown to have some efficacy
in treating behavioral or psychiatric symptoms of Alzheimer's
Disease, e.g., citalopram (Celexa), fluoxetine (Prozac), paroxeine
(Paxil), sertraline (Zoloft), trazodone (Desyrel), lorazepam
(Ativan), oxazepam (Serax), aripiprazole (Abilify), clozapine
(Clozaril), haloperidol (Haldol), olanzapine (Zyprexa), quetiapine
(Seroquel), risperidone (Risperdal), and ziprasidone (Geodon).
[0092] In some instances, the methods are practiced in conjunction
with one or more additional non-TIMP polypeptides active agents,
where such non-TIMP polypeptide active agent exhibit a desirable
anti-aging associated condition activity, e.g., as described above.
Examples of such non-TIMP polypeptide active agents include, but
are not limited to: chemokine (C--C motif) ligand 2 (CCL2) (i.e.,
MCP1) and C--C motif chemokine 11 (i.e., chemotactic protein or
eotaxin-1) and agonists/mimetics thereof (e.g., as described in
published application no. 20130040844, the disclosure of which is
herein incorporated by reference0; Granulocyte-macrophage
colony-stimulating factor (GM-CSF)(i.e., colony stimulating factor
2 or CSF2); etc. In such instances, the active agent may be any
type of convenient active agent, including those types of agents
discussed above in connection with TIMP active agents, e.g.,
polypeptides and mimetics/fragments thereof, small molecules,
nucleic acids, potentiators, etc.
[0093] In some aspects of the subject methods, the method further
comprises the step of measuring cognition and/or synaptic
plasticity after treatment, e.g., using the methods described
herein or known in the art, and determining that the rate of
cognitive decline or loss of synaptic plasticity have been reduced
and/or that cognitive ability or synaptic plasticity have improved
in the individual. In some such instances, the determination is
made by comparing the results of the cognition or synaptic
plasticity test to the results of the test performed on the same
individual at an earlier time, e.g., 2 weeks earlier, 1 month
earlier, 2 months earlier, 3 months earlier, 6 months earlier, 1
year earlier, 2 years earlier, 5 years earlier, or 10 years
earlier, or more.
[0094] In some embodiments, the subject methods further include
diagnosing an individual as having a cognitive impairment, e.g.,
using the methods described herein or known in the art for
measuring cognition and synaptic plasticity, prior to administering
the subject plasma-comprising blood product. In some instances, the
diagnosing will comprise measuring cognition and/or synaptic
plasticity and comparing the results of the cognition or synaptic
plasticity test to one or more references, e.g., a positive control
and/or a negative control. For example, the reference may be the
result(s) of the test performed by one or more age-matched
individuals that experience aging-associated cognitive impairments
(i.e., positive controls) or that do not experience
aging-associated cognitive impairments (i.e., negative controls).
As another example, the reference may be the result(s) of the test
performed by the same individual at an earlier time, e.g., 2 weeks
earlier, 1 month earlier, 2 months earlier, 3 months earlier, 6
months earlier, 1 year earlier, 2 years earlier, 5 years earlier,
or 10 years earlier, or more.
[0095] In some embodiments, the subject methods further include
diagnosing an individual as having an aging-associated disorder,
e.g., Alzheimer's disease, Parkinson's disease, frontotemporal
dementia, progressive supranuclear palsy, Huntington's disease,
amyotrophic lateral sclerosis, spinal muscular atrophy, multiple
sclerosis, multi-system atrophy, glaucoma, ataxias, muscular
dystrophy, dementia, and the like. Methods for diagnosing such
aging-associated disorders are well-known in the art, any of which
may be used by the ordinarily skilled artisan in diagnosing the
individual. In some embodiments, the subject methods further
comprise both diagnosing an individual as having an
aging-associated disorder and as having a cognitive impairment.
Utility
[0096] The subject methods find use in treating, including
preventing, aging-associated impairments and conditions associated
therewith, such as impairments in the cognitive ability of
individuals. Individuals suffering from or at risk of developing an
aging-associated cognitive impairments include individuals that are
about 50 years old or older, e.g., 60 years old or older, 70 years
old or older, 80 years old or older, 90 years old or older, and
usually no older than 100 years old, i.e., between the ages of
about 50 and 100, e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or
about 100 years old, and are suffering from cognitive impairment
associated with natural aging process, e.g., mild cognitive
impairment (M.C.I.); and individuals that are about 50 years old or
older, e.g., 60 years old or older, 70 years old or older, 80 years
old or older, 90 years old or older, and usually no older than 100
years old, i.e., between the ages of about 50 and 90, e.g., 50, 55,
60, 65, 70, 75, 80, 85, 90, 95 or about 100 years old, that have
not yet begun to show symptoms of cognitive impairment. Examples of
cognitive impairments that are due to natural aging include the
following:
[0097] Mild cognitive impairment (M.C.I.) is a modest disruption of
cognition that manifests as problems with memory or other mental
functions such as planning, following instructions, or making
decisions that have worsened over time while overall mental
function and daily activities are not impaired. Thus, although
significant neuronal death does not typically occur, neurons in the
aging brain are vulnerable to sub-lethal age-related alterations in
structure, synaptic integrity, and molecular processing at the
synapse, all of which impair cognitive function.
[0098] Individuals suffering from or at risk of developing an
aging-associated cognitive impairment that will benefit from
treatment with the subject plasma-comprising blood product, e.g.,
by the methods disclosed herein, also include individuals of any
age that are suffering from a cognitive impairment due to an
aging-associated disorder; and individuals of any age that have
been diagnosed with an aging-associated disorder that is typically
accompanied by cognitive impairment, where the individual has not
yet begun to present with symptoms of cognitive impairment.
Examples of such aging-associated disorders include the
following:
[0099] Alzheimer's disease (AD). Alzheimer's disease is a
progressive, inexorable loss of cognitive function associated with
an excessive number of senile plaques in the cerebral cortex and
subcortical gray matter, which also contains .beta.-amyloid and
neurofibrillary tangles consisting of tau protein. The common form
affects persons >60 yr old, and its incidence increases as age
advances. It accounts for more than 65% of the dementias in the
elderly.
[0100] The cause of Alzheimer's disease is not known. The disease
runs in families in about 15 to 20% of cases. The remaining,
so-called sporadic cases have some genetic determinants. The
disease has an autosomal dominant genetic pattern in most
early-onset and some late-onset cases but a variable late-life
penetrance. Environmental factors are the focus of active
investigation.
[0101] In the course of the disease, synapses, and ultimately
neurons are lost within the cerebral cortex, hippocampus, and
subcortical structures (including selective cell loss in the
nucleus basalis of Meynert), locus caeruleus, and nucleus raphae
dorsalis. Cerebral glucose use and perfusion is reduced in some
areas of the brain (parietal lobe and temporal cortices in
early-stage disease, prefrontal cortex in late-stage disease).
Neuritic or senile plaques (composed of neurites, astrocytes, and
glial cells around an amyloid core) and neurofibrillary tangles
(composed of paired helical filaments) play a role in the
pathogenesis of Alzheimer's disease. Senile plaques and
neurofibrillary tangles occur with normal aging, but they are much
more prevalent in persons with Alzheimer's disease.
[0102] Parkinson's Disease.
[0103] Parkinson's Disease (PD) is an idiopathic, slowly
progressive, degenerative CNS disorder characterized by slow and
decreased movement, muscular rigidity, resting tremor, and postural
instability. Originally considered primarily a motor disorder, PD
is now recognized to also affect cognition, behavior, sleep,
autonomic function, and sensory function. The most common cognitive
impairments include an impairment in attention and concentration,
working memory, executive function, producing language, and
visuospatial function.
[0104] In primary Parkinson's disease, the pigmented neurons of the
substantia nigra, locus caeruleus, and other brain stem
dopaminergic cell groups are lost. The cause is not known. The loss
of substantia nigra neurons, which project to the caudate nucleus
and putamen, results in depletion of the neurotransmitter dopamine
in these areas. Onset is generally after age 40, with increasing
incidence in older age groups.
[0105] Secondary parkinsonism results from loss of or interference
with the action of dopamine in the basal ganglia due to other
idiopathic degenerative diseases, drugs, or exogenous toxins. The
most common cause of secondary parkinsonism is ingestion of
antipsychotic drugs or reserpine, which produce parkinsonism by
blocking dopamine receptors. Less common causes include carbon
monoxide or manganese poisoning, hydrocephalus, structural lesions
(tumors, infarcts affecting the midbrain or basal ganglia),
subdural hematoma, and degenerative disorders, including
striatonigral degeneration.
[0106] Frontotemporal Dementia.
[0107] Frontotemporal dementia (FTD) is a condition resulting from
the progressive deterioration of the frontal lobe of the brain.
Over time, the degeneration may advance to the temporal lobe.
Second only to Alzheimer's disease (AD) in prevalence, FTD accounts
for 20% of pre-senile dementia cases. Symptoms are classified into
three groups based on the functions of the frontal and temporal
lobes affected: Behavioural variant FTD (bvFTD), with symptoms
include lethargy and aspontaneity on the one hand, and
disinhibition on the other; progressive nonfluent aphasia (PNFA),
in which a breakdown in speech fluency due to articulation
difficulty, phonological and/or syntactic errors is observed but
word comprehension is preserved; and semantic dementia (SD), in
which patients remain fluent with normal phonology and syntax but
have increasing difficulty with naming and word comprehension.
Other cognitive symptoms common to all FTD patients include an
impairment in executive function and ability to focus. Other
cognitive abilities, including perception, spatial skills, memory
and praxis typically remain intact. FTD can be diagnosed by
observation of reveal frontal lobe and/or anterior temporal lobe
atrophy in structural MRI scans.
[0108] A number of forms of FTD exist, any of which may be treated
or prevented using the subject methods and compositions. For
example, one form of frontotemporal dementia is Semantic Dementia
(SD). SD is characterized by a loss of semantic memory in both the
verbal and non-verbal domains. SD patients often present with the
complaint of word-finding difficulties. Clinical signs include
fluent aphasia, anomia, impaired comprehension of word meaning, and
associative visual agnosia (the inability to match semantically
related pictures or objects). As the disease progresses, behavioral
and personality changes are often seen similar to those seen in
frontotemporal dementia although cases have been described of
`pure` semantic dementia with few late behavioral symptoms.
Structural MRI imaging shows a characteristic pattern of atrophy in
the temporal lobes (predominantly on the left), with inferior
greater than superior involvement and anterior temporal lobe
atrophy greater than posterior.
[0109] As another example, another form of frontotemporal dementia
is Pick's disease (PiD, also PcD). A defining characteristic of the
disease is build-up of tau proteins in neurons, accumulating into
silver-staining, spherical aggregations known as "Pick bodies".
Symptoms include loss of speech (aphasia) and dementia. Patients
with orbitofrontal dysfunction can become aggressive and socially
inappropriate. They may steal or demonstrate obsessive or
repetitive stereotyped behaviors. Patients with dorsomedial or
dorsolateral frontal dysfunction may demonstrate a lack of concern,
apathy, or decreased spontaneity. Patients can demonstrate an
absence of self-monitoring, abnormal self-awareness, and an
inability to appreciate meaning. Patients with gray matter loss in
the bilateral posterolateral orbitofrontal cortex and right
anterior insula may demonstrate changes in eating behaviors, such
as a pathologic sweet tooth. Patients with more focal gray matter
loss in the anterolateral orbitofrontal cortex may develop
hyperphagia. While some of the symptoms can initially be
alleviated, the disease progresses and patients often die within
two to ten years.
[0110] Huntington's Disease.
[0111] Huntington's disease (HD) is a hereditary progressive
neurodegenerative disorder characterized by the development of
emotional, behavioral, and psychiatric abnormalities; loss of
intellectual or cognitive functioning; and movement abnormalities
(motor disturbances). The classic signs of HD include the
development of chorea--involuntary, rapid, irregular, jerky
movements that may affect the face, arms, legs, or trunk--as well
as cognitive decline including the gradual loss of thought
processing and acquired intellectual abilities. There may be
impairment of memory, abstract thinking, and judgment; improper
perceptions of time, place, or identity (disorientation); increased
agitation; and personality changes (personality disintegration).
Although symptoms typically become evident during the fourth or
fifth decades of life, the age at onset is variable and ranges from
early childhood to late adulthood (e.g., 70 s or 80 s).
[0112] HD is transmitted within families as an autosomal dominant
trait. The disorder occurs as the result of abnormally long
sequences or "repeats" of coded instructions within a gene on
chromosome 4 (4p16.3). The progressive loss of nervous system
function associated with HD results from loss of neurons in certain
areas of the brain, including the basal ganglia and cerebral
cortex.
[0113] Amyotrophic Lateral Sclerosis.
[0114] Amyotrophic lateral sclerosis (ALS) is a rapidly
progressive, invariably fatal neurological disease that attacks
motor neurons. Muscular weakness and atrophy and signs of anterior
horn cell dysfunction are initially noted most often in the hands
and less often in the feet. The site of onset is random, and
progression is asymmetric. Cramps are common and may precede
weakness. Rarely, a patient survives 30 years; 50% die within 3
years of onset, 20% live 5 years, and 10% live 10 years. Diagnostic
features include onset during middle or late adult life and
progressive, generalized motor involvement without sensory
abnormalities. Nerve conduction velocities are normal until late in
the disease. Recent studies have documented the presentation of
cognitive impairments as well, particularly a reduction in
immediate verbal memory, visual memory, language, and executive
function.
[0115] A decrease in cell body area, number of synapses and total
synaptic length has been reported in even normal-appearing neurons
of the ALS patients. It has been suggested that when the plasticity
of the active zone reaches its limit, a continuing loss of synapses
can lead to functional impairment. Promoting the formation or new
synapses or preventing synapse loss may maintain neuron function in
these patients.
[0116] Multiple Sclerosis.
[0117] Multiple Sclerosis (MS) is characterized by various symptoms
and signs of CNS dysfunction, with remissions and recurring
exacerbations. The most common presenting symptoms are paresthesias
in one or more extremities, in the trunk, or on one side of the
face; weakness or clumsiness of a leg or hand; or visual
disturbances, e.g., partial blindness and pain in one eye
(retrobulbar optic neuritis), dimness of vision, or scotomas.
Common cognitive impairments include impairments in memory
(acquiring, retaining, and retrieving new information), attention
and concentration (particularly divided attention), information
processing, executive functions, visuospatial functions, and verbal
fluency. Common early symptoms are ocular palsy resulting in double
vision (diplopia), transient weakness of one or more extremities,
slight stiffness or unusual fatigability of a limb, minor gait
disturbances, difficulty with bladder control, vertigo, and mild
emotional disturbances; all indicate scattered CNS involvement and
often occur months or years before the disease is recognized.
Excess heat may accentuate symptoms and signs.
[0118] The course is highly varied, unpredictable, and, in most
patients, remittent. At first, months or years of remission may
separate episodes, especially when the disease begins with
retrobulbar optic neuritis. However, some patients have frequent
attacks and are rapidly incapacitated; for a few the course can be
rapidly progressive.
[0119] Glaucoma.
[0120] Glaucoma is a common neurodegenerative disease that affects
retinal ganglion cells (RGCs). Evidence supports the existence of
compartmentalized degeneration programs in synapses and dendrites,
including in RGCs. Recent evidence also indicates a correlation
between cognitive impairment in older adults and glaucoma (Yochim B
P, et al. Prevalence of cognitive impairment, depression, and
anxiety symptoms among older adults with glaucoma. J Glaucoma.
2012; 21(4):250-254).
[0121] Myotonic Dystrophy.
[0122] Myotonic dystrophy (DM) is an autosomal dominant multisystem
disorder characterized by dystrophic muscle weakness and myotonia.
The molecular defect is an expanded trinucleotide (CTG) repeat in
the 3' untranslated region of the myotonin-protein kinase gene on
chromosome 19q. Symptoms can occur at any age, and the range of
clinical severity is broad. Myotonia is prominent in the hand
muscles, and ptosis is common even in mild cases. In severe cases,
marked peripheral muscular weakness occurs, often with cataracts,
premature balding, hatchet facies, cardiac arrhythmias, testicular
atrophy, and endocrine abnormalities (e.g., diabetes mellitus).
Mental retardation is common in severe congenital forms, while an
aging-related decline of frontal and temporal cognitive functions,
particularly language and executive functions, is observed in
milder adult forms of the disorder. Severely affected persons die
by their early 50 s.
[0123] Dementia.
[0124] Dementia describes class of disorders having symptoms
affecting thinking and social abilities severely enough to
interfere with daily functioning. Other instances of dementia in
addition to the dementia observed in later stages of the
aging-associated disorders discussed above include vascular
dementia, and dementia with Lewy bodies, described below.
[0125] In vascular dementia, or "multi-infarct dementia", cognitive
impairment is caused by problems in supply of blood to the brain,
typically by a series of minor strokes, or sometimes, one large
stroke preceded or followed by other smaller strokes. Vascular
lesions can be the result of diffuse cerebrovascular disease, such
as small vessel disease, or focal lesions, or both. Patients
suffering from vascular dementia present with cognitive impairment,
acutely or subacutely, after an acute cerebrovascular event, after
which progressive cognitive decline is observed. Cognitive
impairments are similar to those observed in Alzheimer's disease,
including impairments in language, memory, complex visual
processing, or executive function, although the related changes in
the brain are not due to AD pathology but to chronic reduced blood
flow in the brain, eventually resulting in dementia. Single photon
emission computed tomography (SPECT) and positron emission
tomography (PET) neuroimaging may be used to confirm a diagnosis of
multi-infarct dementia in conjunction with evaluations involving
mental status examination.
[0126] Dementia with Lewy bodies (DLB, also known under a variety
of other names including Lewy body dementia, diffuse Lewy body
disease, cortical Lewy body disease, and senile dementia of Lewy
type) is a type of dementia characterized anatomically by the
presence of Lewy bodies (clumps of alpha-synuclein and ubiquitin
protein) in neurons, detectable in post mortem brain histology. Its
primary feature is cognitive decline, particularly of executive
functioning. Alertness and short term memory will rise and fall.
Persistent or recurring visual hallucinations with vivid and
detailed pictures are often an early diagnostic symptom. DLB it is
often confused in its early stages with Alzheimer's disease and/or
vascular dementia, although, where Alzheimer's disease usually
begins quite gradually, DLB often has a rapid or acute onset. DLB
symptoms also include motor symptoms similar to those of
Parkinson's. DLB is distinguished from the dementia that sometimes
occurs in Parkinson's disease by the time frame in which dementia
symptoms appear relative to Parkinson symptoms. Parkinson's disease
with dementia (PDD) would be the diagnosis when dementia onset is
more than a year after the onset of Parkinson's. DLB is diagnosed
when cognitive symptoms begin at the same time or within a year of
Parkinson symptoms.
[0127] Progressive Supranuclear Palsy.
[0128] Progressive supranuclear palsy (PSP) is a brain disorder
that causes serious and progressive problems with control of gait
and balance, along with complex eye movement and thinking problems.
One of the classic signs of the disease is an inability to aim the
eyes properly, which occurs because of lesions in the area of the
brain that coordinates eye movements. Some individuals describe
this effect as a blurring. Affected individuals often show
alterations of mood and behavior, including depression and apathy
as well as progressive mild dementia. The disorders long name
indicates that the disease begins slowly and continues to get worse
(progressive), and causes weakness (palsy) by damaging certain
parts of the brain above pea-sized structures called nuclei that
control eye movements (supranuclear). PSP was first described as a
distinct disorder in 1964, when three scientists published a paper
that distinguished the condition from Parkinson's disease. It is
sometimes referred to as Steele-Richardson-Olszewski syndrome,
reflecting the combined names of the scientists who defined the
disorder. Although PSP gets progressively worse, no one dies from
PSP itself.
[0129] Ataxia.
[0130] People with ataxia have problems with coordination because
parts of the nervous system that control movement and balance are
affected. Ataxia may affect the fingers, hands, arms, legs, body,
speech, and eye movements. The word ataxia is often used to
describe a symptom of incoordination which can be associated with
infections, injuries, other diseases, or degenerative changes in
the central nervous system. Ataxia is also used to denote a group
of specific degenerative diseases of the nervous system called the
hereditary and sporadic ataxias which are the National Ataxia
Foundation's primary emphases.
[0131] Multiple-System Atrophy.
[0132] Multiple-system atrophy (MSA) is a degenerative neurological
disorder. MSA is associated with the degeneration of nerve cells in
specific areas of the brain. This cell degeneration causes problems
with movement, balance, and other autonomic functions of the body
such as bladder control or blood-pressure regulation. The cause of
MSA is unknown and no specific risk factors have been identified.
Around 55% of cases occur in men, with typical age of onset in the
late 50 s to early 60 s. MSA often presents with some of the same
symptoms as Parkinson's disease. However, MSA patients generally
show minimal if any response to the dopamine medications used for
Parkinson's.
[0133] In some embodiments, the subject methods and compositions
find use in slowing the progression of aging-associated cognitive
impairment. In other words, cognitive abilities in the individual
will decline more slowly following treatment by the disclosed
methods than prior to or in the absence of treatment by the
disclosed methods. In some such instances, the subject methods of
treatment include measuring the progression of cognitive decline
after treatment, and determining that the progression of cognitive
decline is reduced. In some such instances, the determination is
made by comparing to a reference, e.g., the rate of cognitive
decline in the individual prior to treatment, e.g., as determined
by measuring cognition prior at two or more time points prior to
administration of the subject blood product.
[0134] The subject methods and compositions also find use in
stabilizing the cognitive abilities of an individual, e.g., an
individual suffering from aging-associated cognitive decline or an
individual at risk of suffering from aging-associated cognitive
decline. For example, the individual may demonstrate some
aging-associated cognitive impairment, and progression of cognitive
impairment observed prior to treatment with the disclosed methods
will be halted following treatment by the disclosed methods. As
another example, the individual may be at risk for developing an
aging-associated cognitive decline (e.g., the individual may be
aged 50 years old or older, or may have been diagnosed with an
aging-associated disorder), and the cognitive abilities of the
individual are substantially unchanged, i.e., no cognitive decline
can be detected, following treatment by the disclosed methods as
compared to prior to treatment with the disclosed methods.
[0135] The subject methods and compositions also find use in
reducing cognitive impairment in an individual suffering from an
aging-associated cognitive impairment. In other words, cognitive
ability is improved in the individual following treatment by the
subject methods. For example, the cognitive ability in the
individual is increased, e.g., by 2-fold or more, 5-fold or more,
10-fold or more, 15-fold or more, 20-fold or more, 30-fold or more,
or 40-fold or more, including 50-fold or more, 60-fold or more,
70-fold or more, 80-fold or more, 90-fold or more, or 100-fold or
more, following treatment by the subject methods relative to the
cognitive ability that is observed in the individual prior to
treatment by the subject methods. In some instances, treatment by
the subject methods and compositions restores the cognitive ability
in the individual suffering from aging-associated cognitive
decline, e.g., to their level when the individual was about 40
years old or less. In other words, cognitive impairment is
abrogated.
Combination Therapies
[0136] Active agents of the invention can be administered to a
subject alone or in combination with an additional, i.e., second,
active agent. As such, in some cases, the subject method further
comprises administering to the subject at least one additional
compound. Any convenient agents may be utilized. For example, TIMP
active agents can be supplied alone or in conjunction with one or
more other drugs, such as drugs employed in the treatment of aging
associated conditions, e.g., cholinesterase inhibitors (e.g.,
Donepezil, Rivastigmine, Galantamine, Tacrine), Memantine, Vitamin
E, citalopram (Celexa), fluoxetine (Prozac), paroxeine (Paxil),
sertraline (Zoloft), trazodone (Desyrel), lorazepam (Ativan),
oxazepam (Serax), aripiprazole (Abilify), clozapine (Clozaril),
haloperidol (Haldol), olanzapine (Zyprexa), quetiapine (Seroquel),
risperidone (Risperdal), and ziprasidone (Geodon); non-TIMP
polypeptide active agents; e.g., chemokine (C--C motif) ligand 2
(CCL2) (i.e., MCP1); C--C motif chemokine 11 (i.e., chemotactic
protein or eotaxin-1); Granulocyte-macrophage colony-stimulating
factor (GM-CSF)(i.e., colony stimulating factor 2 or CSF2);
etc.
[0137] The terms "co-administration" and "in combination with"
include the administration of two or more therapeutic agents either
simultaneously, concurrently or sequentially within no specific
time limits. In one embodiment, the agents are present in the cell
or in the subject's body at the same time or exert their biological
or therapeutic effect at the same time. In one embodiment, the
therapeutic agents are in the same composition or unit dosage form.
In other embodiments, the therapeutic agents are in separate
compositions or unit dosage forms. In certain embodiments, a first
agent can be administered prior to (e.g., minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes,
30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12
hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3
weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second therapeutic agent.
[0138] "Concomitant administration" of a known therapeutic drug
with a pharmaceutical composition of the present invention means
administration of the drug and nucleoside agent at such time that
both the known drug and the composition of the present invention
will have a therapeutic effect. Such concomitant administration may
involve concurrent (i.e. at the same time), prior, or subsequent
administration of the drug with respect to the administration of a
subject nucleoside agent. Routes of administration of the two
agents may vary, where representative routes of administration are
described in greater detail below. A person of ordinary skill in
the art would have no difficulty determining the appropriate
timing, sequence and dosages of administration for particular drugs
and nucleoside agents of the present invention.
[0139] In some embodiments, the compounds are administered to the
subject within twenty-four hours of each other, such as within 12
hours of each other, within 6 hours of each other, within 3 hours
of each other, or within 1 hour of each other. In certain
embodiments, the compounds are administered within 1 hour of each
other. In certain embodiments, the compounds are administered
substantially simultaneously. By administered substantially
simultaneously is meant that the compounds are administered to the
subject within about 10 minutes or less of each other, such as 5
minutes or less, or 1 minute or less of each other.
Pharmaceutical Preparations
[0140] Also provided are pharmaceutical preparations of the subject
compounds. The subject compounds can be incorporated into a variety
of formulations for administration to a subject. More particularly,
the compounds of the present invention can be formulated into
pharmaceutical compositions by combination with appropriate,
pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or
gaseous forms, such as tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols. The formulations may be designed for administration via a
number of different routes, including oral, buccal, rectal,
parenteral, intraperitoneal, intradermal, transdermal, intracheal,
etc., administration.
[0141] In pharmaceutical dosage forms, the compounds may be
administered in the form of their pharmaceutically acceptable
salts, or they may also be used alone or in appropriate
association, as well as in combination, with other pharmaceutically
active compounds. The following methods and excipients are merely
exemplary and are in no way limiting.
[0142] The pharmaceutical compositions containing the active
ingredient may be in a form suitable for oral use, for example, as
tablets, troches, lozenges, aqueous or oily suspensions,
dispersible powders or granules, emulsions, hard or soft capsules,
or syrups or elixirs. Compositions intended for oral use may be
prepared according to any method known to the art for the
manufacture of pharmaceutical compositions and such compositions
may contain one or more agents selected from the group consisting
of sweetening agents, flavoring agents, coloring agents and
preserving agents in order to provide pharmaceutically elegant and
palatable preparations. Tablets contain the active ingredient in
admixture with non-toxic pharmaceutically acceptable excipients
which are suitable for the manufacture of tablets. These excipients
may be for example, inert diluents, such as calcium carbonate,
sodium carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example, magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monostearate or glyceryl distearate may be
employed. They may also be coated by the technique described in the
U.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic
therapeutic tablets for control release.
[0143] Formulations for oral use may also be presented as hard
gelatin capsules wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredients is mixed with water or an oil medium, for example
peanut oil, liquid paraffin, or olive oil.
[0144] Aqueous suspensions contain the active material in admixture
with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethyl-cellulose, methylcellulose,
hydroxy-propylmethycellulose, sodium alginate,
polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or
wetting agents may be a naturally-occurring phosphatide, for
example lecithin, or condensation products of an alkylene oxide
with fatty acids, for example polyoxyethylene stearate, or
condensation products of ethylene oxide with long chain aliphatic
alcohols, for example heptadecaethylene-oxycetanol, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived
from fatty acids and hexitol anhydrides, for example polyethylene
sorbitan monooleate. The aqueous suspensions may also contain one
or more preservatives, for example ethyl, or n-propyl,
p-hydroxybenzoate, one or more coloring agents, one or more
flavoring agents, and one or more sweetening agents, such as
sucrose, saccharin or aspartame.
[0145] Oily suspensions may be formulated by suspending the active
ingredient in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as those set forth above, and flavoring agents may be added to
provide a palatable oral preparation. These compositions may be
preserved by the addition of an anti-oxidant such as ascorbic
acid.
[0146] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents and suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0147] The pharmaceutical compositions of the invention may also be
in the form of an oil-in-water emulsions. The oily phase may be a
vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for example liquid paraffin or mixtures of these. Suitable
emulsifying agents may be naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0148] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol or sucrose. Such
formulations may also contain a demulcent, a preservative and
flavoring and coloring agents. The pharmaceutical compositions may
be in the form of a sterile injectable aqueous or oleagenous
suspension. This suspension may be formulated according to the
known art using those suitable dispersing or wetting agents and
suspending agents which have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parenterally-acceptable diluent or
solvent, for example as a solution in 1,3-butane diol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid find use in the
preparation of injectables.
[0149] The compounds can be formulated into preparations for
injection by dissolving, suspending or emulsifying them in an
aqueous or nonaqueous solvent, such as vegetable or other similar
oils, synthetic aliphatic acid glycerides, esters of higher
aliphatic acids or propylene glycol; and if desired, with
conventional additives such as solubilizers, isotonic agents,
suspending agents, emulsifying agents, stabilizers and
preservatives.
[0150] The compounds can be utilized in aerosol formulation to be
administered via inhalation. The compounds of the present invention
can be formulated into pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen and the like.
[0151] Furthermore, the compounds can be made into suppositories by
mixing with a variety of bases such as emulsifying bases or
water-soluble bases. The compounds of the present invention can be
administered rectally via a suppository. The suppository can
include vehicles such as cocoa butter, carbowaxes and polyethylene
glycols, which melt at body temperature, yet are solidified at room
temperature.
[0152] The compounds of this invention and their pharmaceutically
acceptable salts which are active on topical administration can be
formulated as transdermal compositions or transdermal delivery
devices ("patches"). Such compositions include, for example, a
backing, active compound reservoir, a control membrane, liner and
contact adhesive. Such transdermal patches may be used to provide
continuous or discontinuous infusion of the compounds of the
present invention in controlled amounts. The construction and use
of transdermal patches for the delivery of pharmaceutical agents is
well known in the art. See, e.g., U.S. Pat. No. 5,023,252, issued
Jun. 11, 1991, herein incorporated by reference in its entirety.
Such patches may be constructed for continuous, pulsatile, or on
demand delivery of pharmaceutical agents.
[0153] Optionally, the pharmaceutical composition may contain other
pharmaceutically acceptable components, such a buffers,
surfactants, antioxidants, viscosity modifying agents,
preservatives and the like. Each of these components is well-known
in the art. See, for example, U.S. Pat. No. 5,985,310, the
disclosure of which is herein incorporated by reference.
[0154] Other components suitable for use in the formulations of the
present invention can be found in Remington's Pharmaceutical
Sciences, Mace Publishing Company, Philadelphia, Pa., 17th ed.
(1985). In an embodiment, the aqueous cyclodextrin solution further
comprise dextrose, e.g., about 5% dextrose.
[0155] Dosage levels of the order of from about 0.01 mg to about
140 mg/kg of body weight per day are useful in representative
embodiments, or alternatively about 0.5 mg to about 7 g per patient
per day. For example, inflammation may be effectively treated by
the administration of from about 0.01 to 50 mg of the compound per
kilogram of body weight per day, or alternatively about 0.5 mg to
about 3.5 g per patient per day. Those of skill will readily
appreciate that dose levels can vary as a function of the specific
compound, the severity of the symptoms and the susceptibility of
the subject to side effects. Dosages for a given compound are
readily determinable by those of skill in the art by a variety of
means.
[0156] The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a formulation intended for the oral
administration of humans may contain from 0.5 mg to 5 g of active
agent compounded with an appropriate and convenient amount of
carrier material which may vary from about 5 to about 95 percent of
the total composition. Dosage unit forms will generally contain
between from about 1 mg to about 500 mg of an active ingredient,
typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600
mg, 800 mg, or 1000 mg.
[0157] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination and the severity of the particular disease undergoing
therapy.
[0158] As such, unit dosage forms for oral or rectal administration
such as syrups, elixirs, and suspensions may be provided wherein
each dosage unit, for example, teaspoonful, tablespoonful, tablet
or suppository, contains a predetermined amount of the composition
containing one or more inhibitors. Similarly, unit dosage forms for
injection or intravenous administration may comprise the
inhibitor(s) in a composition as a solution in sterile water,
normal saline or another pharmaceutically acceptable carrier. The
term "unit dosage form," as used herein, refers to physically
discrete units suitable as unitary dosages for human and animal
subjects, each unit containing a predetermined quantity of
compounds of the present invention calculated in an amount
sufficient to produce the desired effect in association with a
pharmaceutically acceptable diluent, carrier or vehicle. The
specifications for the novel unit dosage forms of the present
invention depend on the particular peptidomimetic compound employed
and the effect to be achieved, and the pharmacodynamics associated
with each compound in the host.
Kits & Systems
[0159] Also provided are kits and systems that find use in
practicing embodiments of the methods, such as those described as
described above. The term "system" as employed herein refers to a
collection of two or more different active agents, present in a
single composition or in disparate compositions, that are brought
together for the purpose of practicing the subject methods. The
term "kit" refers to a packaged active agent or agents. For
example, kits and systems for practicing the subject methods may
include one or more pharmaceutical formulations. As such, in
certain embodiments the kits may include a single pharmaceutical
composition, present as one or more unit dosages, where the
composition may include one or more expression/activity inhibitor
compounds. In yet other embodiments, the kits may include two or
more separate pharmaceutical compositions, each containing a
different active compound.
[0160] Also of interest are kits and systems finding use in assays
of the invention, e.g., as described above. Such kits and systems
may include one or more components of the assays, e.g., vectors
encoding fusion proteins, enzyme substrates, buffers, etc.
[0161] In addition to the above components, the subject kits may
further include instructions for practicing the subject methods.
These instructions may be present in the subject kits in a variety
of forms, one or more of which may be present in the kit. One form
in which these instructions may be present is as printed
information on a suitable medium or substrate, e.g., a piece or
pieces of paper on which the information is printed, in the
packaging of the kit, in a package insert, etc. Yet another means
would be a computer readable medium, e.g., diskette, CD, portable
flash drive, etc., on which the information has been recorded. Yet
another means that may be present is a website address which may be
used via the internet to access the information at a removed site.
Any convenient means may be present in the kits.
[0162] The following examples are provided by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0163] Recombinant TIMP2 protein was delivered at a concentration
of 50 .mu.g/kg four times over the course of a week by
intraperitoneal injection in aged wildtype (C57Bl/6J) mice. Brains
of treated mice revealed elevated levels of active neurons, namely
those expressing the immediate early gene c-Fos in the dentate
gyrus subregion of the hippocampus. Eight (long-term)
intraperitoneal injections (50 .mu.g/kg) of TIMP2 were given to
aged wildtype mice every other day prior to Barnes maze, nesting,
and fear conditioning assessment. Behavioral testing revealed
significantly improved performance in all three tasks in
TIMP2-treated mice. Seven (long-term) intraperitoneal injections
(50 .mu.g/kg) of TIMP2 were given to aged wildtype mice every other
day alone or in combination with another cognition-promoting
factor, CSF2. Brains of TIMP2 and TIMP2+CSF2 mice revealed
significantly elevated levels of c-Fos+ (active) neurons in the
dentate gyrus.
[0164] The results demonstrated that TIMP2 was sufficient to
elevate the number of c-Fos-expressing cells in the hippocampus of
aged mice. When administered systemically over a longer timecourse,
TIMP2 is sufficient to increase the number of c-Fos-expressing
cells, reverse learning and memory deficits in several behavioral
paradigms, and restore impairments in nesting ability. The above
results demonstrate that TIMP2 is suitable to provide cognitive
(learning and memory) benefits to patients suffering from
age-related cognitive impairments or neurodegenerative diseases
that decrease synaptic function, e.g. Alzheimer's disease. Our
discovery represents the first description of a protein (TIMP2)
that declines with age in blood that could be used to reverse
impairments in synaptic plasticity and age-related learning and
memory deficits. TIMP2, when administered systemically for a period
of only .about.2 weeks, is able to confer enhanced plasticity and
memory and learning function without the need for direct to
delivery to the brain.
[0165] Systemic TIMP2 treatment in aged mice robustly improves
long-term potentiation, a cellular correlate of learning and
memory. Brain slices isolated from aged wildtype mice that were
treated with recombinant TIMP2 (i.p., 50 .mu.g/kg) display enhanced
long-term potentiation (LTP) as compared to a control (vehicle).
Shown in FIG. 1A are population spike amplitudes (PSA) within
dentate gyrus following stimulation in the perforant path of the
hippocampus. Quantification of the maintenance phase of the PSA
shown in FIG. 1A is provided in FIG. 1B. (Mean+/-SEM; Student's t
test; *P<0.05.)
[0166] Systemic TIMP2 is necessary for hippocampal-dependent
spatial memory, as assessed in a novel location recognition task.
TIMP2 levels in young wildtype mice were targeted for approximately
one month using an antibody-mediated neutralization approach (60
.mu.g/kg) prior to assessment of object location displacement
discrimination 24 hours after training. (Mean+/-SEM; 2-way ANOVA,
followed by Tukey's post hoc test; ****P<0.0001.) The results
are shown in FIG. 2.
[0167] There are currently no effective treatments for the
significant decline in synaptic plasticity, learning/memory, and
other cognitive ability associated with normal brain aging or
neurodegeneration. We have identified a youth-derived protein,
TIMP2, with strong rejuvenating activity. TIMP2, when administered
systemically for a relatively short course of treatment, is able to
confer enhanced plasticity and memory and learning function without
the need for direct delivery to the brain. TIMP2 is not a growth
factor, nor is it a canonical immune signaling molecule, which
highlights an additional advantage, namely that TIMP2 affords the
ability to target brain aging processes without supplying a
potentially tumorigenic or proinflammatory molecule that may harm
other organ systems. Moreover, utilizing a protein naturally
produced and found in blood to limit cognitive dysfunction is
unlikely to produce harmful side effects compared to conventional
small molecule drug design.
[0168] No effective therapeutic agents exist to treat the
age-related outcomes of CNS aging. Our approach allows for facile,
systemic treatment of elderly patients or those otherwise suffering
from cognitive impairment with TIMP2, a protein normally produced
by the body.
[0169] Notwithstanding the appended clauses, the disclosure is also
defined by the following clauses:
1. A method of treating an adult mammal for an aging-associated
condition, the method comprising:
[0170] enhancing a TIMP activity in the mammal in a manner
sufficient to treat the adult mammal for the aging-associated
condition.
2. The method according to Clause 1, wherein the TIMP activity is a
TIMP1, TIMP2, TIMP3 or TIMP4 activity. 3. The method according to
Clause 2, wherein the TIMP activity is a TIMP2 activity. 4. The
method according to any of Clauses 1 to 3, where the method
comprises enhancing a systemic TIMP activity. 5. The method
according to any of Clauses 1 to 4, wherein the method comprises
increasing a systemic level of a TIMP active agent in the mammal.
6. The method according to Clause 5, wherein the systemic level of
a TIMP active agent is increased by administering a TIMP active
agent to the mammal. 7. The method according to Clause 6, wherein
the TIMP active agent is a TIMP polypeptide or mimetic thereof. 8.
The method according to Clause 7, wherein the TIMP active agent is
a TIMP polypeptide. 9. The method according to Clause 8, wherein
the TIMP polypeptide has a sequence that is at least 60% identical
to any of SEQ ID NOS: 1 to 4. 10. The method according to any of
clauses 1 to 5, wherein the method comprises enhancing expression
of an endogenous TIMP coding sequence. 11. The method according to
Clauses 1 to 4, wherein the method comprises potentiating TIMP in
the subject. 12. The method according to any of the preceding
clauses, wherein the mammal is a primate. 13. The method according
to Clause 12, wherein the primate is a human. 14. The method
according to any of the preceding clauses, wherein the adult mammal
is an elderly mammal. 15. The method according to Clause 14,
wherein the elderly mammal is a human that is 60 years or older.
16. The method according to any of the preceding clauses, wherein
the aging-associated condition comprises a cognitive impairment.
17. The method according to any of the preceding clauses, wherein
the adult mammal suffers from an aging associated disease
condition. 18. The method according to any of the preceding
clauses, wherein the aging associated disease condition is a
cognitive decline disease condition.
[0171] The preceding merely illustrates the principles of the
invention. It will be appreciated that those skilled in the art
will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention and the concepts contributed by the
inventors to furthering the art, and are to be construed as being
without limitation to such specifically recited examples and
conditions. Moreover, all statements herein reciting principles,
aspects, and embodiments of the invention as well as specific
examples thereof, are intended to encompass both structural and
functional equivalents thereof. Additionally, it is intended that
such equivalents include both currently known equivalents and
equivalents developed in the future, i.e., any elements developed
that perform the same function, regardless of structure. The scope
of the present invention, therefore, is not intended to be limited
to the exemplary embodiments shown and described herein. Rather,
the scope and spirit of the present invention is embodied by the
appended claims.
Sequence CWU 1
1
221220PRTHomo sapiens 1Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala
Leu Gly Leu Leu Leu1 5 10 15Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala
Cys Ser Cys Ser Pro Val 20 25 30His Pro Gln Gln Ala Phe Cys Asn Ala
Asp Val Val Ile Arg Ala Lys 35 40 45Ala Val Ser Glu Lys Glu Val Asp
Ser Gly Asn Asp Ile Tyr Gly Asn 50 55 60Pro Ile Lys Arg Ile Gln Tyr
Glu Ile Lys Gln Ile Lys Met Phe Lys65 70 75 80Gly Pro Glu Lys Asp
Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser Ala 85 90 95Val Cys Gly Val
Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile 100 105 110Ala Gly
Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp 115 120
125Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu
130 135 140Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg
Cys Pro145 150 155 160Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu
Cys Leu Trp Met Asp 165 170 175Trp Val Thr Glu Lys Asn Ile Asn Gly
His Gln Ala Lys Phe Phe Ala 180 185 190Cys Ile Lys Arg Ser Asp Gly
Ser Cys Ala Trp Tyr Arg Gly Ala Ala 195 200 205Pro Pro Lys Gln Glu
Phe Leu Asp Ile Glu Asp Pro 210 215 2202207PRTHomo sapiens 2Met Ala
Pro Phe Glu Pro Leu Ala Ser Gly Ile Leu Leu Leu Leu Trp1 5 10 15Leu
Ile Ala Pro Ser Arg Ala Cys Thr Cys Val Pro Pro His Pro Gln 20 25
30Thr Ala Phe Cys Asn Ser Asp Leu Val Ile Arg Ala Lys Phe Val Gly
35 40 45Thr Pro Glu Val Asn Gln Thr Thr Leu Tyr Gln Arg Tyr Glu Ile
Lys 50 55 60Met Thr Lys Met Tyr Lys Gly Phe Gln Ala Leu Gly Asp Ala
Ala Asp65 70 75 80Ile Arg Phe Val Tyr Thr Pro Ala Met Glu Ser Val
Cys Gly Tyr Phe 85 90 95His Arg Ser His Asn Arg Ser Glu Glu Phe Leu
Ile Ala Gly Lys Leu 100 105 110Gln Asp Gly Leu Leu His Ile Thr Thr
Cys Ser Phe Val Ala Pro Trp 115 120 125Asn Ser Leu Ser Leu Ala Gln
Arg Arg Gly Phe Thr Lys Thr Tyr Thr 130 135 140Val Gly Cys Glu Glu
Cys Thr Val Phe Pro Cys Leu Ser Ile Pro Cys145 150 155 160Lys Leu
Gln Ser Gly Thr His Cys Leu Trp Thr Asp Gln Leu Leu Gln 165 170
175Gly Ser Glu Lys Gly Phe Gln Ser Arg His Leu Ala Cys Leu Pro Arg
180 185 190Glu Pro Gly Leu Cys Thr Trp Gln Ser Leu Arg Ser Gln Ile
Ala 195 200 2053211PRTHomo sapiens 3Met Thr Pro Trp Leu Gly Leu Ile
Val Leu Leu Gly Ser Trp Ser Leu1 5 10 15Gly Asp Trp Gly Ala Glu Ala
Cys Thr Cys Ser Pro Ser His Pro Gln 20 25 30Asp Ala Phe Cys Asn Ser
Asp Ile Val Ile Arg Ala Lys Val Val Gly 35 40 45Lys Lys Leu Val Lys
Glu Gly Pro Phe Gly Thr Leu Val Tyr Thr Ile 50 55 60Lys Gln Met Lys
Met Tyr Arg Gly Phe Thr Lys Met Pro His Val Gln65 70 75 80Tyr Ile
His Thr Glu Ala Ser Glu Ser Leu Cys Gly Leu Lys Leu Glu 85 90 95Val
Asn Lys Tyr Gln Tyr Leu Leu Thr Gly Arg Val Tyr Asp Gly Lys 100 105
110Met Tyr Thr Gly Leu Cys Asn Phe Val Glu Arg Trp Asp Gln Leu Thr
115 120 125Leu Ser Gln Arg Lys Gly Leu Asn Tyr Arg Tyr His Leu Gly
Cys Asn 130 135 140Cys Lys Ile Lys Ser Cys Tyr Tyr Leu Pro Cys Phe
Val Thr Ser Lys145 150 155 160Asn Glu Cys Leu Trp Thr Asp Met Leu
Ser Asn Phe Gly Tyr Pro Gly 165 170 175Tyr Gln Ser Lys His Tyr Ala
Cys Ile Arg Gln Lys Gly Gly Tyr Cys 180 185 190Ser Trp Tyr Arg Gly
Trp Ala Pro Pro Asp Lys Ser Ile Ile Asn Ala 195 200 205Thr Asp Pro
2104224PRTHomo sapiens 4Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Trp
Val Leu Leu Leu Arg1 5 10 15Leu Leu Ala Leu Leu Arg Pro Pro Gly Leu
Gly Glu Ala Cys Ser Cys 20 25 30Ala Pro Ala His Pro Gln Gln His Ile
Cys His Ser Ala Leu Val Ile 35 40 45Arg Ala Lys Ile Ser Ser Glu Lys
Val Val Pro Ala Ser Ala Asp Pro 50 55 60Ala Asp Thr Glu Lys Met Leu
Arg Tyr Glu Ile Lys Gln Ile Lys Met65 70 75 80Phe Lys Gly Phe Glu
Lys Val Lys Asp Val Gln Tyr Ile Tyr Thr Pro 85 90 95Phe Asp Ser Ser
Leu Cys Gly Val Lys Leu Glu Ala Asn Ser Gln Lys 100 105 110Gln Tyr
Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys Val Phe Ile 115 120
125His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser Leu Val Gln
130 135 140Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly Cys
Gln Ile145 150 155 160Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser
Ala Pro Asn Glu Cys 165 170 175Leu Trp Thr Asp Trp Leu Leu Glu Arg
Lys Leu Tyr Gly Tyr Gln Ala 180 185 190Gln His Tyr Val Cys Met Lys
His Val Asp Gly Thr Cys Ser Trp Tyr 195 200 205Arg Gly His Leu Pro
Leu Arg Lys Glu Phe Val Asp Ile Val Gln Pro 210 215 220518PRTHomo
sapiens 5Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser
Ser Ala1 5 10 15Tyr Ser66PRTHomo sapiens 6Arg Gly Val Phe Arg Arg1
575PRTHomo sapiens 7Gly Ser Gly Gly Ser1 584PRTHomo sapiens 8Gly
Gly Gly Ser195PRTHomo sapiens 9Gly Ser Gly Ser Gly1 5105PRTHomo
sapiens 10Gly Ser Gly Gly Ser1 5115PRTHomo sapiens 11Gly Ser Gly
Ser Gly1 5124PRTHomo sapiens 12Gly Gly Gly Ser1134PRTHomo sapiens
13Gly Gly Ser Gly1145PRTHomo sapiens 14Gly Gly Ser Gly Gly1
5155PRTHomo sapiens 15Gly Ser Gly Ser Gly1 5165PRTHomo sapiens
16Gly Ser Gly Gly Gly1 5175PRTHomo sapiens 17Gly Gly Gly Ser Gly1
5185PRTHomo sapiens 18Gly Ser Ser Ser Gly1 5194PRTHomo sapiens
19Arg Gly Arg Arg1206PRTHomo sapiens 20Arg Lys Arg Lys Lys Arg1
5214PRTHomos sapiens 21Arg Lys Lys Arg1226PRTHomo sapiens 22Arg Arg
Arg Lys Lys Arg1 5
* * * * *