U.S. patent application number 13/045967 was filed with the patent office on 2011-08-18 for combination therapy for preventing or treating alzheimer's disease, and kit therefor.
This patent application is currently assigned to AFFIRIS Forschungs-und Entwicklungs GmbH. Invention is credited to Frank Mattner, Walter Schmidt.
Application Number | 20110201987 13/045967 |
Document ID | / |
Family ID | 35115768 |
Filed Date | 2011-08-18 |
United States Patent
Application |
20110201987 |
Kind Code |
A1 |
Mattner; Frank ; et
al. |
August 18, 2011 |
COMBINATION THERAPY FOR PREVENTING OR TREATING ALZHEIMER'S DISEASE,
AND KIT THEREFOR
Abstract
The invention relates to a method for preventing or treating
Alzheimer's disease (AE). According to said method, a means for
inducing a sequestration of amyloid .beta. (A.beta.) into a plasma
is administered to a person, and the person is treated by means of
an apheresis device comprising a fixed carrier that can come into
contact with the blood or plasma flow and comprises a receptor
binding an amyloid-.beta.-precurser-protein (APP), the APP being
removed from the blood of the person by means of the apheresis
device. The invention also relates to a set for carrying out said
method.
Inventors: |
Mattner; Frank; (Vienna,
AT) ; Schmidt; Walter; (Vienna, AT) |
Assignee: |
AFFIRIS Forschungs-und Entwicklungs
GmbH
Vienna
AT
|
Family ID: |
35115768 |
Appl. No.: |
13/045967 |
Filed: |
March 11, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11571970 |
Jan 11, 2007 |
7935348 |
|
|
PCT/EP05/53224 |
Jul 6, 2005 |
|
|
|
13045967 |
|
|
|
|
Current U.S.
Class: |
604/6.01 ;
530/329 |
Current CPC
Class: |
A61K 2039/6056 20130101;
A61P 25/28 20180101; A61K 39/0007 20130101; A61K 38/1709 20130101;
A61M 1/3679 20130101; A61K 38/08 20130101 |
Class at
Publication: |
604/6.01 ;
530/329 |
International
Class: |
A61M 1/36 20060101
A61M001/36; C07K 7/06 20060101 C07K007/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 13, 2004 |
AT |
A 1185/2004 |
Claims
1. A kit for preventing or treating Alzheimer's Disease (AD),
comprising an agent for inducing a sequestration of amyloid .beta.
(A.beta.) in plasma, and an apheresis device that comprises a solid
carrier which can be brought into contact with the blood or with
the plasma flux, which carrier has a receptor that binds the
amyloid-.beta.-precursor protein (APP).
2. The kit according to claim 1, characterized in that the
APP-binding receptor is selected from anti-APP antibodies,
A.beta.-binding receptors, in particular anti-A.beta.40 antibodies
or anti-A.beta.42 antibodies, APP-binding proteins, in particular
gelsolin, apoJ or apoE, APP-binding peptides, APP binding
glycolipids, preferably gangliosides, in particular GM1, or
APP-binding nucleic acids, in particular aptamers, or mixtures of
said receptors.
3. The kit according to claim 1 or 2, characterized in that the
carrier is a sterile and pyrogen-free column.
4. The kit according to any one of claims 1 to 3, characterized in
that the agent for inducing a sequestration of amyloid .beta.
(A.beta.) in plasma is selected from agents with a high affinity to
A.beta., in particular gelsolin or GM1, A.beta.-specific
antibodies, peptides or nucleic acids, or a A.beta.-specific active
or passive vaccine.
5. The kit according to any one of claims 1 to 4, characterized in
that the A.beta.-specific active vaccine is an A.beta.-derivative
or an A.beta.-mimotope.
6. The kit according to claim 5, characterized in that the
A.beta.-derivative is selected from peptides which partially or
fully consist of D amino acids and/or non-natural amino acids.
7. The kit according to claim 5, characterized in that the
A.beta.-mimotope consists of a peptide or comprises a peptide of
formula X1X2X3X4X5X6, wherein X1 is an amino acid, C excepted, X2
is an amino acid, except C, X3 is an amino acid, except C, X4 is an
amino acid, except C, X5 is an amino acid, except C, X6 is an amino
acid, except C, and wherein X1X2X3X4X5X6 is not DAEFRH, the peptide
having a binding capacity to an antibody which is specific for the
natural N-terminal A.beta.42 sequence DAEFRH, and 5-mers thereof
having a binding capacity for the antibody being specific for the
natural N-terminal A.beta.42 sequence DAEFRH.
8. The kit according to claim 7, characterized in that in the
formula X1X2X3X4X5X6 there: X1 is G or an amino acid with a
hydroxyl group or a negatively charged amino acid, preferably E, Y,
S or D, X2 is a hydrophobic amino acid or a positively charged
amino acid, preferably I, L, V, K, W, R, Y, F or A, X3 is a
negatively charged amino acid, preferably D or E, X4 is an aromatic
amino acid or L, preferably Y, F or L, X5 is H, K, Y, F or R,
preferably H, F or R, and X6 is S, T, N, Q, D, E, R, I, K, Y or G,
preferably T, N, D, R, I or G, in particular EIDYHR, ELDYHR,
EVDYHR, DIDYHR, DLDYHR, DVDYHR, DIDYRR, DLDYRR, DVDYRR, DKELRI,
DWELRI, YREFFI, YREFRI, YAEFRG, EAEFRG, DYEFRG, ELEFRG, DRELRI,
DKELKI, DRELKI, GREFRN, EYEFRG, DWEFRDA, SWEFRT, DKELR, DAEFRWP,
DNEFRSP, SAEFRTQ, SAEFRAT, SWEFRNP, SWEFRLY, SWELRQA, SVEFRYH,
SYEFRHH, SQEFRTP, SSEFRVS, DWEFRD, DAELRY, DWEL RQ, SLEFRF, GPEFRW,
GKEFRT or SFEFRG.
9. A method for preventing or treating Alzheimer's Disease (AD),
wherein an agent for inducing a sequestration of amyloid .beta.
(A.beta.) into plasma is administered to a person and the person is
treated with an apheresis device that comprises a solid carrier
which can be brought into contact with the blood or the plasma
flux, said carrier having an
amyloid-.beta.-precursor-protein(APP)-binding receptor, wherein APP
is removed from the blood of the person by means of the apheresis
device.
10. The method according to claim 9, wherein a kit according to any
one of claims 1 to 8 is used.
11. Use of an A.beta.-mimotope, as defined in claim 7 or 8, for
producing an agent which is to be used in a combination therapy, as
defined in claim 1, for preventing or treating AD.
Description
[0001] The invention relates to a combination therapy for the
prevention or treatment of the Alzheimer's Disease as well as a kit
for implementing said combination therapy.
[0002] Amyloid-.beta. peptide (A.beta.) plays a central role in the
neuropathology of Alzheimer's disease (AD) (Roher et al 1993:
".beta.-Amyloid-(1-42) is a major component of cere-brovascular
amyloid deposits: Implications for the pathology of Alzheimer
disease" PNAS 90:10836). Familial forms of the disease have been
linked to mutations in the amyloid precursor protein (APP) and the
presenilin genes. Disease-linked mutations in these genes result in
increased production of the 42-amino acid form of the peptide
(A.beta.42), which is the predominant form found in the amyloid
plaques of Alzheimer's disease. An animal model for the disease is
commercially available. The PDAPP transgenic mouse, which
over-expresses mutant human APP (in which the amino acid at
position 717 is F instead of V), progressively develops many of the
neuropathological hallmarks of Alzheimer's disease in an age- and
brain-dependent manner (Games et al 1995: "Alzheimer-type
neuropathology in transgenic mice overexpressing V717F
.beta.-amyloid precursor protein" Nature 373:523).
[0003] Vaccination studies with a "normal", not mimotope-based
vaccine have already been performed. Transgenic animals were
immunized with aggregated A.beta.42, either before the onset of
AD-type neuropathologies (6 weeks) or at an older age (11 months):
Immunization of young animals prevented the development of plaque
formation, neuritic dystrophy and astrogliosis. Treatment of older
animals markedly reduced AD-like neuropathologies. This
experimental vaccination approach induced the development of
antibodies against A.beta.42 able to cross the blood-brain barrier
and attack amyloid plaques (Schenk et al 1999: "Immunization with
amyloid-.beta. attenuates Alzheimer-disease-like pathology in the
PDAPP mouse" Nature 400:173). The plaques are subsequently removed
by several mechanisms, including Fc-receptor mediated phagocytosis
(Bard et al 2000: "Peripherally administered antibodies against
amyloid .beta.-peptide enter the central nervous system and reduce
pathology in a mouse model of Alzheimer disease" Nature Med 6:916).
This vaccine was also able to delay memory deficits (Janus et al
2000: "A.beta. peptide immunization reduces behavioural impairment
and plaques in a model of Alzheimer's disease" Nature 408:979).
[0004] A highly promising immunization therapy for AD has been in
clinical trials since late 1999. Immunization is presumed to
trigger the immune system to attack the plaques and clear these
deposits from the affected human brain, although the precise
mechanism underlying needs to be characterized in more detail.
[0005] These clinical trials were conducted by the pharmaceutical
company Elan in conjunction with its corporate partner, American
Home Products (therapeutic vaccine AN-1792, QS21 as adjuvant).
Phase I trials were successfully completed in 2000. Phase II trials
were begun late 2001 to test efficacy in a panel of patients with
mild to moderate AD.
[0006] Now these phase II trials have been permanently discontinued
due to neuroinflammation in several patients (Editorial 2002
"Insoluble problem?" Nature Med 8:191). The symptoms included
aseptic meningoencephalitis leading to the immediate halt of these
world-wide trials. In the worst case scenario, affected patients
will be shown to have mounted an autoimmune response--a risk
inherent in many immunotherapies. Autoimmune complications could
have been anticipated given the ubiquity of APP, which of course
bears antigenic determinants in common with its proteolytic
product. More recently, additional studies concentrated on the
nature of aggregated A.beta.42 immunization-induced antibodies (in
humans and mice) revealing that most antibodies recognize a small
domain between amino acid 4 and 10 of A.beta.42 (.beta.A4-10). The
mouse antibodies were able to block A.beta. fibrillogenesis and
disrupted pre-existing A.beta. fibers (McLaurin et al 2002:
"Therapeutically effective anti-bodies against amyloid-.beta.
peptide target amyloid-.beta. residues 4-10 and inhibit
cytotoxicity and fibrillogenesis" Nature Med 8:1263). Of note, the
human antibodies do not react with APP exposed on the surface of
cells or any other non-aggregated proteolytic product of the
precursor (Hock et al 2002: "Generation of antibodies specific for
.beta.-amyloid by vaccination of patients with Alzheimer disease"
Nature Med 8:1270). A clear difference was observed between human
and mouse sera: In contrast to human antibodies, mouse antibodies
detect monomeric, oligomeric, and fibrillar A.beta.. This is of
importance and may be a prerequisite for the therapeutic potency
since evidence is accumulating that small oligomers of A.beta.,
which are not recognized by human anti-A.beta., are the major toxic
players in the disease (Walsh et al 2002: "Naturally secreted
oligomers of amyloid .beta. protein potently inhibit hippocampal
long-term potentiation in vivo" Nature 416:535). Thus, a potential
new strategy is the immunization with a vaccine containing
Th-amyloid amino acids 4-10 (instead of aggregated A.beta.42).
Despite unknown efficacy this strategy may also face autoimmune
problems since patients shall be directly immunized with a (linear
B cell) "self" epitope.
[0007] Despite these disappointing developments in recent AD
vaccination strategies, an A.beta. vaccine is still regarded as the
most promising way for combatting AD. However, there is an urgent
need for improvements and new strategies in AD vaccination.
Especially, such a vaccine should not induce autoreactive T and/or
B cells.
[0008] Nevertheless, also more and more other therapeutics are
being developed which should prevent amyloid-.beta. production,
amyloid-.beta.-aggregation or neurotoxic events triggered by said
aggregates. The therapeutic strategies with respect to AD which
have so far been explored are summarized in the survey article of
Wolfe (Nature Reviews Drug Discovery 1 (2002 859-866).
[0009] The basis for the formation of amyloid-.beta. plaques is the
so-called amyloid-.beta. precursor protein (APP) which is an
integral transmembrane protein (for which no known physiological
function has been clearly proven either; however, most recent
research results suggest that APP acts as so-called membrane cargo
receptor for kinesin I). APP is proteolytically cleaved by
so-called secretases, wherein in particular an A.beta. peptide of
40 amino acids in length (A.beta.40) is physiologically formed.
Other, shorter and longer forms of A.beta. also develop, especially
a 42-amino-acid version (A.beta.42) having high aggregation
potential. Consequently, said A.beta.42 form is the form which
occurs most in amyloid plaques. This is why one possible treatment
strategy for AD is mainly focussed on attacking secretases which
are responsible for said different cleavages (.alpha.- and
especially .beta.- and .gamma.-secretase). Thus, it has been tried
to use modulators and inhibitors, respectively, for said enzymes in
AD treatment (such as, e.g., benzodiazepines, sulphonamides,
benzocaprolactames).
[0010] A further gene which is associated with AD is apolipoprotein
E, wherein therefor three allele variants exist (APOE2, APOE3 and
APOE4). It has been shown that persons with one or two copies of
APOE4 run a greater risk of getting AD than carriers of APOE2,
compared with the total population. It has also been shown that
persons taking statins, i.e. medicaments inhibiting cholesterol
biosynthesis, run a significantly reduced risk of getting AD. This
is why a further treatment strategy for AD focusses one inhibiting
cholesterol biosynthesis, e.g. with statins.
[0011] A further aspect in treating AD is the inhibition of amyloid
aggregation in cerebral plaques which could, i.a., be realized by
secretase inhibitors as well. It has further been suggested to
reduce the zinc content, since zinc, if present in physiologically
relevant concentrations, can induce the aggregation of A.beta..
[0012] Further treatment strategies for AD which have been proposed
in the prior art concern the prevention of APP expression and the
increase in A.beta. clearance, wherein for said prevention
substances were searched for which interact with the APP promoter
region. With respect to A.beta. clearance, an increase in the
activity of certain proteases, such as the insulin-degrading enzyme
and neprolysin, or the periphal application of anti-A.beta.
antibodies was suggested (De Mattos et al., PNAS 98 (15) (2001),
8850-8855). Such tests, however, already led to contradicting
results in the mouse model (Wolfe, (2002)). Finally, it was
attempted to redissolve already existing amyloid plaques, e.g. by
reducing the amyloid-.beta. level in the serum of AD patients. In
this context it was also proposed to reduce plaque deposits of
.beta.-amyloid proteins in the brain by employing apheresis methods
(U.S. Pat. No. 6,551,266, wherein it is proposed to remove
macromolecules with a molecular weight of more than 500 kD by
apheresis), yet without demonstrating it in AD. Nevertheless,
dissolution of already existing plaques in brain cells is not
directly possible by apheresis methods (plaques or molecules with
>500 kd cannnot cross the blood/brain barrier).
[0013] As mentioned, the presence of .beta.-amyloid (A.beta.40 and
A.beta.42) plaques is the most striking pathological feature of AD.
This is why the reduction of A.beta. is regarded as the primary
pharmaceutical aim in AD prophylaxis and therapy. Despite the
described amyloid removal by induction of anti-A.beta. antibodies
by means of active immunization, clinical tests have so far failed
in said immunization due to severe side effects which has led to a
stop of the treatment. More recent preclinical results showed that
antibodies may (also) lead to the peripheral reduction of A.beta.
and may thus possibly change the A.beta. periphery brain
dynamics.
[0014] It has further been shown that a peripheral treatment with
an agent which has a high affinity to A.beta. (such as, e.g.
gelsolin or GM1) leads to a reduction of the A.beta. amount in the
brain (Masouka et al., Journal of Neuroscience 2003: 29-33).
Accordingly, compounds have been proposed as a general approach
which can reduce the A.beta. content in the plasma and reduce or
prevent amyloidose in the brain. Based thereon, new therapeutic
agents could be developed, the activity of which does not depend on
crossing the blood/brain barrier.
[0015] A method-depending effect on the A.beta. content in the
plasma has been shown for said plasma-A.beta.-sequestration-induced
A.beta. efflux from the brain: the A.beta. content in the plasma
was not reduced by gelsolin; instead, administration of gelsolin
and passive immuniation with anti-A.beta. monoclonal antibodies led
to an increased A.beta. content in the plasma. The A.beta. load in
the brain, however, was reduced only when using relatively young
APP transgenic mice in the experiment; when using mice older than 6
months, the treatment turned out to ineffective. This could be
ascribed to the increased insolubility of A.beta. in the brain of
older mice. On the other hand, a longer term of treatment could
possibly be successful, yet neither the administration of gelsolin
or GM1, nor the passive immunization are suitable for long-term
administration.
[0016] It is therefore the aim of the present invention to provide
a new treatment and prevention strategy for Alzheimer's Disease, in
particular a strategy which is also based on a successful
immunization.
[0017] Accordingly, the present invention provides a combination
therapy comprising an A.beta.-efflux-inducing agent and an
A.beta.-peptide-specific apheresis. According to the invention, the
A.beta. efflux is induced (by agents, such as, e.g., gelsolin, GM1,
an A.beta.-specific active or passive vaccine) and said efflux is
sustained by an A.beta. apheresis. In this context, even an active
immunization effected once or twice with a vaccine, which contains
A.beta., A.beta. derivatives or A.beta. mimotopes, is sufficient to
induce a IgM and/or IgG-mediated sequestration of plasma
A.beta..
[0018] This is why an aspect of the invention which is of
particular priority concerns a kit for preventing or treating
Alzheimer's Disease (AD), comprising [0019] an agent for inducing a
sequestration of amyloid .beta. (A.beta.) in plasma, and [0020] an
apheresis device comprising a solid carrier which can be brought
into contact with the blood or with the plasma flux, and having a
receptor that binds the amyloid-.beta.-precursor protein (APP).
[0021] In the inventive kit the APP-binding receptor is preferably
selected from anti-APP antibodies, (soluble) A.beta.-binding
receptors, such as, e.g. anti-A.beta.40 antibodies or
anti-A.beta.42 antibodies, APP-binding proteins, in particular
gelsolin, apoJ or apoE, APP-binding peptides, APP-binding
gangliosides, in particular GM1, or APP-binding nucleic acids, in
particular aptamers, or mixtures of said receptors.
[0022] In the kit, a sterile and pyrogen-free column is preferably
used as apheresis carrier.
[0023] In the kit, the agent for inducing a sequestration of
amyloid .beta. (A.beta.) in plasma is preferably selected from
agents having a high affintiy to A.beta., in particular gel-solin
or GM1, an A.beta.-specific peptide ligand or nucleic acid ligand,
an A.beta.-specific active or passive vaccine or A.beta.-specific
humanized monoclonal antibodies.
[0024] The A.beta.-specific active vaccine preferably is an A.beta.
derivative or an A.beta. mimotope.
[0025] Particularly preferred A.beta. derivatives are selected from
peptides which partly or entirely consist of D-amino acids and/or
which do not consist of natural amino acids.
[0026] A.beta. mimotopes preferably consist of or comprise a
peptide of formula
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6, [0027] wherein X.sub.1
is an amino acid, except C, [0028] X.sub.2 is an amino acid, except
C, [0029] X.sub.3 is an amino acid, except C, [0030] X.sub.4 is an
amino acid, except C, [0031] X.sub.5 is an amino acid, except C,
[0032] X.sub.6 is an amino acid, except C, [0033] and wherein
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6 is not DAEFRH, said
peptide having a binding capacity to an antibody being specific for
the natural N-terminal A.beta.42 sequence DAEFRH, and 5-mers
thereof having a binding capacity to said antibody being specific
for the natural N-terminal A.beta.42 sequence DAEFRH.
[0034] In particularly preferred peptides of formula X1X2X3X4X5X6
there: [0035] X1 is G or an amino acid with a hydroxyl group or a
negatively charged amino acid, preferably E, Y, S or D, [0036] X2
is a hydrophobic amino acid or a positively charged amino acid,
preferably I, L, V, K, W, R, Y, F or A, [0037] X3 is a negatively
charged amino acid, preferably D or E, [0038] X4 is an aromatic
amino acid or L, preferably Y, F or L, [0039] X5 is H, K, Y, F or
R, preferably H, F or R, and [0040] X6 is S, T, N, Q, D, E, R, I,
K, Y or G, preferably T, N, D, R, I or G.
[0041] In this context, the 20 amino acids which naturally occur in
proteins can be replaced by chemical analogues or by D-amino acids;
e.g. L, I and V can be replaced by Nle, Nva, Cha or alpha amino
acids with other linear or cyclic aliphatic side chains, W and F by
aromatic amino acids and R and K by alkaline amino acids, such as,
e.g. ornithine or homoarginine. Serine and threonine are suitable
for the substitution by amino acids with aliphatic and/or aromatic
side chains with terminal OH group. Efficiency and effectiveness of
such an exchange can be checked easily with the experimental model
which is described, e.g. in PCT/EP04/00162. Additionally, steric
considerations can also be taken into account (by the aid of
computor models with respect to the binding of the antibody to the
peptide.
[0042] Particularly suitable epitopes are selected from at least
one of the following epitopes: EIDYHR, ELDYHR, EVDYHR, DIDYHR,
DLDYHR, DVDYHR, DIDYRR, DLDYRR, DVDYRR, DKELRI, DWELRI, YREFFI,
YREFRI, YAEFRG, EAEFRG, DYEFRG, ELEFRG, DRELRI, DKELKI, DRELKI,
GREFRN, EYEFRG, DWEFRDA, SWEFRT, DKELR, SFEFRG, DAEFRWP, DNEFRSP,
GSEFRDY, GAEFRFT, SAEFRTQ, SAEFRAT, SWEFRNP, SWEFRLY, SWELRQA,
SVEFRYH, SYEFRHH, SQEFRTP, SSEFRVS, DWEFRD, DAELRY, DWELRQ, SLEFRF,
GPEFRW, GKEFRT, AYEFRH, DICE(N1e)R, DKE (Nva)R or DKE (Cha) R.
[0043] According to the invention an A.beta.42 mimotope is used for
vaccination against AD: The mimotope induces the production of
antibodies against A.beta.42 but not against the native APP. The
mimotope may be identified with a (monoclonal) antibody and
(commercially available) peptide libraries (e.g. according to
Reineke et al. 2002: "Identification of distinct antibody epitopes
and mimotopes from a peptide array of 5520 randomly generated
sequences" J Immunol Methods 267:37). A (monoclonal) antibody is
used that does not recognize APP but detects only different A.beta.
species with amino-terminal aspartic acid (an example of such an
antibody is described in Johnson-Wood et al 1997: "Amyloid
precursor protein processing and A.beta.42 deposition in a
transgenic mouse model of Alzheimer disease" PNAS 94:1550). Such an
antibody has been proven to be an ideal tool to identify
vaccine-suitable mimotopes in the course of the present invention.
Although such monoclonal antibodies were shown to have beneficial
effects in a mouse model of AD when directly administered to mice
(Bard et al 2000: "Peripherally administered antibodies against
amyloid .beta.-peptide enter the central nervous system and reduce
pathology in a mouse model of Alzheimer disease" Nature Med 6:916),
these antibodies have never been proposed to be used as mimotope
search tools for isolating AD vaccine compounds.
[0044] In the prior art, all efforts were concentrated on the
naturally occurring A.beta. peptide. As mentioned above, A.beta.
peptide vaccine clinical trials were stopped due to
neuroinflammatory events. Indeed, T cell epitope prediction
programs (BIMAS for MHC class I-restricted epitopes and TEPITOPE
for MHC class II-restricted epitopes) propose high score (self)
epitopes within the sequence. This could imply that the
neuroinflammatory events are due to autoimmune reactions which
would make such a vaccine unsuitable for a general application.
[0045] In contrast to such A.beta. vaccines proposed by the prior
art, no autoimmune reactions are expected to occur during treatment
with a vaccine containing a mimotope according to the present
invention, because the (monoclonal) antibody used for mimotope
identification according to the present invention does not
recognize APP and the mimotope sequence is different from
A.beta.42-derived self sequences that have been used in trials so
far or shall be used in future trials.
[0046] The antibody used for the mimotope identification according
to the present invention detects the A.beta.-derived amino acid
sequence DAEFRH (=original epitope) with a free amino terminal
aspartic acid, thus it does not recognize native APP. The antibody
may be a monoclonal or polyclonal antibody preparation or any
antibody part or derivative thereof, the only prerequisite is that
the antibody molecule specifically recognizes the DAEFRH epitope,
i.e. that it does not bind to the natural N-terminally prolonged
forms of the amyloid precursor protein, which means that the
binding capacity to the DAEFRH epitope is at least 100 times,
preferably at least 1000 times, more preferred at least 10.sup.5
times, higher than to the APP molecule. The antibody may be an
antibody showing the same or a higher binding capacity to the
DAEFRH sequence as the antibody described by Johnson-Wood et al.,
1997. Of course, also antibodies with a lower binding capacity may
be used (>10%, >50% or >80% of the binding capacity of the
Johnson-Wood et al. antibody), although the higher binding capacity
is more preferred.
[0047] The compounds according to the invention bind to those
antibodies with comparable specificity as the DAEFRH sequence.
[0048] The mimotope to be used according to the invention has a
preferred length of 5 to 15 amino acids. Said compound may be
present in the vaccine in an isolated (peptide)form or may be
coupled to other molecules or may be complexed, such as
pharmaceutical carrier substances or polypeptide, lipid or
carbohydrate structures. The mimotopes according to the invention
preferably have a (minimum) length of between 5 and 15, 6 and 12
amino acid residues, specifically between 9 and 11. The mimotopes
can, however, be (covalently or non-covalently) coupled to
unspecific linkers or carriers, in particular peptide linkers or
protein carriers. Furthermore, the peptide linkers or protein
carriers may consist of T cell helper epitopes or contain the
same.
[0049] The pharmaceutically acceptable carrier preferably is KLH,
tetanustoxoid, albumin-binding protein, bovine serum albumin, a
dendrimer (MAP; Biol. Chem. 358: 581) as well as the adjuvant
substances described in Singh et al., Nat. Biotech. 17 (1999),
1075-1081 (in particular those indicated in table 1 of said
document) and in O'Hagan et al., Nature review, Drug Discovery 2
(9) (2003), 727-735 (in particular the endogenous
immunopotentiating compounds and dispensing systems described
therein) or mixtures thereof. Moreover, the vaccine composition may
contain aluminum hydroxide.
[0050] A vaccine which comprises the present compound (mimotope)
and the pharmaceutically acceptable carrier can be administered in
any suitable way of application, e.g. i.v., i.p., i.m.,
intranasally, orally, subcutaneously, etc., and in any suitable
dispensing device (O'Hagan et al., Nature Reviews, Drug Discovery 2
(9), (2003), 727-735). The vaccine typically contains the inventive
compound in an amount of between 0.1 ng and 10 mg, preferably 10 ng
and 1 mg, in particular 100 ng and 100 .mu.g or, alternatively,
e.g. between 100 fMol and 10.mu.-Mol, preferably 10 pMol and 1
.mu.Mol, in particular 100 pMol and 100 nMol. The vaccine may also
contain typical adjuvants, e.g. buffers, stabilizers, etc.
[0051] According to the present invention, an apheresis device is
provided for maintaining the A.beta. efflux after initiation in the
course of the combination therapy, said device comprising a solid
carrier which can be brought into contact with the blood or plasma
flux, said carrier comprising an
amyloid-.beta.-precursor-protein(APP)-binding receptor. With the
present apheresis device AD patients and persons running the risk
of getting AD may be specifically cleared from APP or APP
decomposition products, in particular A.beta.40 or A.beta.42, by
means of apheresis and, thus, the effect of A.beta. sequestration
can be maintained in the first step. It is known that there is a
dynamic equilibrium of A.beta.42 between the central nervous system
(CNS) and the plasma. As mentioned above, it could be shown in the
mouse model (DeMattos PNAS 2001, see above) that peripheral
application of anti-A.beta. antibodies influences the CNS and
plasma A.beta.42 clearance and reduces the A.beta.42 load in the
brain, without anti-A.beta. antibodies crossing the blood/brain
barrier. Matsuoka et al. (Journal of Neuroscience 2003: 29-33)
confirmed said results by peripherally applying other
A.beta.42-binding molecules (gelsolin and GM1). With this the
process of plaque development can be prevented at a very good
accessible site in the brain, namely already in the blood, i.e.
then said proteins and decomposition peptides, respectively, cannot
return to the brain any longer and cannot aggregate there. The
process of plaque development in the brain can also be prevented by
capturing A.beta.42 in the blood. In doing so, it is not critical
whether the receptors in the apheresis device, which are brought
into contact with the blood or plasma of the patient, are specific
for A.beta.42 or other decomposition forms of APP, the only
essential thing is that APP and its (proteolytic) de-composition
products, in particular A.beta.42, are eliminated from the blood by
said specific adsorption, so that no "wrong" protein decomposition
(namely to A.beta.42) occurs or no plaques develop. Consequently,
the present invention is based on a completely different
application approach for apheresis as compared to U.S. Pat. No.
6,551,266, namely on eliminating already potential structural
plaque elements and not the plaques themselves. Besides,
elimination of plaques by apheresis can be excluded a priori as not
being effective for treating AD by apheresis, since the blood
apheresis cannot reach the regions in the brain where plaques
develop.
[0052] On the other hand, compared to other methods which lead to
depletion of A.beta. in the body itself (such as, e.g., in DeMattos
et al., PNAS 98(15) (2001), 8850-8855 with peripheral anti-A.beta.
antibodies) and which are conducted over a longer period of time,
the inventive combination therapy involves the decisive advantage
that no autoimmune responses can be triggered. Furthermore,
according to the invention no substances which can act only in the
body (possibly only after having been transportetd to a specific
site) have to be supplied to the patient, but the pathogenic agent
is selectively removed, i.e. the cause of the disease is
specifically removed in an extracorporeal manner, eliminating
reaction products in the body not being necessary.
[0053] According to the invention, already existing and known
apheresis devices in all embodiments can be easily adapted to the
present invention. In particular, when choosing the solid carrier
(and the apheresis device) its/their medical suitability should be
taken into consideration. Such carriers, methods or devices are
described i.a. in U.S. Pat. No. 5,476,715, U.S. Pat. No. 6,036,614,
U.S. Pat. No. 5,817,528 or U.S. Pat. No. 6,551,266. Corresponding
commercial apheresis apparatuses are i.a. distributed by Fresenius,
Plasmaselect, ASAHI, Kaneka, Braun etc., offering, e.g., the
systems LDL-Therasorb.RTM., Immunosorba.RTM., Prosorba.RTM.,
Globafin.RTM., Ig-Therasorb.RTM., Immusorba.RTM., Liposorba.RTM.,
HELP.RTM., DALI.RTM., Bilirubin-Bile-Acid-Absorber BR-350,
Prometheus.RTM. detoxication, MARS.RTM., ADAsorb of Medicap or
Plasma FLO. Although all these systems in their commercially
available form are not always primarily directed on the specific
elimination of a single protein, a person skilled in the art of
apheresis can adapt them easily to the present invention, e.g. as
immuno apheresis and/or by installing the inventive solid carrier
(e.g. as column) into the apheresis device.
[0054] Therefore, according to the invention, by "APP-binding
receptors" all substances are understood which have an affinity to
the ligand APP and its biological by-products, in particular
A.beta.42, and which are capable of removing said polypeptides from
the blood or plasma of AD patients or persons running the risk of
getting AD. Said APP and A.beta.42 receptors, respectively,
preferably are (poly or monoclonal) antibodies, proteins, peptides,
gangliosides or nucleic acids.
[0055] Anti-APP antibodies, anti-A.beta.40 antibodies or
anti-A.beta.42 antibodies, APP-binding proteins, especially
gel-solin, apoJ or apoE, APP-binding peptides, APP-binding
gangliosides, especially GM1, or APP-binding nucleic acids,
especially aptamers, or mixtures of said receptors, are
particularly preferred.
[0056] Examples of such antibodies are 3D6 (A.beta.1-5), 2H3
(A.beta.1-12), 2G3 (A.beta.33-40), 21F12 (A.beta.33-42), 12H7
(A.beta.33-42) (Johnson-Wood et al., PNAS 1997:1550-1555), 10D5,
16C11 (Bard et al., Nature Medicine 2000:916-919), the antibodies
(m266, m243) described in DeMattos et al. (2001) as well as
antibodies of same specificity. Such antibodies are obtained, e.g.,
when immunizing mammals with vaccine formulations comprising APP,
A.beta.42 or fragments or variants thereof, optionally followed by
cell fusion and clone selection protocols (with monoclonal
antibodies).
[0057] Further examples for APP-binding protein receptors are
gelsolin (Matsuoka et al. 2003, see above), apoJ and apoE (DeMattos
et al., 2001, see above). GM1 is an example of an APP-binding
ganglioside receptor (Matsuoka et al., 2003, see above).
[0058] In this context, peptides serving as APP-binding receptors
may be composed of D or L amino acids or combinations of D and L
amino acids, and may optionally be modified by further
modifications, ring formations or derivatizations. Suitable peptide
receptors for, e.g., A.beta.42, can be provided from commercially
available peptide libraries. These peptides are preferably at least
5, preferably 6 amino acids in length, in particular at least 8
amino acids, wherein the preferred lengths may be up to 10,
preferably up to 14 or 20 amino acids. According to the invention,
however, also longer peptides can be used as APP-binding receptors
without any problems. Moreover, oligomers (such as, e.g.
polyethylenimine and polylysine) are suitable receptors.
[0059] Of course, phage libraries, peptide libraries (see above) or
structure libraries, e.g. obtained by combinatorial chemistry or
high-throughput screening techniques for different structures, are
also suitable for producing such APP-binding receptors.
[0060] Furthermore, APP-binding receptors can be used which are
based on nucleic acids ("aptamers"; but also "decoy"
oligodeoxynucleotides (ds oligonucleotides that constitute binding
sites for transcription factors in terms of their sequence)),
wherein said nucleic acids can be detected by various
(oligonucleotide) libraries (e.g. with 2-160 nucleic acid residues)
(for example, Burgstaller et al., Curr. Opin. Drug Discov. Dev. 5
(5) (2002), 690-700; Famulok et al., Acc. Chem. Res. 33 (2000),
591-599; Mayer et al.; PNAS 98 (2001), 4961-4965; and many others).
The backbone of the nucleic acid can be detected, e.g., by natural
phosphor diester compounds and also by phosphorothioate or
combinations or chemical variations (e.g. as PNA), wherein
according to the invention primarily U, T, A, C, G, H and mC can be
used as bases. The 2' residues of the nucleotides, which can be
used according to the present invention, preferably are H, OH or
other protective groups and modifications at the 2' position,
wherein the nucleic acids can also be modified, e.g. provided with
protective groups, as they are usually used in oligonucleotide
synthesis. By "protective group" an etherization of the oxygen atom
is understood, whereas the --OH-group is replaced by something
different in the 2'-modification. Many different possibilities are
described in the prior art for both versions; methyl, allyl, propyl
and the like protective groups (i.e., e.g., 2'-OCH.sub.3,
2'-O--CH.dbd.CH.sub.3, etc.) are particularly preferred;
particularly preferred modifications are 2'-deoxy, 2'-amino,
2'-fluoro, 2'-bromo, 2'-azido but also metals, such as selenium,
etc. Furthermore, according to the invention also oligonucleotide
stabilizing methods, which have been developed for the antisense
technology (ribozymes, RNAi, etc.), may be used for providing
nucleic acids (compare, e.g., the companies ISIS and Ribozyme
Pharmaceuticals leading in this field, in particular their patent
documents and homepages).
[0061] This is why APP-binding aptamers (which, according to the
invention and as defined above, also include A.beta.42-binding
aptamers) are also preferred APP-binding receptors in the scope of
the present invention.
[0062] Therefore, APP-binding receptors which preferably consist of
peptides, antibodies or nucleic acids, are used as carrier material
for extracorporeally eliminating APP and its proteolytic
decomposition products in Alzheimer patients and those running the
risk of getting Alzheimer.
[0063] When using the present invention in medicinal routine
practice, the carrier is required to be sterile and pyrogen-free so
that every carrier substance and every receptor/carrier
combination, respectively, which meets these characteristics, is
preferred according to the present invention (see, e.g., U.S. Pat.
No. 6,030,614 or U.S. Pat. No. 5,476,715). Among the suitable
examples are porous homopolymers, co- or terpolymers of monomers
containing vinyl (e.g. acrylic acid, such as, e.g. TSK Toyopearl,
Fractogel TSK), carriers with modifications (activations) with
compounds containing oxirane (e.g. epichlorohydrine) and optionally
further reactions with compounds containing NH.sub.3, amino or
carboxyl, or CNBr or CNCL adsorbents as described in EP 110,409 A
and DE 36,17,672 A. Particularly preferred adsorption materials for
therapeutic purposes are suitable for avoiding a loss of blood
cells, do not or only little activate the complementing system and
delay aggregate formation in the extracorporeal circulation as far
as possible. Furthermore, the used carrier materials should
preferably be sufficiently stable against sterilization measures
also in receptor-coupled form, in particular against ethylene oxide
saturation, glutaraldehyde saturation, gamma radiation, treatments
with vapor, UV, solvents and/or detergents, etc. Products based on
sepharose, agarose, acrylic, vinyl and dextran etc., may also be
used, their preferably suitable functional groups for binding to
the APP-binding receptors being already commercially available.
Further suitable carriers also include monoliths (carriers based on
cross-linked glycidylmethacrylate-co-ethyleneglycoldimethacrylate
polmer).
[0064] Chemistry known to the person skilled in the art can be used
for coupling the receptors to the appropriate carriers (e.g.
Bioconjugate Techniques, Greg T Hermanson, Ed., Academic Press,
Inc. San Diego, Calif., 1995, 785 pp).
[0065] According to a further aspect the present invention relates
to the use of the inventive device for providing a treatment of or
a treatment device for Alzheimer's Disease or for preventing such a
disease in the scope of the inventive combination therapy, by
adapting the device to be suitable for the treatment of the
respective patient. When conducting the treatment, a patient is
sufficiently long connected with the apheresis device for
effectively eliminating APP polypeptides, wherein the blood or
plasma flux of the patient is brought into contact with the solid
carrier that comprises the APP-binding receptor, whereupon APP
and/or the proteolytic decomposition products of APP, in particular
A.beta.42, are bound. In the course of the apheresis treatment,
certainly, peripheral or central venous vein access and
arteriovenous fistula are to be ensured, as well as sufficient
anticoagulation, and the required quantification and measure data
are to be recorded. Moreover, most of the apheresis methods require
a primary separation of plasma and blood cells before the plasma
treatment proper. Special persons who require such a prophylactic
measure are persons with a familial factor older persons (>50,
>60 or >70 years) or persons having another risk factor for
AD, in particular genetic factors.
[0066] According to a further central aspect, the present invention
relates to a method for preventing or treating Alzheimer's Disease
(AD), wherein [0067] an agent for inducing a sequestration of
amyloid .beta. (A.beta.) into plasma is administered to a person
and the person is treated with an apheresis device that comprises a
solid carrier which can be brought into contact with the blood or
the plasma flux, said carrier having an
amyloid-.beta.-precursor-protein(APP)-binding receptor, wherein APP
is removed from the blood of the person by means of the apheresis
device.
[0068] Said method is preferably conducted with the inventive
kit.
[0069] Accordingly, the present invention also relates to the use
of A.beta. mimotopes, as defined above, for producing an agent
which is to be used in an inventive combination treatment for
preventing or treating AD.
[0070] The invention will be explained in more detail by way of the
following examples, to which it is, certainly, not restricted.
[0071] 1. Production of the Carrier Carrying the APP Receptor 1.1.
Monolithic Column
[0072] A CIM.RTM. Epoxy Monolithic Column (BIA Separations, SI) is
equilibrated with 0.5 M Na-phosphate buffer at an pH of 8.0
according to the producer's instruction and a monoclonal antibody
against A.beta. peptide is also activated according to the
producer's instruction and is coupled to the CIM column. The column
is washed several times with phosphate buffer (+1 M NaCl) and,
optionally, the surplus epoxy groups are blocked.
[0073] Quality assurance is done by controlling the wash and
equilibration eluate; only columns without active epoxy groups and
without antibody leakage in the eluate are used in the further
process and installed in an apheresis apparatus.
[0074] 1.2 Sepharose Column
[0075] An agarose bulk material (sepharose CL4B) is aseptically
filled into a sterile and pyrogen-free container and the material
is aseptically washed, wherein the gel material is completely dried
under vacuum between every washing step. The sepharose is then
sterilized under vapor in the autoclave for 30 minutes at
115.degree. C.
[0076] After sterilization, the sepharose is taken up in 60%
acetone/water in a sterile container and is activated with CNBr and
triethylamine (14 g CNBr per 96 ml actone; 30 ml triethylamine in
66.2 ml 87%-acetone). Then, an acetone/HCl solution was added (392
ml sterile, pyrogen-free water; 16.3 ml 5 N HCL, 408 ml acetone).
The activated sepharose is washed and supplied to the coupling
reaction within 2 h to prevent hydrolysis of activated groups.
[0077] A sterile-filtered antibody solution (m266 or m243,
respectively) is introduced into the reaction vessel and stirred
for at least 90 min. Finally, the reaction solution is thoroughly
washed (with isotonic phosphate buffer) until no reaction products
are detectable in the eluate, the antibody-coupled sepharose is
filled into sterile and depyrogenized glass columns with glass
sinters and a final quality assurance is conducted (eluate analysis
with respect to reaction products, heavy metals, etc.; particle
analysis, pyrogenity; sterility).
[0078] 2. Animal Model for Apheresis Treatment of Alzheimer
Patients
[0079] In the last years a special extracorporeal system for
experimental apheresis in freely movable small animals has been
developed at the Institute of Diabetes "Gerhard Katsch" in
Karlsburg, Germany. This apheresis therapy can be repeatedly
conducted with one and the same animal. Moreover, the animals used
can also be included in subsequent studies for long-term evaluation
of the apheresis therapy. The use of said experimental apheresis
system has been successfully demonstrated in several rat strains.
Repeated apheresis treatment was well-tolerated by rats with Typ-1
diabetes and collogen typ II-induced arthritis when their body
weight was more than 250 g.
[0080] Before the experimental apheresis therapy starts, the
animals are provided with arterial and venous catheders. In a first
step of the apheresis blood cells and plasma are separated by means
of a plasma filter. While the blood cells are directly reinfused
into the animal (via the venous catheder), the separated plasma is
guided passed the adsorption agent produced in Example 1 (wherein
the ligands are separated from the plasma due to the binding to the
immobilized receptors), before it is resupplied to the animal.
[0081] Alternatively, a whole-blood apheresis may also be
conducted, e.g. analoguous thereto, as is done with the DALI
apheresis for LDL.
[0082] 3. Inventive Combination Therapy in the Animal Model:
[0083] The combination therapy in the animal model can basically be
conducted such that the A.beta. efflux occurs before, during or
after apheresis. Furthermore, the frequency of the application of
the two therapies relative to each other can be varied.
Sequence CWU 1
1
5116PRTArtificial SequenceSynthetic Construct 1Asp Ala Glu Phe Arg
His1 526PRTArtificial SequenceSynthetic Construct 2Glu Ile Asp Tyr
His Arg1 536PRTArtificial SequenceSynthetic Construct 3Glu Leu Asp
Tyr His Arg1 546PRTArtificial SequenceSynthetic Construct 4Glu Val
Asp Tyr His Arg1 556PRTArtificial SequenceSynthetic Construct 5Asp
Ile Asp Tyr His Arg1 566PRTArtificial SequenceSynthetic Construct
6Asp Leu Asp Tyr His Arg1 576PRTArtificial SequenceSynthetic
Construct 7Asp Val Asp Tyr His Arg1 586PRTArtificial
SequenceSynthetic Construct 8Asp Ile Asp Tyr Arg Arg1
596PRTArtificial SequenceSynthetic Construct 9Asp Leu Asp Tyr Arg
Arg1 5106PRTArtificial SequenceSynthetic Construct 10Asp Val Asp
Tyr Arg Arg1 5116PRTArtificial SequenceSynthetic Construct 11Asp
Lys Glu Leu Arg Ile1 5126PRTArtificial SequenceSynthetic Construct
12Asp Trp Glu Leu Arg Ile1 5136PRTArtificial SequenceSynthetic
Construct 13Tyr Arg Glu Phe Phe Ile1 5146PRTArtificial
SequenceSynthetic Construct 14Tyr Arg Glu Phe Arg Ile1
5156PRTArtificial SequenceSynthetic Construct 15Tyr Ala Glu Phe Arg
Gly1 5166PRTArtificial SequenceSynthetic Construct 16Glu Ala Glu
Phe Arg Gly1 5176PRTArtificial SequenceSynthetic Construct 17Asp
Tyr Glu Phe Arg Gly1 5186PRTArtificial SequenceSynthetic Construct
18Glu Leu Glu Phe Arg Gly1 5196PRTArtificial SequenceSynthetic
Construct 19Asp Arg Glu Leu Arg Ile1 5206PRTArtificial
SequenceSynthetic Construct 20Asp Lys Glu Leu Lys Ile1
5216PRTArtificial SequenceSynthetic Construct 21Asp Arg Glu Leu Lys
Ile1 5226PRTArtificial SequenceSynthetic Construct 22Gly Arg Glu
Phe Arg Asn1 5236PRTArtificial SequenceSynthetic Construct 23Glu
Tyr Glu Phe Arg Gly1 5247PRTArtificial SequenceSynthetic Construct
24Asp Trp Glu Phe Arg Asp Ala1 5256PRTArtificial SequenceSynthetic
Construct 25Ser Trp Glu Phe Arg Thr1 5265PRTArtificial
SequenceSynthetic Construct 26Asp Lys Glu Leu Arg1
5276PRTArtificial SequenceSynthetic Construct 27Ser Phe Glu Phe Arg
Gly1 5287PRTArtificial SequenceSynthetic Construct 28Asp Ala Glu
Phe Arg Trp Pro1 5297PRTArtificial SequenceSynthetic Construct
29Asp Asn Glu Phe Arg Ser Pro1 5307PRTArtificial SequenceSynthetic
Construct 30Gly Ser Glu Phe Arg Asp Tyr1 5317PRTArtificial
SequenceSynthetic Construct 31Gly Ala Glu Phe Arg Phe Thr1
5327PRTArtificial SequenceSynthetic Construct 32Ser Ala Glu Phe Arg
Thr Gln1 5337PRTArtificial SequenceSynthetic Construct 33Ser Ala
Glu Phe Arg Ala Thr1 5347PRTArtificial SequenceSynthetic Construct
34Ser Trp Glu Phe Arg Asn Pro1 5357PRTArtificial SequenceSynthetic
Construct 35Ser Trp Glu Phe Arg Leu Tyr1 5367PRTArtificial
SequenceSynthetic Construct 36Ser Trp Glu Leu Arg Gln Ala1
5377PRTArtificial SequenceSynthetic Construct 37Ser Val Glu Phe Arg
Tyr His1 5387PRTArtificial SequenceSynthetic Construct 38Ser Tyr
Glu Phe Arg His His1 5397PRTArtificial SequenceSynthetic Construct
39Ser Gln Glu Phe Arg Thr Pro1 5407PRTArtificial SequenceSynthetic
Construct 40Ser Ser Glu Phe Arg Val Ser1 5416PRTArtificial
SequenceSynthetic Construct 41Asp Trp Glu Phe Arg Asp1
5426PRTArtificial SequenceSynthetic Construct 42Asp Ala Glu Leu Arg
Tyr1 5436PRTArtificial SequenceSynthetic Construct 43Asp Trp Glu
Leu Arg Gln1 5446PRTArtificial SequenceSynthetic Construct 44Ser
Leu Glu Phe Arg Phe1 5456PRTArtificial SequenceSynthetic Construct
45Gly Pro Glu Phe Arg Trp1 5466PRTArtificial SequenceSynthetic
Construct 46Gly Lys Glu Phe Arg Thr1 5476PRTArtificial
SequenceSynthetic Construct 47Ala Tyr Glu Phe Arg His1
5485PRTArtificial SequenceSynthetic Construct 48Asp Lys Glu Xaa
Arg1 5495PRTArtificial SequenceSynthetic Construct 49Asp Lys Glu
Xaa Arg1 5505PRTArtificial SequenceSynthetic Construct 50Asp Lys
Glu Xaa Arg1 5516PRTArtificial SequenceSynthetic Construct 51Xaa
Xaa Xaa Xaa Xaa Xaa1 5
* * * * *