U.S. patent application number 17/221675 was filed with the patent office on 2021-12-02 for means and methods for treating copper-related diseases.
The applicant listed for this patent is HELMHOLTZ ZENTRUM MUNCHEN - DEUTSCHES FORSCHUNGSZENTRUM FUR GESUNDHEIT UND UMWELT (GMBH), IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC., THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to Alan Angelo DISPIRITO, Josef LICHTMANNEGGER, Jeremy David SEMRAU, Hans ZISCHKA.
Application Number | 20210369809 17/221675 |
Document ID | / |
Family ID | 1000005768011 |
Filed Date | 2021-12-02 |
United States Patent
Application |
20210369809 |
Kind Code |
A1 |
ZISCHKA; Hans ; et
al. |
December 2, 2021 |
MEANS AND METHODS FOR TREATING COPPER-RELATED DISEASES
Abstract
The present invention relates to the field of (bio-)medicine,
and more particularly to the treatment of copper-related diseases.
Novel means and methods for depleting (excess) copper from organs
and/or the circulation are provided. Agents with a high copper
binding affinity and stabilized forms thereof are provided, as well
as a novel treatment regimen. The means and methods of the present
invention are particularly useful for treatment of Wilson Disease,
but also for treatment of other conditions.
Inventors: |
ZISCHKA; Hans; (Munich,
DE) ; LICHTMANNEGGER; Josef; (Neuching, DE) ;
DISPIRITO; Alan Angelo; (Ames, IA) ; SEMRAU; Jeremy
David; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
HELMHOLTZ ZENTRUM MUNCHEN - DEUTSCHES FORSCHUNGSZENTRUM FUR
GESUNDHEIT UND UMWELT (GMBH)
THE REGENTS OF THE UNIVERSITY OF MICHIGAN
IOWA STATE UNIVERSITY RESEARCH FOUNDATION, INC. |
Neuherberg
Ann Arbor
Ames |
MI
IA |
DE
US
US |
|
|
Family ID: |
1000005768011 |
Appl. No.: |
17/221675 |
Filed: |
April 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16063220 |
Jun 15, 2018 |
11000568 |
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PCT/EP2016/081407 |
Dec 16, 2016 |
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17221675 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/16 20180101; A61K
38/164 20130101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61P 1/16 20060101 A61P001/16 |
Goverment Interests
GOVERNMENT STATEMENT OF INTEREST
[0002] This invention was made with government support under grant
DE-SC0006630 awarded by the U.S. Department of Energy. The
government has certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2015 |
EP |
15201070.8 |
Feb 19, 2016 |
LU |
92979 |
Claims
1. A pharmaceutical composition comprising a stabilized
methanobactin, wherein said methanobactin complexes Zn(I) and/or
Zn(II) and/or said pharmaceutical composition is provided at a pH
.gtoreq.9.
2. The pharmaceutical composition of claim 1 comprising a
stabilized methanobactin complexing Zn(I) and/or Zn(II), being
prepared contacting an amount of Zn(I) and/or Zn(II) and an amount
of methanobactin in a ratio of 1:1 in aqueous solution.
3. The pharmaceutical composition of claim 1, being essentially
stable at 37.degree. C. for at least 20, 50, 75, 100, 125, 150
hours or more.
4. A kit comprising the pharmaceutical composition of claim 1.
5. The pharmaceutical composition of claim 1 further comprising an
active agent.
6. The pharmaceutical composition of claim 5, wherein the active
agent is selected from the group consisting of copper chelators
d-penicillamine (D-PA), trientine (TETA), tetrathiomolybdate (TTM),
zinc salts, chemotherapeutic agents, levodopa and derivatives
thereof, dopamine agonists, MAO-B inhibitors,
catechol-O-methyltransferase (COMT) inhibitors, anticholinergics,
amantadine, cholinesterase inhibitors, memantine and riluzole.
7. The pharmaceutical composition of claim 1, which is administered
to a subject parentally or orally.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 16/063,220 filed on Jun. 15, 2018 which is a
U.S. national phase application of International PCT Patent
Application No. PCT/EP2016/081407, which was filed on Dec. 16,
2016, which claims priority to Luxembourg Application No. 92979,
filed Feb. 19, 2016, and European Application No. 15201070.8, filed
Dec. 18, 2015. These applications are incorporated herein by
reference in their entireties.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The Sequence Listing associated with this application is
provided in text format in lieu of a paper copy, and is hereby
incorporated by reference into the specification. The name of the
text file containing the Sequence Listing is
SCHI_007_01US_ST25.txt. The text file is 7 KB, created on Apr. 2,
2021, and is being submitted electronically via EFS-Web.
BACKGROUND
[0004] Copper is an essential trace element for eukaryotes and most
prokaryotes that plays an important role in critical biological
functions such as enzyme activity, oxygen transport and cell
signaling. However, due to its high redox activity and its ability
to catalyze the production of free radicals, copper can have
detrimental effects on lipids, proteins, DNA and other
biomolecules. Particularly, mitochondria are thought to be the
major targets for oxidative damage resulting from copper toxicity.
Moreover, copper can interfere with proteins and can displace other
metals such as zinc from metalloproteins, thereby inhibiting their
activity. In order to prevent copper from exerting its potentially
toxic effects, it usually does not exist in free form, but only as
a complex. In the human body, approximately 95% of the copper in
plasma is bound to proteins such as ceruloplasmin, a multicopper
ferroxidase that is synthesized and secreted by hepatocytes. It is
estimated that less than 1 atom of free copper is present per
cell.
[0005] Due to its ambivalent role in metabolism, any imbalance in
copper bioavailability inevitably leads to deficiency or toxicity,
and all organisms have evolved mechanisms that regulate its
absorption, excretion and bioavailability. In mammals, copper
absorption occurs in the small intestine via enterocyte uptake,
followed by its transfer into the blood by the copper transporter
ATP7A. The liver plays a critical role in copper metabolism,
serving both as the site of copper storage and regulating its
distribution to serum and tissues and excretion of excess copper
into the bile. Particularly, hepatocytes transport and regulate
physiological copper via the specialized transporter ATP7B.
[0006] ATP7A and ATP7B are closely related in structure and
function, with approximately 60% amino acid sequence identity. They
undergo ATP-dependent cycles of phosphorylation and
dephosphorylation to catalyze the translocation of copper across
cellular membranes for the metallation of many essential
cuproenzymes, as well as for the removal of excess cellular copper
to prevent toxicity.
[0007] ATP7B mutations result in a major impairment in the ability
of hepatocytes to maintain copper homeostasis at the cellular and
systemic levels, resulting in impaired biliary copper excretion and
persistent copper accumulation in the liver, a condition known as
Wilson disease (WD). This can lead--most likely due to the
spillover of liver copper (Bandmann et al., The Lancet. Neurology
14, 103-113 (2015))--to deleterious effects on the brain and in
many cases to chronic liver disease but also to fulminant hepatic
failure (Gitlin, Gastroenterology 125, 1868-1877 (2003).
[0008] Untreated Wilson Disease is universally fatal, with most
patients dying from liver disease. In order to restore body copper
homeostasis, the clinically used copper chelators D-penicillamine
(D-PA) and trientine (TETA) or the candidate drug
tetrathiomolybdate (TTM) are administered daily (Gitlin J D,
Gastroenterology. 2003 December; 125(6):1868-77). This lifelong
therapy is effective only if commenced before the onset of advanced
hepatic or neurologic disease (Roberts et al., Am J Clin Nutr 88,
851S-854S (2008)). The same holds true for zinc salts, which are
primarily used in mild cases of WD to decrease copper absorption
via the gastrointestinal tract or as copper maintenance therapy in
chelator treated WD patients (Gitlin J D, loc. cit.). However, in
circumstances of acute liver failure--caused by either delayed
diagnosis, treatment failure, or rapidly developing fulminant
hepatitis--death is almost certain unless liver transplantation is
performed (Gitlin J D, loc. cit.). All of the currently
FDA/EMA-approved copper chelators have severe adverse effects,
including bone marrow toxicity, nephrotoxicity, hepatotoxicity,
anemia and triggering of autoimmune disease (Gitlin J D, loc.
cit.). Due to the toxicity of D-PA, discontinuation of treatment is
required in almost one third of WD patients (Weiss & Stremmel,
Current gastroenterology reports 14, 1-7 (2012)).
[0009] Currently approved pharmacological treatments usually fail
to restore copper homeostasis in acute WD, thus rendering liver
transplantation the only viable treatment option. Given these
issues, there is a clear unmet medical need for an alternative and
innovative treatment of WD and other copper-related disease. The
technical problem underlying the present application is to comply
with the unmet medical need for an alternative and innovative
treatment of copper-related disease, such as WD, particularly acute
WD.
SUMMARY
[0010] The present inventors, for the first time, suggest--based on
the unexpected capability of methanobactin to massively deplete
copper from hepatocytes and hepatocyte mitochondria--(1) a novel
treatment regimen involving phases of copper depletion followed by
phases of non-treatment, (2) a novel treatment of (previously
difficult-to-treat or untreatable) acute phase Wilson Disease and
(3) a stabilized form of methanobactin that retains the superior
capabilities of unstabilized Methanobactin (and is thus suitable
for use in accordance with the treatment regimen and medical
indication set out above) but offers the benefit of increased
stability at body temperature.
[0011] Methanobactins are low molecular mass copper-binding
molecules produced by many methanotrophic bacteria and have been
demonstrated to mediate copper acquisition from the environment
(Semrau et al., 2010. FEMS Microbiol. Rev 34:496-531). For the
first time, the present inventors have demonstrated that
methanobactins hold considerable potential for treatment of a
variety of copper-related diseases and conditions, and, due to
their excellent copper binding affinities (Choi et al., 2006.
Biochemistry 45: 1442-1453) and tolerance in vivo, are promising
new agents for a massive and fast depletion of excess copper levels
in patients in need thereof. Due to their beneficial properties,
methanobactins are considered to be particularly useful for acute
de-coppering therapy in Wilson Disease patients.
[0012] Thus, in a first aspect, the present invention relates to a
copper-binding methanobactin for use in a method of treatment of
Wilson Disease in a subject, wherein treatment comprises a
treatment cycle of (a) a first phase of methanobactin
administration followed by (b) a second phase of non-treatment,
wherein the second phase exceeds the first phase. Said first phase
may last for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more
consecutive days, and may involve administration of methanobactin
in single doses once daily, twice daily, three times daily, four
times daily, every other day or continuously. The second phase may
last for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or
more. At least one further treatment cycle may follow on the second
phase of non-treatment of the treatment cycle. Particularly,
treatment according to the method of the invention may comprise
continuous treatment cycles.
[0013] In a further aspect, the present invention also relates to
copper-binding methanobactin for use in a method of treatment of
acute phase Wilson Disease in a subject.
[0014] In any event, the methanobactin for the uses of the
invention may comprise or consist of the following general formula
(I):
R.sup.1-(X).sub.2-5-(R.sup.2) (I)
wherein R1 and R2 are each a 5-membered heterocycle comprising N
and associated with an enethiolate; and each X is independently
selected from any amino acid.
[0015] It is further contemplated that the methanobactin may be
derived from bacteria, including methanotroph and non-methanotroph
bacteria, such as Methylocystis spec., Methylosinus spec.,
Methylomicrobium spec. and Methylococcus spec. For instance, the
methanobactin may be selected from (a) a Methylosinus trichosporium
OB3b methanobactin (mb-OB3b) (b) a Methylocystis strain SB2
methanobactin (mb-SB2), (c) a Methylococcus capsulatus Bath
methanobactin (mb-Bath) (d) a Methylomicrobium album BG8
methanobactin (mb-BG8), (e) a Methylocystis strain M methanobactin,
(f) a Methylocystis hirsuta CSC1 methanobactin and (g) a
Methylocystis rosea methanobactin (mb-rosea), (h) a Methylosinus
sp. strain LW3 methanobactin (mb-LW3), (i) a Methylosinus sp.
strain LW4 methanobactin (mb-LW4), (j) a Methylocystis sp. strain
LW5 (mb-LW5), (k) a Methylosinus sp. strain PW1 methanobactin
(mb-PW1), (I) a Methylocystis parvus OBBP methanobactin (mb-OBBP),
(m) a Cupriavidus basiliensis B-8 methanobactin (mb-B-8), (n) a
Pseudomonas extremaustralis 14-3 methanobactin (mb-14-3), (o) a
Azospirillum sp. stain B510 methanobactin (mb-B510), (p) a
Tistrella mobilis KA081020-065 (mb-mobilis) methanobactin and (q) a
Comamonas composti DSM 21721 methanobactin (mb-21721).
[0016] The methanobactin for the uses of the invention is envisaged
to bind copper, in particular Cu(I), with a K.sub.d of 10.sup.-15
or less.
[0017] Said methanobactin may be complexing Zn(I) and/or
Zn(II).
[0018] In a further aspect, the present invention provides a
pharmaceutical composition comprising a stabilized methanobactin.
Said pharmaceutical composition may be stable at 37.degree. C. for
at least 20 hours or more. Stabilization may be achieved by a)
providing the pharmaceutical composition at a pH of .gtoreq.9
and/or by providing the methanobactin in the form of a complex with
Zn(I) and/or Zn(II). Said zinc:methanobactin complex may be
prepared by contacting an amount of Zn(I) and/or Zn(II) and an
amount of methanobactin in a ratio of 1:1 in aqueous solution. The
pharmaceutical composition of the present invention is envisaged to
be useful for treating a variety of diseases, including Wilson
Disease, cancer, neurodegenerative diseases, diabetes, bacterial
infections, inflammatory diseases, fibrosis, cirrhosis, familiar
amyotrophic lateral sclerosis, lead and/or mercury poisoning.
DESCRIPTION OF THE FIGURES
[0019] FIG. 1: Liver disease in the LPP rat mirrors acute liver
failure in WD patients by a devastating mitochondrial copper
overload.
[0020] (A) Histopathological comparison (HE staining) of liver
damage in diseased LPP.sup.-/- rats (upper panel) and untreated WD
patients with acute liver failure (lower panel). Tissue necrosis
with resorptive inflammation as well as repair (fibrosis) is
detectable (black arrowhead), proliferation of bile ducts (circle),
anisokaryosis (black arrow), and several inflammatory infiltrations
(white arrow) are marked. Insert shows apoptosis (white asterisk)
and nodules with ballooned hepatocytes (black asterisk). Scale bar:
100 .mu.m.
[0021] (B) Mitochondrial structure impairments in diseased
LPP.sup.-/- rats (both left panels) and untreated WD livers with
acute liver failure (both right panels). Transparent vacuoles of
varying sizes (asterisk), cristae dilations (arrow), marked
differences in electron densities, and separated inner and outer
membranes (arrowhead) can be identified. Scale bar: 500 nm.
[0022] (C) Comparable copper burden in whole liver homogenates and
in purified liver mitochondria from LPP.sup.-/- rats and untreated
WD patients with acute liver failure. Control heterozygous
LPP.sup.+/- (N=17); affected LPP.sup.-/- with strongly elevated
copper but AST <200 U/L, bilirubin <0.5 mg/dl (N=13); disease
onset LPP.sup.-/- with AST >200 U/L, bilirubin <0.5 mg/dl
(N=8); diseased LPP.sup.-/- with AST >200 U/L, bilirubin >0.5
mg/dl (N=10). .sup.*Significant to control, .sup.#significant to
affected, .sup..dagger.significant to disease onset,
.sup.*p<0.05, .sup.**p<0.01, .sup.***p<0.001.
[0023] FIG. 2: Increasing copper load severely attacks the
mitochondrial membrane integrity.
[0024] (A) The progressive disease states in LPP.sup.-/- rats are
paralleled by a decrease in normally structured mitochondria (type
1 and 2) and an increase in structurally altered mitochondria (type
3 and 4). Scale bar: 500 nm. Control LPP+/-82-89 d, N=4, n=766;
affected LPP.sup.-/-82-93 d, N=6, n=886; disease onset
LPP.sup.-/-81-93 d, N=4, n=784; diseased LPP.sup.-/-104-107 d, N=5,
n=939. N=number of rats, n=number of analyzed mitochondria.
*Significant to control, .dagger.significant to disease onset,
.sup.*p<0.05, .sup.**p<0.01.
[0025] (B) Fluorescence polarization demonstration of physical
alterations in mitochondrial membrane properties at the
protein-lipid interface (TMA-DPH) but not at the membrane inner
lipid phase (DPH) in LPP.sup.-/-vs. control mitochondria. N=number
of rats, n=number of measurements. .sup.*Significant to control,
.sup.*p<0.05, .sup.**p<0.01, .sup.***p<0.001.
[0026] (C) Upon calcium or copper induced MPT isolated LPP.sup.+/-
mitochondria undergo large amplitude swelling, which is
significantly reduced in LPP.sup.-/- mitochondria from diseased and
disease onset rats. (N=2-3, n=4-6). .sup.*Significant to control,
.sup.#significant to affected, .sup..dagger.significant to disease
onset, .sup.*p<0.05, .sup.**p<0.01, .sup.***p<0.001.
[0027] (D) Calcium-induced (100 .mu.M) MPT can be efficiently
inhibited by Cys-A (5 .mu.M). This blocking effect is severely
impaired in mitochondria from diseased and disease onset
LPP.sup.-/- rats. Table (left) shows mean values and standard
deviations whereas curves (right) depict one exemplary measurement.
.sup.*Significant to control, .sup.#significant to affected,
.sup..dagger.significant to disease onset, .sup.*p<0.05,
.sup.**p<0.01.
[0028] (E) LPP.sup.-/- mitochondria lose their membrane potential
at earlier time points compared to control mitochondria. Table
(left) shows mean values and standard deviations whereas curves
(right) depict one exemplary measurement. .sup.*Significant to
control, .sup.#significant to affected, .sup..dagger.significant to
disease onset, .sup.*p<0.05, .sup.**p<0.01,
.sup.***p<0.001.
[0029] FIG. 3: Methanobactin (MB) driven copper depletion from
liver mitochondria, hepatocytes and whole liver.
[0030] (A) MB driven copper extraction from freshly isolated copper
burdened LPP.sup.-/- mitochondria vs. copper extraction by the
copper chelators D-PA, TETA, and TTM (2 mM each, 30 min incubation,
Co=buffer treated control, N=3). .sup.*Significant to control,
.sup.**p<0.01.
[0031] (B) Toxicity of MB to the copper-dependent mitochondrial
respiratory complex IV activity, versus toxicity observed with TTM
(MB: N=3, n=9; TTM: N=1, n=3). .sup.*Significant to buffer control,
.sup.#significant to respective concentration of MB,
.sup.*p<0.05, .sup.**p<0.01, .sup.***p<0.001.
[0032] (C) Copper preloaded HepG2 (N=3) and WD patient-derived
hepatocyte like (HLC) cells (one out of two independent
experiments) are highly efficiently de-coppered by MB ((+) 24 h MB
treated, (-) untreated control). Significant to untreated control,
.sup.***p<0.001.
[0033] (D) Dose-dependent intracellular MB uptake into HepG2 cells
(given in .mu.g MB per mg cellular protein) at 2 and 24 hours
(N=3).
[0034] (E) Cellular (HepG2) toxicity of MB in comparison to TTM
(N=3, n=9). The ALP dissipating protonophor CCCP served as positive
control. .sup.*Significant to buffer control, .sup.#significant to
respective concentration of MB, .sup.**p<0.01,
.sup.***p<0.001. Arrows indicate cells with low
.DELTA..PSI..
[0035] (F) MB (500 .mu.M) treated HepG2 cells show only
intermediate phases of mitochondrial membrane potential loss (250
.mu.M CCCP, N=2). Staining indicates nuclei (blue), mitochondria
with .DELTA..PSI. (orange-red) and mitochondria without
.DELTA..PSI. (green). Scale bar: 50 .mu.m.
[0036] (G) Cumulative copper excretion into bile upon a two hour
LPP.sup.-/- liver perfusion. MB (0.7 .mu.M) forces tenfold higher
copper amounts into bile in comparison to TTM (0.8 .mu.M) (please
note the different scales for MB (right, blue axis) and D-PA, TETA
and TTM (left, black axis)). D-PA (2.2 .mu.M) and TETA (1.8 .mu.M)
did not bring copper into bile (N=3, Co=Krebs-Ringer buffer
control).
[0037] (H) Copper concentration in the perfusate during a two hour
LPP.sup.-/- liver perfusion. All chelators except TTM transport
copper to the perfusate (concentrations as in G, N=2).
[0038] (I) Two hour LPP.sup.-/- liver perfusion. MB significantly
reduces the liver copper content in contrast to D-PA, TETA, TTM and
Krebs-Ringer buffer perfused controls (Concentrations of chelators
as in G, N=3). .sup.*Significant to control, .sup.*p<0.05.
[0039] FIG. 4: Acute liver failure is efficiently avoided by a
short-term in vivo treatment with methanobactin (MB).
[0040] (A) Reductions in histopathological features of overt liver
damage were found in LPP.sup.-/- livers treated for 3 or 5 days by
MB but not upon four days D-PA or TETA treatment (scale bar: 100
.mu.m, HE staining, legend to symbols as in FIGS. 1, 6). Daily
doses were 150 mg (130 .mu.mol) MB/kg bw, 100 mg (540 .mu.mol)
D-PA/kg bw or 480 mg (2190 .mu.mol) TETA/kg bw.
[0041] (B) In contrast to untreated LPP.sup.-/- controls (U, N=6)
and D-PA (no. 8,9) or TETA (no. 10, 11) treated LPP.sup.-/- rats,
short-term MB treated LPP.sup.-/- animals (no. 1-7) presented with
markedly decreased AST levels, returning to normal.
[0042] (C) Upon i.p. injection, MB is only detectable in the serum
for half an hour, indicating a very short systemic residence time
(n=2).
[0043] (D) Short-term MB treated LPP.sup.-/- rats (N=3, each)
presented with a progressive but minor reduction at the whole liver
copper level but a significant reduction at the mitochondrial
copper level, in contrast to untreated LPP.sup.-/- controls (N=4)
and D-PA or TETA (N=2, each) treated LPP.sup.-/- rats. *Significant
to untreated controls, *p<0.05.
[0044] (E) In contrast to mitochondria isolated from untreated
LPP.sup.-/- controls (FIG. 2A) and D-PA or TETA treated LPP.sup.-/-
rats, massively reduced numbers in mitochondria with severely
impaired structure (type 4) were isolated from short-term MB
treated LPP.sup.-/- rats. (N=2, each, quantification in FIG. 9A,
scale bar: 1 .mu.m).
[0045] FIG. 5: The protection against acute liver failure by
methanobactin lasts several weeks.
[0046] (A) Short-term MB treated LPP.sup.-/- rats stay healthy for
at least two weeks, thereafter serum AST and bilirubin (not shown)
levels rise again. At the time of analysis one animal (no 1) is
still healthy and two animals (no 2, 3) are diseased.
[0047] (B, C, D) In this order (rat 1-3) the mitochondrial copper
content increases but not the whole liver copper content (B), the
typical histological features of overt liver damage with increased
frequency (C), and increased severity of mitochondrial structure
impairments (D). Scale bar: 100 .mu.m in (C) and 500 nm in (D).
[0048] FIG. 6:
[0049] (A) Masson trichrome staining demonstrated signs of fibrosis
(stained blue) in diseased LPP.sup.-/- rat liver (left panel) but
marked fibrosis in explanted WD patient liver (right panel). Scale
bar: 100 .mu.m.
[0050] (B) Histopathological analysis (HE staining) of LPP rat
livers at different disease states shows increasing alterations
during progression of the disease (Scale bar: 100 .mu.m; white
asterisk: (different stages of) apoptosis, black arrow:
anisokaryosis, black asterisk: ballooned hepatocytes, white arrow:
inflammatory infiltrates; white arrowhead: cytoplasmic
condensation).
[0051] (C) Electron micrographs of LPP rat liver mitochondria in
situ corresponding to the disease states as in B (Scale bar: 500
nm). Separated inner and outer membranes are indicated by
arrows.
[0052] (D) Liver damage in explanted livers of WD patients with
D-PA treatment failure. Left panel: HE stain reveals
histopathological features of liver damage and massive fibrosis
(black arrowhead, Scale bar: 100 .mu.m). Middle panels: Some areas
presented with relatively intact mitochondria (I), others
demonstrated severe structural impairments (II, III, cf. FIG. 1B,
Scale bar: 500 nm). Right panel: In comparison to untreated WD
patients with acute liver failure (cf. FIG. 1C), lower total copper
contents were determined in tissue homogenates and in isolated
mitochondria from livers of WD patients with D-PA treatment
failure.
[0053] FIG. 7:
[0054] (A) Copper loading of isolated control mitochondria
(LPP.sup.+/-). Mitochondria (4 mg/ml) were pre-incubated with DTT
(1 mM), challenged with concentrated (20 mM) or diluted (2 mM)
copper stock solutions, subsequently re-purified by density
gradient centrifugation, and their copper load determined
(N=4).
[0055] (B) Copper pre-loaded LPP.sup.+/- mitochondria from (A) were
incubated with copper chelators (2 mM) for 30 min and subsequently
re-purified by density gradient centrifugation. (N=5,
.sup.*significant to control, .sup.*p<0.05,
.sup.**p<0.01).
[0056] (C) Comparison of the effect of MB to LPP.sup.+/- control
mitochondria vs. LPP.sup.-/- mitochondria on copper-dependent
mitochondrial respiratory complex IV activity (LPP.sup.+/-: N=3,
n=9, LPP.sup.-/-: N=2, n=6, .sup.*significant to buffer control,
.sup.#significant to respective concentration of LPP.sup.+/-,
.sup.*p<0.05, .sup.**p<0.01, .sup.***p<0.001).
[0057] (D) MB treatment causes a 50% reduction of copper in HepG2
cells with basic copper load (N=3, (+) 24 h MB treated, (-)
untreated control, .sup.*significant to untreated control
.sup.*p<0.05),
[0058] (E) Dose dependent toxicity (neutral red) of histidine bound
copper on HepG2 cells (N=5; .sup.*significant to control,
.sup.*p<0.05)
[0059] FIG. 8:
[0060] (A) Bile flow during two hour LPP.sup.-/- liver perfusion.
Displayed are mean values of three independent experiments.
[0061] (B) Cumulative biliary copper excretion during two hour
LPP.sup.-/- liver perfusion by MB (N=3).
[0062] (C) Parallel LDH and copper release into the perfusate
during two hour LPP.sup.-/- liver perfusion.
[0063] FIG. 9:
[0064] (A) Quantification to FIG. 4E. In contrast to mitochondria
isolated from untreated and D-PA or TETA treated LPP.sup.-/-
animals, massively reduced numbers in mitochondria with severely
impaired structure (type 4) were isolated from short-term MB
treated LPP.sup.-/- animals (N=number of rats, n=number of
mitochondria; Affected (A): N=6, n=886; Disease onset (Do): N=4,
n=784; 3 d MB: N=2, n=324; 5 d MB: N=2, n=527; D-PA: N=2, n=252;
TETA: N=2, n=366).
[0065] (B) Respiratory analysis of mitochondria from the MB treated
LPP.sup.-/- rats in FIG. 5 after MB drug holiday. At the time of
analysis mitochondria from the still healthy animal (no. 1) are as
intact as control mitochondria (respiratory control ratio with
succinate as substrate, RCRs), whereas mitochondria from the two
diseased animals (no. 2, 3) are impaired.
[0066] (C) Stability analysis of metal free MB and Zn-loaded MB
followed by absorbance measurements of their two metastable
oxazolone rings (OxaA/ZnA at 394 nm and OxaB/ZnB at 340 nm).sup.57
at 37.degree. C. In contrast to metal free methanobactin, Zn-MB is
time stable at 37.degree. C.
[0067] (D) Histopathological analysis of untreated (left) and MB
treated (right) moribund LPP.sup.-/- rats. Liver damage was present
in both tissues, however less severe in the MB treated animal
indicating liver regeneration (Scale bar: 100 .mu.m, HE staining,
symbols as in FIGS. 1, 6).
[0068] (E) Mitochondria from the animals described in D, either
isolated (left) or in situ (right). In contrast to the untreated
animal, only minor structural alterations were observed in the MB
treated LPP.sup.-/- rat (Scale bar: 500 nm).
[0069] (F) Progressively impaired ATP production of mitochondria
isolated from LPP.sup.-/- rats at different disease states. Short
term MB treatments reverse this impairment.
[0070] FIG. 10:
[0071] Different application routes or treatment regimens can
further enhance MB induced mitochondrial de-coppering.
[0072] Short term treatments of one week by either 5.times. MB i.p,
5.times. MB i.v., and especially by 16.times. MB i.p. (one week,
twice daily) drastically reduce the mitochondrial copper load.
[0073] FIG. 11:
[0074] Chemical structures of full-length mbs from M. trichosporium
OB3b (A) (144, 155), Methylocystis strain sp.M (B) (136), M.
hirsuta CSC1 (C) (136), M. rosea (D) (136) and Methylocystis strain
sp. SB2 (E) (135).
[0075] FIG. 12: Mb precursor peptides.
[0076] Sequences detected in bacteria of known genome sequence from
methanotrophs with structurally characterized mbs are shown in red,
sequences detected in bacteria of known genome sequence from
methanotrophs are shown in blue and sequences detected in bacteria
of known genome sequence from non-methanotrophs are shown in green.
Bar above amino acids represent the amino acid pair that is or
proposed to be post-translationally modified into an oxazolone,
imidazolone or pyrazinedione group. Abbreviations: methanobactin
from Methylosinus trichosporium OB3b (mb-OB3b), Methylosinus sp.
strains LW3 (mb-LW3), LW4 (mb-LW4), PW1 (mb-PW1), Methylocystis
parvus OBBP (mb-OBBP), Methylocystis rosea (mb-rosea),
Methylocystis strains SB2 (mb-SB2), SC2 (mb-SC2), and LW5 (mb-LW5),
Cupriavidus basiliensis B-8 (mb-B-8), Pseudomonas extremaustralis
14-3 (mb-14-3), Azospirillum sp. stain B510 (mb-B510), Tistrella
mobilis KA081020-065 (mb-mobilis) and Comamonas composti DSM 21721
(mb-21721).
[0077] FIG. 13: Mb gene clusters.
[0078] Gene clusters of complete genomes of methanotrophs M.
trichosporium OB3b, Methylocystis sp. SB2 and Methylocystis
rosea.
[0079] FIG. 14: Repetetive treatment regimen with recurrent
de-coppering phases.
[0080] LPP-/- rats were subjected to the first treatment cycle
consisting of three daily MB injections (i.p.) for five days
followed by a period of non-treatment. Recurrent treatment cycles
resulted in marked reduction in mitochondrial and liver copper load
and doubling of time before disease onset as compared to untreated
animals.
[0081] FIG. 15: A structurally and chemically different
methanobactin (MB) peptide such as mb-SB2 from Methylocystis
strains SB2 exhibits similar therapeutic potential in depleting
copper from hepatocyte mitochondria.
[0082] MB-SB2 from Methylocystis strains SB2 acts as a promising
cooper chelator compared to existing clinically approved cooper
chelators such as D-PA on freshly isolated mitochondria from three
different LPP.sup.-/- rats (1 mM of D-PA, Ob3b, SB2, 30 min
incubation, Control=buffer treated control, N=3). MB peptide mb-SB2
from Methylocystis strains SB2 is structurally and chemically
deviating from other MB peptides (f.e. from mb-OB3b derived from
Methylosinus trichosporium OB3b).
DETAILED DESCRIPTION
[0083] Wilson Disease (WD), an autosomal recessively inherited
copper overload disorder, is a yet incurable disease that is fatal
when left untreated. The overall therapeutic approach is the
restoration and maintenance of normal copper homeostasis, either by
medical therapy or by liver transplantation. Copper chelators (such
as D-penicillamine, trientine and tetrathiomolybdate) and/or zinc
salts presently represent the gold standard of WD treatment.
Regardless of the specific approach chosen, treatment must be
continued throughout the patient's lifetime, because abnormal
copper accumulation cannot be controlled by a low copper diet.
Importantly, non-adherence or discontinuation of medical therapy is
associated with the risk of intractable hepatic or neurologic
deterioration.
[0084] Presently available treatment options are, unfortunately,
only of limited efficacy in terms of reducing copper levels; and
are moreover incapable of restoring physiological copper excretion
via the bile. Along with severe side effects, the need for high
dosages and repeated administration (often several times a day),
results in a severe impairment of quality of life and overall poor
patient compliance.
[0085] Moreover, commonly prescribed WD therapeutics fail to
restore liver function once WD manifests as advanced liver
failure--e.g., due to delayed diagnosis, poor compliance, or rapid,
fulminant hepatitis. In this case, liver transplantation with all
its inherent risks and detriments presently remains the only viable
option. Although liver transplantation is effective to restore
normal biliary copper excretion (thereby preventing disease
recurrence) and promotes removal of copper from extrahepatic sites,
given the chronic shortage of suitable donor organs and the
substantial morbidity and mortality associated with the procedure,
it is considered as a treatment option only in life-threatening
circumstances.
[0086] Novel means and methods for WD treatment are thus urgently
needed. The surprising findings underlying the present invention
show that methanobactins, methanotroph-derived chalkophores, are
surprisingly potent and well-tolerated de-coppering agents.
Unexpectedly, the present inventors found that due to their
superior copper binding affinity, methanobactins (in contrast to
other copper chelators, being far less efficient) can
advantageously be used for massive depletion of (excess) copper
with a long-term effect--thereby allowing for a novel treatment
regimen that is expected to markedly improve patient compliance
and, consequently, overall therapeutic success. Moreover, the
present inventors found that methanobactins are even capable of
removing accumulated mitochondrial copper--which has recently been
suggested as a crucial causative factor for oxidative stress
underlying tissue and organ damage in a number of diseases.
Therefore, methanobactins are not only promising agents for
treatment of WD, but also for a number of unrelated diseases that
have been linked to increased copper levels in the blood, in whole
cells and/or in mitochondria within.
Wilson disease
[0087] Wilson Disease (WD) is an inherited disorder associated with
mutations in the copper transporting ATPase ATP7B, resulting in
impaired, non-functional or impaired ATP7B protein activity. More
than 500 mutations in ATP7B have been identified, most of which are
low-abundance mutations.
[0088] WD is typically characterized by severe impairment (or even
complete absence) of biliary copper excretion, resulting in hepatic
copper overload and, eventually, copper spillover into the
circulation and/or central nervous system.
[0089] A variety of signs and symptoms reflecting cellular injury
from excess copper may be present in affected patients. Many types
of liver disease may be encountered in patients with Wilson
Disease, and presenting symptoms of liver disease can be highly
variable, ranging from asymptomatic, with only biochemical
abnormalities, to overt cirrhosis. Wilson Disease may also present
as acute liver failure as described elsewhere herein. Other
manifestations include Coombs positive hemolytic anemia,
cardiomyopathy, and endocrine dysfunction. Neurologic signs, more
common in the second or third decade of life, are variable, and
most often include tremor, ataxia, and dystonia, consistent with
neuropathologic findings of basal ganglia involvement. The most
common psychiatric features are abnormal behaviour (typically
increased irritability or disinhibition), personality changes,
anxiety, and depression.
[0090] Diagnosis of WD typically requires a combination of tests
that are reflected by the diagnostic score that was proposed by the
Working Party at the 8.sup.th International Meeting on WD, Leipzig
2001 (Ferenci et al. Liver Int. 2003; 23(3):139-42) and is now
included in the European Association for the Study of the Liver
(EASL) clinical practice guidelines for Wilson Disease (EASL
Clinical Practice Guidelines: Wilson's disease, J Hepatol. 2012
March; 56(3):671-85). Often, the combination of Kayser-Fleischer
rings and a low serum ceruloplasmin decreased by 50% of the lower
normal value, typically 0.1 g/L or less, is sufficient to establish
a diagnosis. Kayser-Fleischer rings are caused by deposition of
copper in Descemet's membrane of the cornea and can be assessed by
slit lamp examination. ATP7B loss-of-function and consequent
failure to incorporate copper during ceruloplasmin biosynthesis
results in the secretion of an apoprotein that is devoid of
enzymatic activity and rapidly degraded, accounting for low serum
concentrations of enzymatically active ceruloplasmin and thus
proportionally low total serum concentrations of copper typically
seen in WD patients, except in cases of severe liver injury or
acute liver failure, when there are high serum concentrations of
non-ceruloplasmin-bound copper due to its sudden release from the
liver.
[0091] Other important diagnostic parameters according to the EASL
Clinical Practice Guidelines (loc. cit.) include increased urinary
copper excretion (>1.6 .mu.mol/24 h or >0.64 .mu.mol/24 h in
children), non-ceruloplasmin-bound copper ("free copper") levels
>1.6 .mu.mol/L and a hepatic parenchymal copper content of >4
.mu.mol/g dry weight. Direct genetic testing for ATP7B mutations
are also increasingly available to confirm clinical WD
diagnosis.
[0092] Notably, methanobactin treatment according to the present
invention is in general envisaged for WD manifesting by any of the
aforementioned signs and symptoms. Due to their superior copper
binding affinity, methanobactins are considered useful in any form
of WD. Unless noted otherwise, the term "Wilson Disease" or "WD"
thus includes acute and non-acute forms of WD, presenting with
hepatic and/or neurological deficits, early onset WD in infancy and
late-onset WD in adults, previously treated and untreated WD.
Advantageously, methanobactins, particularly administered according
to the treatment regimen provided herein, are also considered to be
effective when otherwise liver transplantation would be indicated,
including WD patients with acute liver failure as the first
presentation of disease, non-responders to conventional copper
chelator therapy, those who present with end-stage liver disease
(ESLD) and severe hepatic insufficiency, and patients with
neurological WD in the absence of liver failure as reviewed by
Schilsky M L, Ann N Y Acad Sci. 2014 May; 1315:45-9. Also
encompassed by the term are related copper-overload diseases in
non-human mammalian subjects, including dogs. The term WD also
includes animal models of WD, such LPP-/- rats carrying an ATP7B
mutation that completely abolishes its hepatic copper transport
activity.
[0093] In general, patients presenting with any of the
manifestations mentioned in the foregoing are envisaged to benefit
from methanobactin therapy. Particularly, (recurring) treatment
cycles of massive copper depletion as a result of methanobactin
administration according to the treatment regimen described herein
are envisaged as an effective, well-tolerated and patient-compliant
treatment option for WD presenting with any of the aforementioned
signs and symptoms.
Treatment regimen
[0094] Accordingly, in a first aspect, the present invention
provides a copper-binding methanobactin for use in a method of
treatment of Wilson Disease in a subject, wherein treatment
comprises at least one treatment cycle of (a) a first phase of
methanobactin administration followed by (b) a second phase of
non-treatment, wherein the second phase exceeds the first phase.
"Non-treatment" refers to a period of time during which no
methanobactin is administered. Optionally and advantageously,
"non-treatment" may include that no other WD therapeutics (in
particular copper chelators) are administered. Surprisingly, it
turned out that methanobactins as described herein are extremely
efficient and well-tolerated de-coppering agents that allow for
(recurrent) treatment phases of massive copper depletion with a
long-term effect. I.e., the present inventors discovered that
steady administration (as with copper chelators known in the art)
is not necessarily required when using methanobactins for WD
treatment, but that patients can rather undergo (recurrent) phases
of methanobactin treatment for removing excess copper, followed by
phases that preferably do not require administration of WD
therapeutics at all. This is a significant advantage over currently
known WD therapeutics which often require life-long, steady
administration in high dosages. The treatment regimen according to
the present invention is therefore expected to markedly improve
quality of life of WD patients, and, thereby, patient compliance
and overall therapeutic success.
[0095] Particularly, the first phase of the inventive treatment
regimen is envisaged to last for a period of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or more consecutive days. During the first phase of
treatment ("de-coppering phase"), the methanobactin may be
administered as described elsewhere herein either in single doses
once daily, twice daily, three times daily, four times daily, five
times daily, every other day, or continuously. The first phase of
administration of the methanobactin is followed by a second phase
of non-treatment. Advantageously, said second phase is even thought
to exceed the first phase of methanobactin administration as
demonstrated in the appended examples, and is hence envisaged to
last for at least 1, 2, 3, 4 or 5 weeks or even longer. As will be
readily acknowledged by the skilled practitioner, the duration of
the second phase will depend on several factors, e.g. nutritional
copper intake, body constitution, severity of WD, etc.
Nevertheless, a minimum period of non-treatment of at least 1 week
is envisaged herein after the first phase of methanobactin
administration.
[0096] It will be readily understood that recurrent treatment
cycles are envisaged, i.e. several treatment cycles as described in
the foregoing may follow one another. Specifically, a phase of
non-treatment may be followed by a phase of treatment (de-coppering
phase), and a subsequent phase of non-treatment may be followed by
another de-coppering phase, and so on. Treatment cycles may be
reiterated in intervals, over several weeks, months, years, or even
life-long. The treatment regimen of the present invention provides
for prophylactic depletion of copper on a regular basis (i.e.
before signs and symptoms of WD occur) and/or for acute and
optionally recurrent de-coppering treatment whenever necessary. The
skilled practitioner will readily be able to assess when
methanobactin treatment according to the invention is
indicated.
Acute WD
[0097] As explained previously, the present invention provides a
novel and effective treatment regimen that allows for (optionally
repeated) copper depletion in WD patients. Another surprising
insight underlying the present invention is the fact that
methanobactins are effective for treatment of acute WD presenting
as acute liver failure (ALF); a condition that was, to date,
invariably fatal unless liver transplantation was conducted.
[0098] Acute WD is defined herein as WD manifesting as acute liver
failure (ALF), which may be the initial presentation of WD or can
occur when WD treatment is stopped. Known copper chelators
presently used for WD therapy are, by far, not able to bind to and
remove enough excess copper to remedy the rapid deterioration of
liver function seen in WD patients presenting with ALF. In
contrast, methanobactins as described herein have surprisingly been
found to be capable of depleting copper so efficiently that even WD
patients presenting with acute WD--manifesting as ALF--are
envisaged to be effectively treatable without the need of emergency
liver transplantation.
[0099] Acute liver failure is defined as the rapid development of
hepatocellular dysfunction (i.e. within less than 26 weeks from the
onset of the first hepatic symptoms), optionally accompanied by
coagulopathy and hepatic encephalopathy in a patient. Hepatic
encephalopathy may present as deficits in higher brain function
(e.g. mood, concentration in grade I) to deep coma (grade IV).
Coagulopathy typically manifests as a prolongation in prothrombin
time (usually an International Normalized Ratio (INR) .gtoreq.1.5),
and progressive thrombocytopenia (detectable in a full blood
count).
[0100] Diagnosis of ALF is based on physical exam, laboratory
findings and patient history. On laboratory testing, liver function
can be assessed by evaluating aspartate transaminase (AST), alanine
transaminase (ALT), alkaline phosphatase (ALP), gamma glutamyl
transpeptidase (GGT), total bilirubin and/or albumin levels.
Subjects with ALF due to WD often present with a nonimmune
(Coombs-negative) hemolytic anemia that may precede the development
of liver failure or occur concurrently with the liver injury. Decay
of liver cells may result in the release of large amounts of stored
copper into the circulation, thereby increasing "free"
(non-ceruloplasmin bound) copper levels. An increase in the
alkaline phosphatase (ALP) to bilirubin ratio of less than 4:1
owing to the relative decrease in alkaline phosphatase (ALP) and
increased bilirubin (resulting from hemolysis and hepatic
dysfunction), a ratio of aspartate transaminase (AST) to alanine
transaminase (ALT) of greater than 2.2:1, and increases in serum
copper, typically above 200 .mu.g/dL, are suggestive of ALF due to
acute WD. Guidance as to how to identify such patients is i.a.
provided by Schilsky M L, Ann NY Acad Sci. 2014 May; 1315:45-9 and
Bermann et al. Gastroenterology. 1991 April; 100(4):1129-34.
Particularly, if both the alkaline phosphatase (ALP) to bilirubin
ratio is greater than 4:1 and the AST to ALT ratio is above 2.2
concurrently, ALF due to WD can be assumed.
[0101] Diagnosis can be confirmed by evaluating other signs and
symptoms suggesting Wilson Disease, including clinical symptoms
(e.g. deep jaundice) and the conventional WD diagnostic parameters
(ceruloplasmin, serum or urinary copper as described elsewhere
herein). The diagnosis has to be ascertained by determining the
hepatic copper content by liver biopsy and/or mutation analysis as
described previously.
[0102] The clinical presentation of acute WD typically progresses
rapidly from hepatic to renal failure and, when untreated, leads to
almost 95% mortality unless emergency liver transplantation is
available. The present inventors were the first to acknowledge that
methanobactins can be used as an effective remedy of the severe
clinical manifestations of acute WD. It is contemplated that copper
depletion by administration of methanobactins may even render liver
transplantation in acute WD patients obsolete. Methanobactin
treatment of acute WD can be carried out according to the treatment
regimen described elsewhere herein, or according to any other
treatment scheme that the skilled practitioner considers
appropriate. Typically, acute WD treatment will involve a phase of
massive copper depletion by administration of a sufficient amount
of methanobactin, that may be ended once the signs and symptoms of
acute WD subside; and/or laboratory values improve. Subsequently,
treatment according to the regimen of the present invention may
follow.
Methanobactin
[0103] As set out elsewhere herein, the present inventors were the
first to acknowledge the therapeutic potential of methanobactins as
safe and effective copper depleting agents for WD treatment
according to a novel treatment regimen, and for treatment of acute
WD that was to date considered to be irreversible by drug therapy.
The term "methanobactin" or "mb" as used herein generally refers to
a copper-binding (and Cu(II)-reducing) peptide derived from
bacteria, particularly methanotroph bacteria. Unless denoted
otherwise, "copper" is used herein to refer to both Cu(I) and
Cu(II). Naturally occurring methanobactins are thought to be
secreted to the extracellular media where they function as
chalkophores by binding to Cu(II) or Cu(I) and shuttling the copper
into the cell.
[0104] The term "methanobactin" as used herein in particular
encompasses modified peptides characterized by the presence of one
oxazolone ring and a second oxazolone, imidazolone or pyrazinedione
ring. The two rings are separated by 2-5 amino acid residues. Each
ring has an adjacent thioamide group. Structurally, mbs can be
divided into two groups that are both envisaged for the uses
according to the present invention (FIGS. 11, 12). One type (Group
I) is represented by mb from Methylosinus trichosporium OB3b. Based
on sequence similarity and alignments, the putative mbs from
Methylosinus sp. strain LW3 (mb-LW3), Methylosinus sp. strain LW4
(mb-LW4), Methylosinus sp. strain PW1 (mb-PW1), Methylocystis
strain LW5 (mb-LW5) and one of the two mbs from Methylocystis
parvus OBBP (mb-OBBP(2)) would also fall within this group (FIG.
12). In this group the rings are separated by 4 or 5 amino acids
and the mb contains 2 or more Cys not involved in ring
formation.
[0105] The second group (group II) is represented by the
structurally characterized mbs from Methylocystis strains SB2,
rosea and SC2 (FIGS. 11, 12). This mb group lack the Cys in the
core peptide, are smaller and probably less rigid, due to the
absence of the disulfide bond found in mb-OB3b. In this group the
rings are separated by two amino acids. In contrast to the other
members of group II mbs, mb-B-8, mb-14-3, mb-B510 and mb-21721
contain 4 Cys. However, based on the location of the Cys we predict
all 4 Cys are modified into the heterocyclic rings. Mbs from the
structurally characterized members in this group contain a sulfate
group, which may aid in the formation of a tight bend by making a
hydrogen bond with the backbone amide of Ser2. The sulfate group
also increases Cu.sup.2+/1+ affinity (E I Ghazouani et al., 2012.
Proc. Nat. Acad.Sc. 109: 8400). The conserved T/S adjacent to the
C-terminal ring suggests that the other members of this group also
contain a sulfate group.
[0106] It was discovered that the genome region of the putative mb
precursor matching sequence in M. trichosporium OB3b had a number
of distinctive and striking features (FIG. 13). These include (a) a
precursor peptide translationally modified peptide; (b) a potential
cleavage site between the leader and core peptide, suggestive of
secretion; (c) genes upstream and downstream of the mb gene cluster
encoding protein sequences compatible with possible roles in
maturation of the mb precursor sequence, transport, and regulation
of mb biosynthesis. Elaboration on this initial search revealed a
series of genomes containing gene clusters with characteristics
matching that of the M. trichosporium OB3b mb gene cluster, e.g. in
Methylocystis parvus OBBP Methylosinus sp. LW3 as well as
non-methanotrophs Azospirillum sp. B510, Azospirillum sp. B506,
Pseudomonas extremaustralis Pseudomonas extremaustralis substrain
laumondii TT01 Tistrella mobilis, Gluconacetobacter sp. SXCC.
Gluconacetobacter oboediens Methylobacterium sp. B34 , Cupriavidus
basilensis B-8, Photorhabdus luminescens and Vibrio caribbenthicus
BAA-2122.
[0107] At present the only genes in the Methylosinus trichosporium
OB3b mb gene cluster with a known function are the structural gene
for mb-OB3b, MbnA, and TonB-transporter (MbnT) which is responsible
for Cu.sup.+-mb-OB3b uptake (Semrau et al., unpublished results).
The cytochrome c peroxidase MbnH, and the FAD.sup.+-dependent
oxidoreductase, present instead or sometimes in addition to MbnH in
methanotroph gene clusters MbnF are likely candidates to be
involved in the oxidation steps required for ring formation. In
addition, the aminotransferase MbnN found in the mb-OB3b, but not
the mb-SB2 gene cluster may be involved in formation of the
N-terminal keto-isopropyl group, and the sulfotransferase MbnS
found in the mb-SB2 and mb-rosea, but not the mb-OB3b gene cluster
may catalyse sulfonation of the threonine. One other gene product,
the multidrug and toxin extrusion (MATE) protein has been suggested
to be involved in secretion of mature mbs.
[0108] Generally, the present invention encompasses methanobactins
encoded by a mb gene, preferably a Methylosinus trichosporium OB3b
mb gene or variant or ortholog thereof. The term "variant" in
reference to a nucleic acid sequence refers to polymorphisms, i.e.
the exchange, deletion, or insertion of one or more nucleotides,
respectively, as compared to the "parent" nucleic acid sequence
that the variant is derived from. "Orthologs", or orthologous
genes, are genes in different species that evolved from a common
ancestral gene by speciation. As used herein a variant or ortholog
encodes a copper-binding methanobactin preferably exhibiting the
same advantageous properties as the mb evaluated in the appended
examples. It is envisaged that the variant or ortholog of the
mb-OB3b gene comprises or consists of a nucleic acid sequence
having at least about 60%, such as at least about 65%, 70%, 75%,
80%, 85%, 90%, 95% or 99% sequence identity to the mb gene.
[0109] The mb OB3b gene, variant or ortholog thereof is envisaged
to encode an mb precursor peptide that comprises or consists of an
amino acid sequence that has at least about 60%, such as at least
about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to
the amino acid sequence of the known mb-OB3b precursor peptide with
UniProt Acc. No. E3YBA4 (entry version No. 15 of Jun. 24, 2015) and
as depicted in SEQ ID No. 1 (FIG. 12). Particularly, and as
described in more detail below, the mb OB3b gene was found to
encode a precursor peptide including a leader peptide and a core
peptide, separated by a potential cleavage site. Preferred %
sequence identities for the overall precursor peptide are indicated
above. Moreover, the encoded (i.e., non-translationally modified)
methanobactin (i.e., core peptide) is envisaged to comprise or
consist of an amino acid sequence that has at least about 60%, such
as at least about 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence
identity to the amino acid sequence of the known mb-OB3b core
precursor peptide as depicted in SEQ ID No. 1 (FIG. 12).
[0110] Generally, the term "sequence identity" indicates the extent
to which two (nucleotide or amino acid) sequences have identical
residues at the same positions in an alignment, and is often
expressed as a percentage. Preferably, identity is determined over
the entire length of the sequences being compared. Thus, two copies
of exactly the same sequence have 100% identity, but sequences that
are less highly conserved, and have deletions, additions, or
replacements, may have a lower degree of identity. Those skilled in
the art will recognize that several algorithms are available for
determining sequence identity using standard parameters, for
example Blast (Altschul, et al. (1997) Nucleic Acids Res.
25:3389-3402), Blast2 (Altschul, et al. (1990) J. Mol. Biol.
215:403-410), Smith-Waterman (Smith, et al. (1981) J. Mol. Biol.
147:195-197) and ClustalW. Accordingly, for instance, the amino
acid sequences of SEQ ID No: 1 may serve as "subject sequence" or
"reference sequence", while the amino acid sequence or nucleic acid
sequence of a polypeptide or polynucleotide different therefrom can
serve as "query sequence".
[0111] A high affinity for copper is a common feature of
methanobactins. Therefore, methanobactins of the invention are
envisaged to bind copper--specifically Cu(I)--with high binding
affinity. The term "affinity" or "binding affinity" refers to the
strength of the binding of a ligand, such as a methanobactin to
Cu(I). The affinity of the binding of a given ligand to its target
is often determined by measurement of the on-rate constant
(k.sub.on) and off-rate constant (k.sub.off) and calculating the
quotient of k.sub.off to k.sub.on to obtain the equilibrium
dissociation constant K.sub.d(K.sub.d=k.sub.off/k.sub.on) which is
inversely related to the binding affinity, i.e. the lower the
K.sub.d value, the higher the binding affinity. Preferred
methanobactins of the invention bind Cu(I) with an equilibrium
dissociation constant or K.sub.d in the nanomolar range, i.e.
10.sup.-7, 10.sup.-8, 10.sup.-8, in the picomolar range, i.e.
10.sup.-10, 10.sup.-11, 10.sup.-12, or in the femtomolar range,
i.e. 10.sup.-13, 10.sup.-14, 10.sup.-15. Preferably, methanobactins
of the invention bind Cu(I) with a K.sub.d in the femtomolar range,
and are particularly envisaged to bind Cu(I) with a K.sub.d of
10.sup.-15 or less. A number of different methods have been used to
determine metal binding affinity constants for mbs. All measurement
methods show Cu(II)/(I) and Cu(I) affinities of .about.10.sup.21
M.sup.-1 or greater, and is one of the highest known for biological
systems. With respect to this proposal, mb-OB3b has been shown to
remove Cu from metallothionein in both in vitro and in vivo
experiments. Mbs have been shown to solubilize and bind insoluble
forms of Cu(I) under anaerobic conditions, and to extract Cu from
copper minerals, humic materials, and glass Copper (Cu(I)) binding
affinity can for example be measured according the ESI-MS approach
of Banci et al. (Nature. 2010 Jun. 3; 465(7298):645-8) which relies
on the simultaneous monitoring of the variation in the
metallated/non-metallated Cu(I) binding ligand ratios at increasing
concentrations of a competing ligand, namely dithiothreitol (DTT)
or diethyl-dithio-carbamate (DETC). Alternatively, Cu(I) binding
affinities can for example be determined from competition
titrations with the chromophoric copper chelator bathocuproine
disulfonate (BCS) as described by E I Ghazoiani A et al., Proc Natl
Acad Sci USA. 2012 May 29; 109(22):8400-4. Measuring Cu(I) binding
affinities with this method, methanobactins encompassed by the
present invention will also exhibit a K.sub.d of 10.sup.-15 or
less, such as 10.sup.-16, 10.sup.-17, 10.sup.-18, 10.sup.-19,
10.sup.-20, 10.sup.-21 or less.
[0112] As set out previously herein, methanobactins exhibiting high
copper binding affinities (and binding Cu(I) with a K.sub.d of
10.sup.-15 or less, "high-affinity mb") are particularly envisaged
for the uses according to the present invention, and in particular
for massive copper depletion in (acute) WD therapy. However,
methanobactins with a higher K.sub.d (i.e. binding Cu(I) with a
lower affinity) can also be successfully employed in treatment of a
variety of diseases. E.g., in cases when a less extensive and/or
quick copper depletion is desired, methanobactins with a lower
binding affinity towards Cu(I) ("low-affinity mb") can be utilized.
It is also contemplated to combine methanobactins with different
Cu(I) binding affinities for treatment. E.g., one or more treatment
cycles with a high-affinity mb for extensive removal of copper from
a patient can be followed by one or more treatment cycles with a
low-affinity mb for maintenance therapy in order to keep copper
levels low without excessively depleting copper. Vice versa,
treatment could also be started with low-affinity mb and, after one
or more treatment cycles, optionally gradually be continued using
mbs with a higher Cu(I) binding affinity.
[0113] The term "methanobactin" includes naturally occurring
methanobactins and functional variants, fragments and derivatives
thereof which retain the capability of complexing copper (i.e.,
Cu(I) and Cu(II)), and preferably bind Cu(I) with a binding
affinity that is comparable or even higher than that of the
naturally occurring methanobactins.
[0114] As set forth previously, the methanobactin of the invention
may be derived from bacteria listed in FIG. 12, including
Methylocystis spec., Methylosinus spec., Methylomicrobium spec. and
Methylococcus spec. Particularly, the methanobactin may be selected
from (a) a Methylosinus trichosporium OB3b methanobactin (mb-OB3b)
(b) a Methylocystis. strain SB2 methanobactin (mb-SB2), (c) a
Methylococcus capsulatus Bath methanobactin (mb-Bath) (d) a
Methylomicrobium album BG8 methanobactin (mb-BG8), (e) a
Methylocystis strain M methanobactin, (f) a Methylocystis hirsute
CSC1 methanobactin and (g) a Methylocystis rosea methanobactin
(mb-rosea), (h) a Methylosinus sp. strain LW3 methanobactin
(mb-LW3), (i) a Methylosinus sp. strain LW4 methanobactin (mb-LW4),
(j) a Methylocystis sp. strain LW5 (mb-LW5), (k) a Methylosinus sp.
strain PW1 methanobactin (mb-PW1), (I) a Methylocystis parvus OBBP
methanobactin (mb-OBBP), (m) a Cupriavidus basiliensis B-8
methanobactin (mb-B-8), (n) a Pseudomonas extremaustralis 14-3
methanobactin (mb-14-3), (o) a Azospirillum sp. strain B510
methanobactin (mb-B510), (p) a Tistrella mobilis KA081020-065
(mb-mobilis) methanobactin and (q) a Comamonas composti DSM 21721
methanobactin (mb-21721).
[0115] Methanobactins selected for the uses according to the
present invention preferably have the same advantageous properties
as the mb evaluated in the appended examples and/or as described
elsewhere herein.
[0116] In general, methanobactin of the invention may comprise, or
consist of, the following general formula (I):
R.sup.1-(X).sub.2-5-R.sup.2 (I)
wherein R.sup.1 and R.sup.2 are each a 5-membered heterocycle
comprising N and associated with an enethiolate; and each X is
independently selected from any amino acid.
[0117] The term "amino acid" or "amino acid residue" typically
refers to an amino acid having its recognized definition such as an
amino acid selected from the group consisting of: alanine (Ala or
A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp
or D); cysteine (Cys or C); glutamine (Gln or Q); glutamic acid
(Glu or E); glycine (Gly or G); histidine (His or H); isoleucine
(Ile or I): leucine (Leu or L); lysine (Lys or K); methionine (Met
or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser
or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr
or Y); and valine (Val or V), although modified, synthetic, or rare
amino acids may be used as desired. Generally, amino acids can be
grouped as having a nonpolar side chain (e.g., Ala, Ile, Leu, Met,
Gly, Phe, Pro, Val); a negatively charged side chain (e.g., Asp,
Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an
uncharged polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Trp, and
Tyr). The term encompasses naturally occurring and synthetic amino
acids, as well as amino acid analogs and amino acid mimetics that
function in a manner similar to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that function in a
manner similar to a naturally occurring amino acid.
[0118] Particularly, where the methanobactin is mb-OB3b, it is
contemplated to comprise or consist of the formula (11)
R.sup.1GSCYR.sup.2SCM (II),
wherein R.sup.1 is selected from
(N-2-isopropylester-(4-thionyl-5-hydroxy-imidazole) and
N-2-isopropylester-(4-thiocarbonyl-5-hydroxy-imidazolate), and
R.sup.2 is selected from
pyrrolidine-(4-hydroxy-5-thionyl-imidazole) and
pyrrolidine-(4-hydroxy-5-thiocarbonyl-imidazolate). Said mb-OB3b
may in particular comprise or consist of the formula (IV):
##STR00001##
[0119] When complexing zinc or copper, said mb-OB3b is envisaged to
have the following structure (VI)
##STR00002##
wherein Y is selected from copper (Cu(I) or Cu(II)) or zinc (Zn(I)
or Zn(II)).
[0120] Where the methanobactin is mb-SB2, it is envisaged to be of
the formula (III)
R.sup.1ASR.sup.2AA (III)
wherein R.sup.1 is 4-guanidinobutanoyl-imidazole and R.sup.2 is
1-amino-2-hydroxy-oxazolone.
[0121] Said mb-SB2 may in particular be of the formula (V):
##STR00003##
[0122] When complexing zinc or copper, said mb-SB2 is envisaged to
have the following structure (VII):
##STR00004##
wherein Y is selected from copper (Cu(I) or Cu(II)) or zinc (Zn(I)
or Zn(II).
[0123] This specific MB peptide mb-SB2 from Methylocystis strains
SB2 acts most effectively as a promising copper chelator compared
to existing clinically approved cooper chelators such as D-PA.
mb-SB2 even depletes copper at least as effective as another MB
peptide mb-OB3b derived from Methylosinus trichosporium OB3b, as
mentioned above.
[0124] MB peptide mb-SB2 from Methylocystis strains SB2 is
structurally and chemically deviating from other MB peptides,
especially from mb-OB3b derived from Methylosinus trichosporium
OB3b). Yet, a structurally different and less heavy peptide within
the MB-family exhibits a similar therapeutic potential as a copper
chelator.
[0125] When used herein, the terms "complexing" and "binding" are
used interchangeably, i.e. for instance a methanobactin "binding"
copper is to be understood as a methanobactin "complexing" copper,
and vice versa. The term "complexing" generally means forming a
complex consisting of a central ion and surrounding array of
molecules that are known as ligands or complexing agents. For the
present invention, the central ion will be copper (i.e. Cu(I) or
Cu(II)), or zinc (i.e. Zn(I) or Zn(II)) and the ligand will be
methanobactin. One methanobactin will typically complex one copper
or zinc ion, forming a methanobactin-copper complex or a
methanobactin-zinc complex, respectively. The person skilled in the
art will readily understand that methanobactin-copper complexes
will typically form after administration of the methanobactin to
the subject, when methanobactin complexes and thereby depletes
(excess) copper in the subject's body. Methanobactin-zinc complexes
are envisaged herein as stabilized forms of methanobactin as
described below.
[0126] As set out elsewhere herein, methanobactin fragments,
variants and derivatives are also envisioned for the uses described
herein.
[0127] "Methanobactin fragments" are "functional" or
"copper-binding" peptides that retain the copper-binding region of
the "parent" methanobactin they are derived from. It is for
instance envisaged to provide particularly small methanobactin
fragments that are capable of crossing the blood-brain-barrier in
order to effectively treat neurological WD or other conditions
associated with copper overload in the CNS.
[0128] The term "methanobactin variant" refers to methanobactins
having the general methanobactin formula of a "parent"
methanobactin (FIG. 12), but containing at least one amino acid
substitution, deletion, or insertion as compared to the parent
methanobactin, provided that the variant retains the desired
copper-binding affinity and/or biological activities described
herein.
[0129] "Methanobactin derivatives" are chemically modified
methanobactins. Generally, all kind of modifications are comprised
by the present invention as long as they do not abolish the
beneficial effects of the methanobactins. That is, methanobactin
derivatives preferably retain the copper-binding affinity and/or
biological activity of the methanobactins they are derived from.
Methanobactin derivatives also include stabilized methanobactins as
described in the following.
[0130] Possible chemical modifications in the context of the
present invention include acylation, acetylation or amidation of
the amino acid residues. Other suitable modifications include,
e.g., extension of an amino group with polymer chains of varying
length (e.g., XTEN technology or PASylation.RTM.), N-glycosylation,
O-glycosylation, and chemical conjugation of carbohydrates, such as
hydroxyethyl starch (e.g., HESylation.RTM.) or polysialic acid
(e.g., PolyXen.RTM. technology). Chemical modifications such as
alkylation (e. g., methylation, propylation, butylation),
arylation, and etherification may be possible and are also
envisaged. Further chemical modifications envisaged herein are
ubiquitination, conjugation to therapeutic or diagnostic agents,
labeling (e.g., with radionuclides or various enzymes), and
insertion or substitution by chemical synthesis of non-natural
amino acids.
[0131] Other possible modifications may involve replacement of
oxazolone group with the more stable imidazolone or pyrazinedione
group. Gene additions and/or deletions of genes from the operons of
Group II methanobactins into Group I or vice versa should result in
alteration may result in a change in the type of ring. Replacement
of oxazolone group(s) with either imidazolone or pyrazinedione
group(s) should increase the stability of methanobactin to the
point where oral administration may be possible.
[0132] For the purpose of the invention the methanobactin as
defined above also includes the pharmaceutically acceptable salt(s)
thereof. The phrase "pharmaceutically acceptable salt(s)", as used
herein, means those salts of methanobactins that are safe and
effective for treatment. Pharmaceutically acceptable salts include
those formed with anions such as those derived from hydrochloric,
phosphoric, acetic, oxalic, tartaric acids, choline etc., and those
formed with cations such as those derived from sodium, potassium,
ammonium, calcium, ferric hydroxides, isopropylamine,
triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0133] As set forth previously, the methanobactin fragments,
variants and derivatives preferably retain the advantageous
capabilities of the methanobactins evaluated in the appended
examples.
Biological effect
[0134] As explained previously, methanobactins according to the
present invention are envisaged to elicit the desired biological
effects as described herein, i.e. they are preferably capable of
binding copper with a high binding affinity, and effecting its
depletion from the system and preferably its excretion via the
bile. Without wishing to be bound by specific theory, the present
inventors have established that mitochondrial impairment due to an
increased copper load progressively increases with disease state in
livers from LPP-/- rats, a model of WD. As shown in the appended
examples, methanobactins are able to rapidly deplete mitochondrial
and hepatocellular copper. It is envisaged that methanobactins as
described herein preferably exhibit the same advantageous
characteristics.
[0135] Thus, methanobactins are envisaged for the use of treatment
of Wilson Disease, wherein the treatment reduces (i) whole liver
copper levels, (ii) overall hepatocyte copper levels and/or (iii)
hepatocyte mitochondrial copper levels. Moreover, methanobactins
will preferably effect excretion of (excess) copper via the
bile.
Therapeutic effect
[0136] Administration of methanobactins to subjects in need thereof
(in particular WD patients) is expected to elicit a therapeutic
effect. The term "therapeutic effect" as used herein generally
refers to a desirable or beneficial impact of a treatment, e.g.
amelioration or remission of the disease manifestations. The term
"manifestation" of a disease is used herein to describe its
perceptible expression, and includes both clinical manifestations,
hereinafter defined as indications of the disease that may be
detected during a physical examination and/or that are perceptible
by the patient (i.e., symptoms), and pathological manifestations,
meaning expressions of the disease on the cellular and molecular
level. Amelioration or remission of WD manifestations can be
assessed by using the same tests as described for diagnosis of WD.
Additionally or alternatively it is also possible to evaluate the
general appearance of the respective patient (e.g., fitness,
well-being) which will also aid the skilled practitioner to
evaluate whether a therapeutic effect has been elicited. The
skilled person is aware of numerous other ways which are suitable
to observe a therapeutic effect of the compounds of the present
invention.
Stabilized methanobactin
[0137] In a further aspect, the present inventors have discovered
ways to provide methanobactins in stabilized form.
[0138] Without wishing to be bound by specific theory, it was
discovered that mb-OB3b is susceptible to time- and/or
temperature-dependent decay. Thus, in order to allow an increased
biological half-life and/or plasma concentration of methanobactins
in the subject's body during (and after) treatment, and therefore
preferably improve therapeutic efficacy and provide for a long-term
effect of methanobactin treatment, it is envisaged to provide
stabilized forms of methanobactin. Generally, any form of chemical
modification is conceivable that enables stabilization of the
methanobactins (see also methanobactin derivatives). Specifically,
the present invention provides stabilized forms of methanobactins
complexing zinc, i.e. Zn(I) or Zn(II). Unless denoted otherwise,
the term "zinc" generally refers to Zn(I) and/or Zn(II).
Furthermore, the present inventors found that methanobactins can be
stabilized when provided at a pH of .gtoreq.9. Thus, it is
envisaged to provide stabilized forms of methanobactins, i.e.
methanobactins complexing Zn(I) or Zn(II) and/or being provided at
a pH of 9, 10, or 11, for the uses and methods of the present
invention. In particular, such stabilized forms of methanobactins
can be used for treatment of WD according to the treatment regimen
set out elsewhere herein, and/or for treatment of acute phase WD.
Stabilized forms of methanobactins as described herein have not
been used as medication before. The present invention thus also
comprises a pharmaceutical composition comprising a stabilized
methanobactin, wherein said methanobactin complexes Zn(I) and/or
Zn(II) and/or is provided at a pH .gtoreq.9. The skilled
practitioner will readily understand that when the methanobactin is
provided at a pH .gtoreq.9 for reasons of stabilization, the
pharmaceutical composition comprising methanobactin (optionally
complexing zinc) is required to have a pH .gtoreq.9, too.
[0139] A pharmaceutical composition comprising a methanobactin
complexing Zn(I) or Zn(II) can be provided by contacting an amount
of Zn(I) and/or Zn(II) and an amount of methanobactin in a ratio of
1:1 in aqueous solution. Use of equimolar amounts of zinc and
methanobactin may be beneficial in order to avoid an excess amount
of free zinc ions in the pharmaceutical composition.
Pharmaceutical composition
[0140] As set out in the foregoing, a pharmaceutical composition
comprising methanobactin, in particular in stabilized form, is also
envisaged herein. In particular, said pharmaceutical composition is
envisaged for the use of treatment of Wilson Disease, wherein the
treatment reduces (i) whole liver copper levels, (ii) overall
hepatocyte copper levels and/or (iii) hepatocyte mitochondrial
copper levels. I.e., the pharmaceutical composition preferably
comprises methanobactins complexing Zn(I) or Zn(II) and/or are
provided in a pH .gtoreq.9. Said composition may be stable at
37.degree. C. for at least 20, 50, 75, 100, 125, 150 hours or more.
Accordingly, further aspects of the invention include a
pharmaceutical composition comprising (in particular, stabilized)
methanobactin as described herein and the use of the said
(stabilized) methanobactin for the manufacture of a pharmaceutical
composition. The term "pharmaceutical composition" particularly
refers to a composition suitable for administering to a human.
However, compositions suitable for administration to non-human
animals are also envisaged herein.
[0141] The pharmaceutical composition and its components (i.e.
active ingredients and optionally excipients or carriers) are
preferably pharmaceutically acceptable, i.e. capable of eliciting
the desired therapeutic effect without causing any undesirable
local or systemic effects in the recipient. Pharmaceutically
acceptable compositions of the invention may in particular be
sterile and/or pharmaceutically inert. Specifically, the term
"pharmaceutically acceptable" may mean approved by a regulatory
agency or other generally recognized pharmacopoeia for use in
animals, and more particularly in humans.
[0142] The (stabilized) methanobactin described herein is
preferably present in the pharmaceutical composition in a
therapeutically effective amount. By "therapeutically effective
amount" is meant an amount of methanobactin that elicits the
desired therapeutic effect. The exact amount dose will depend on
the purpose of the treatment, and will be ascertainable by one
skilled in the art using known techniques. Therapeutic efficacy and
toxicity can be determined by standard pharmaceutical procedures in
cell cultures or experimental animals, e.g., ED.sub.50 (the dose
therapeutically effective to 50% of the population) and LD.sub.50
(the dose lethal to 50% of the population). The dose ratio between
therapeutic and toxic effects is the therapeutic index, and it can
be expressed as the ratio, ED.sub.50/LD.sub.50. Pharmaceutical
compositions that exhibit large therapeutic indices are generally
preferred.
[0143] The pharmaceutical composition is envisaged to comprise a
methanobactin as described herein, particularly in stabilized form,
and preferably in a therapeutically effective amount, optionally
together with one or more carriers, excipients and/or additional
active agents.
[0144] "Excipients" include fillers, binders, disintegrants,
coatings, sorbents, antiadherents, glidants, preservatives,
antioxidants, flavoring, coloring, sweeting agents, solvents,
co-solvents, buffering agents, chelating agents, viscosity
imparting agents, surface active agents, diluents, humectants,
carriers, diluents, preservatives, emulsifiers, stabilizers and
tonicity modifiers. Exemplary suitable carriers for use in the
pharmaceutical composition of the invention include saline,
buffered saline, dextrose, and water.
Additional active agents
[0145] The pharmaceutical composition may also comprise further
active agents effective for treatment of the particular disease
concerned. By way of example, active agents presently used for
treatment of WD include the copper chelators d-penicillamine
(D-PA), trientine (TETA) and tetrathiomolybdate (TTM), as well as
zinc salts. For treatment of cancer, useful additional active
agents include known chemotherapeutic agents, including alkylating
agents, antimetabolites, anti-microtubule agents, topoisomerase
inhibitors; cytotoxic antibiotics, and monoclonal antibodies.
Active agents for treatment of neurodegenerative disorders include,
without limitation, levodopa and derivatives thereof, dopamine
agonists, MAO-B inhibitors, catechol-O-methyltransferase (COMT)
inhibitors, anticholinergics, amantadine, cholinesterase
inhibitors, memantine and riluzole. It is within the knowledge of
the skilled person to choose suitable additional agents for
treatment of a specific disease.
Formulation
[0146] The pharmaceutical compositions of the invention can be
formulated in various forms, e.g. in solid, liquid, gaseous or
lyophilized form and may be, inter alia, in the form of an
ointment, a cream, transdermal patches, a gel, powder, a tablet,
solution, an aerosol, granules, pills, suspensions, emulsions,
capsules, syrups, liquids, elixirs, extracts, tincture or fluid
extracts or in a form which is particularly suitable for the
desired method of administration. Processes known per se for
producing medicaments are indicated in Forth, Henschler, Rummel
(1996) Allgemeine und spezielle Pharmakologie und Toxikologie,
Urban & Fischer.
Administration
[0147] A variety of routes are conceivable for administration of
the methanobactins and pharmaceutical compositions according to the
present invention. Typically, administration will be accomplished
parentally, but oral administration is also envisaged. Methods of
parenteral delivery include topical, intra-arterial, intramuscular,
subcutaneous, intramedullary, intrathecal, intraventricular,
intravenous, intraperitoneal, intrauterine, intravaginal,
sublingual or intranasal administration.
Cancer treatment
[0148] The methanobactins and pharmaceutical composition disclosed
herein is also envisaged for treatment of various cancers. Many
cancer types exhibit increased intratumoral copper and/or altered
systemic copper distribution. It has been acknowledged that copper
serves as a limiting factor for multiple aspects of tumor
progression, including growth, angiogenesis and metastasis.
Methanobactins and pharmaceutical compositions described herein are
thus promising tools to inhibit these processes.
[0149] As reviewed by Denoyer et al., Metallomics. 2015 Nov. 4;
7(11):1459-76, high serum copper concentrations are reportedly
associated with a variety of cancers including lymphoma, reticulum
cell sarcoma, bronchogenic and laryngeal squamous cell carcinomas,
cervical, breast, stomach and lung cancers, and elevated serum
copper has been found to correlate with the stage of the disease
and its progression in colorectal and breast cancers as well as
hematological malignancies, including chronic lymphoid leukemia,
non-Hodgkin's lymphoma, multiple myeloma and Hodgkin's lymphoma.
Elevated copper in malignant tissues has also been established in a
range of cancer types, including breast, ovarian, cervical, lung,
stomach and leukemia. The role of copper in cancer development and
progression remains to be elucidated. Elevated levels of redox
active copper may lead to oxidative stress and chronic inflammation
which are intrinsically linked to malignant transformation of
cells. Therefore, it has been proposed that elevated copper in
tissues or serum may be a risk factor for carcinogenesis.
Methanobactins and pharmaceutical compositions described herein
could be used to reduce overall copper levels and thereby minimize
the risk for developing cancer.
[0150] Copper has also been reported to influence various molecular
pathways inducing a pro-angiogenic response. Copper is capable of
directly binding to angiogenic growth factors, and to influence
their secretion and expression via activation of NF.sub..kappa.B.
Moreover, copper has been found to directly influence the ability
of cancerous cells to invade and metastasize.
[0151] Papa et al., Genes Cancer. 2014 April; 5(1-2):15-21 further
reported that the copper-dependent dismutase SOD1 is overexpressed
in many cancers to cope with elevated levels of reactive oxygen
species (ROS) caused by deregulation of the anti-oxidant machinery
of the mitochondrial matrix. Depletion of copper is envisioned to
reduce overall SOD1 activity, and thereby diminishing tumor cell
proliferation and survival. In accordance, methanobactins and
pharmaceutical compositions described herein are also envisaged for
treatment of cancers which overexpress SOD1.
[0152] Known copper-chelating agents (such as D-PA) have been
investigated for their capacity to control angiogenesis and thus by
inference, to impair cancer growth and metastasis. However,
methanobactins and pharmaceutical compositions described herein
have not been elucidated for cancer treatment before. Further
provided herein is therefore the use of methanobactins and
pharmaceutical compositions described herein for treatment of
various cancers, including without limitation, reticulum cell
sarcoma, bronchogenic and laryngeal squamous cell carcinomas,
cervical, breast, colorectal, stomach, lung cancers, liver cancer,
prostate cancer, brain cancer, chronic lymphoid leukemia,
non-Hodgkin's lymphoma, multiple myeloma and Hodgkin's
lymphoma.
Neurodegenerative disorders
[0153] Protein aggregation is a notable feature of various
neurodegenerative disorders, including Parkinson disease, Alzheimer
disease, Prion Disease including Creutzfeldt-Jakob disease (CJD),
fatal familial insomnia (FFI), and Gerstmann-Straussler-Scheinker
syndrome (GSS), familial amyotrophic lateral sclerosis (fALS) and
many others. An increasing number of studies suggest that
transition metals are able to accelerate the aggregation process of
several proteins found in pathological deposits, and that in
particular copper produces a most remarkable acceleration of
aggregation. Hence, copper depletion by methanobactin treatment is
therefore contemplated to reduce protein aggregation, thereby
alleviating or even revert signs and symptoms of the disease.
[0154] It is therefore further envisaged to use methanobactins and
pharmaceutical compositions described herein for treatment of
neurodegenerative diseases including Parkinson Disease, Alzheimer
Disease, Prion Disease, Huntington Disease and fALS.
Diabetes
[0155] Moreover, defective copper regulation has been suggested as
a causative mechanism of organ damage in diabetes which has been
attributed to impaired anti-oxidant defence mechanisms and
oxidative stress. Strikingly, TETA treatment was shown to act on
mitochondrial proteins with roles in energy metabolism in diabetes
patients, and resulted in restoration of cardiac structure and
function (Jullig et al., Proteomics Clin Appl. 2007 April;
1(4):387-99). As demonstrated in example 3 of the present
application, methanobactins are surprisingly capable of efficiently
removing accumulated mitochondrial copper--and are therefore, too,
promising agents for a novel diabetes therapy based on the
depletion of excess copper levels, particularly from the
mitochondria, thereby reducing overall oxidative stress and tissue
damage. In line with previous studies, methanobactins are
particularly envisaged to improve diabetic cardiomyopathy and
arterial and/or renal structure/function and to ameliorate
left-ventricular (LV) hypertrophy in diabetic patients (see Zhang
et al. Cardiovasc Diabetol. 2014 Jun. 14; 13:100).
Other disorders
[0156] Further disease and disorders eligible for treatment with
methanobactin and pharmaceutical compositions described herein
comprise bacterial infections, inflammatory diseases, fibrosis,
cirrhosis, lead and/or mercury poisoning.
[0157] In particular, during bacterial infections macrophages
release copper in an attempt to kill invading microbes through
copper toxicity. This leads to the induction of copper stress
responses in invading microbes (Gleason et al., PNAS 2014 April;
vol. 111, no. 16:5866-5871). According to Gleason et al. (2014)
this high level of host copper is favorable for SOD5 activation of
C. albicans. C. albicans is the most prevalent human fungal
pathogen--a yeast fungus--, which is able to combat the host immune
response (e.g. macrophages) with its expressed superoxide dismutase
5 (SOD5), a monomeric copper-only SOD. Depletion of copper is
therefore more importantly to reduce overall SOD5 activity, thus
reducing human fungal pathogens during bacterial infections. In
accordance, methanobactins and pharmaceutical compositions
described herein are also envisaged for treatment of human fungal
pathogens during bacterial infections such as C. albicans, which
overexpresses SOD5.
[0158] Therefore, the present invention encompasses a
pharmaceutical composition, wherein bacterial infections are
favorable for human fungal pathogens, preferably said human fungal
pathogen is Candida albicans.
Treatment
[0159] The term "treatment" in all its grammatical forms includes
therapeutic or prophylactic treatment of the diseases described
herein, in particular WD. A "therapeutic or prophylactic treatment"
comprises prophylactic treatments aimed at the complete prevention
of clinical and/or pathological manifestations or therapeutic
treatment aimed at amelioration or remission of clinical and/or
pathological manifestations. The term "treatment" thus also
includes the amelioration or prevention of the diseases described
herein, specifically WD.
[0160] The terms "subject" or "individual" or "animal" or "patient"
are used interchangeably herein to refer to any subject,
particularly a mammalian subject, for whom therapy is desired.
Mammalian subjects include humans, non-human primates, dogs, cats,
guinea pigs, rabbits, rats, mice, horses, cattle and the like, with
human subjects being particularly envisaged for treatment according
to the invention.
Dosage
[0161] The exact dose of methanobactin may depend on the purpose of
the treatment (e.g. prophylactic or maintenance therapy vs.
treatment of acute WD), and will be ascertainable by one skilled in
the art using known techniques. Adjustments for route of
administration, age, body weight, general health, sex, diet, time
of administration, drug interaction and the severity of the
condition may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art. In general, dosages of
1 mg/kg body weight (bw) may be capable of eliciting the desired
therapeutic effect as described elsewhere herein. Exemplary dosages
applicable in the uses and methods of the invention include doses
between 1 mg/kg bw and 1000 mg/kg bw, such as between 1 mg/kg bw
and 100 mg/kg bw, and particularly between 1 mg/kg bw and 50 mg/kg
bw, such as 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 mg/kg
bw.
Kit
[0162] It is also envisaged that methanobactins, in particular in
stabilized form, and pharmaceutical compositions described herein
can be provided as part of a kit. Accordingly, in a further aspect,
the present invention also relates to a kit comprising
methanobactins, specifically such in stabilized form, or
pharmaceutical compositions comprising the same for the use of
treatment of Wilson Disease, wherein the treatment reduces (i)
whole liver copper levels, (ii) overall hepatocyte copper levels
and/or (iii) hepatocyte mitochondrial copper levels.
[0163] The kit may be a kit of two or more parts, and comprises the
methanobactins described previously, or a pharmaceutical
composition comprising the same, and further active agents and/or
pharmaceutical excipients. For instance, the kit may comprise one
or more active agents or pharmaceutical compositions comprising the
same useful for treating WD, such as d-penicillamine (D-PA),
trientine (TETA) and tetrathiomolybdate (TTM), and/or zinc salts.
The kit components may be contained in a container or vials. It is
envisaged that the kit components are administered simultaneously,
or sequentially, or separately with respect to the administration
of the methanobactins or pharmaceutical compositions comprising the
same. The present invention further encompasses the application of
the kit components via different administration routes. E.g.,
conventional copper chelators may be administered orally, whereas
the parenteral route of administration can be used for
methanobactins.
[0164] It must be noted that as used herein, the singular forms
"a", "an", and "the", include plural references unless the context
clearly indicates otherwise. Thus, for example, reference to "a
reagent" includes one or more of such different reagents and
reference to "the method" includes reference to equivalent steps
and methods known to those of ordinary skill in the art that could
be modified or substituted for the methods described herein.
[0165] Unless otherwise indicated, the term "at least" preceding a
series of elements is to be understood to refer to every element in
the series. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
present invention.
[0166] The term "and/or" wherever used herein includes the meaning
of "and", "or" and "all or any other combination of the elements
connected by said term".
[0167] The term "about" or "approximately" as used herein means
within 20%, preferably within 10%, and more preferably within 5% of
a given value or range. It includes, however, also the concrete
number, e.g., about 20 includes 20.
[0168] The term "less than" or "greater than" includes the concrete
number. For example, less than 20 means less than or equal to.
Similarly, more than or greater than means more than or equal to,
or greater than or equal to, respectively.
[0169] Throughout this specification and the claims which follow,
unless the context requires otherwise, the word "comprise", and
variations such as "comprises" and "comprising", will be understood
to imply the inclusion of a stated integer or step or group of
integers or steps but not the exclusion of any other integer or
step or group of integer or step. When used herein the term
"comprising" can be substituted with the term "containing" or
"including" or sometimes when used herein with the term
"having".
[0170] When used herein "consisting of" excludes any element, step,
or ingredient not specified in the claim element. When used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim.
[0171] In each instance herein any of the terms "comprising",
"consisting essentially of" and "consisting of" may be replaced
with either of the other two terms.
[0172] It should be understood that this invention is not limited
to the particular methodology, protocols, material, reagents, and
substances, etc., described herein and as such can vary. The
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention, which is defined solely by the claims.
[0173] All publications and patents cited throughout the text of
this specification (including all patents, patent applications,
scientific publications, manufacturer's specifications,
instructions, etc.), whether supra or infra, are hereby
incorporated by reference in their entirety. Nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention. To the
extent the material incorporated by reference contradicts or is
inconsistent with this specification, the specification will
supersede any such material.
EXAMPLES
Materials and Methods
[0174] Patient-derived samples
[0175] Livers from four WD patients with liver failure transplanted
at the University Hospital Heidelberg for Wilson disease were
included in this study. Two patients (No. 1 and 2) had received no
prior copper chelation therapy, while two patients (No. 3 and 4)
presented with liver failure after D-PA treatment. Patients gave
their informed consent, and the study was approved by the ethical
committee of the Medical University of Heidelberg, Germany. Upon
explantation, WD patient livers were shock frozen in LN2 and stored
at -80.degree. C. Thawed samples were immediately fixed for
histological and electron microscopy analyses.
Animals
[0176] The LPP rat strain was provided by Jimo Borjigin, University
of Michigan, Ann Arbor, USA (ahmed et al., log. cit.). Rats were
maintained on an ad lib Altromin 1314 diet (Altromin Spezialfutter
GmbH, Germany) and tap water. The copper content of the diet was 13
mg/kg. All animals were treated under the guidelines for the care
and use of laboratory animals of the Helmholtz Center Munich.
LPP-/- rats are provided with an ATP7b mutation and thus are
ATP7b-/-. Heterozygous LPP+/- rats served as controls in this
study.
Animal treatments
[0177] Animal experiments were approved by the government
authorities of the Regierung von Oberbayern, Munich, Germany.
In vivo treatments
[0178] LPP rats were treated with MB either by daily i.p.
injections for 3 or 5 consecutive days, or twice daily i.p.
injections for 8 consecutive days at a dose of 150 mg/kg bw or 4
days via the drinking water with D-PA at a dose of 100 mg/kg bw/d
or TETA at 480 mg/kg bw/d, respectively (Togashi et al., Hepatology
15, 82-87 (1992)). Based on a mean copper content of 250 .mu.g/g
w.w. in Atp7b-/- rat livers at the age of treatment start (Zischka
et al., log. cit.), a LPP-/- rat liver of 8 g w.w. contains around
31.5 .mu.mol copper. Single MB doses were chosen equimolar to this
copper amount. In case of D-PA the administered dose and chosen
application route has been reported to successfully prevent the
onset of hepatitis in LEC rats in long-term applications (Togashi
et al., log. cit.). Subchronic toxicity studies in rats have
revealed no toxicity of TETA at a dose of 3000 ppm via the drinking
water (Greenman et al., Fundam Appl Toxicol 29, 185-193 (1996)).
Assuming 40 ml water intake per day for a rat weighing 250 g this
translates to a dose of 480 mg/kg bw/d. With respect to the mean
liver copper content in LPP-/- rats aged 85 days, the molar ratios
of the chelators applied was MB 1:D-PA 4.3:TETA 17.4, respectively.
For the intravenous MB application (150 mg/kg bw), catheters
connected to a PinPort.TM. (Instech Laboratories, Inc., USA) were
inserted in the femoral vein of the rat, fixed with non-absorbable
sutures and subcutaneously tunneled and exteriorized through a skin
incision made between the shoulders.
Liver perfusion
[0179] LPP-/- livers (animal age 79-83 days) were perfused in a
single pass manner with Krebs-Ringer bicarbonate solution
containing 5 mM glucose (Beuers et al., J Biol Chem 278,
17810-17818 (2003)). The medium was gassed with 95% O2/5% CO2 and
kept at 37.degree. C. Rat livers were perfused via the portal vein
(Beuers et al., log. cit.), the right lateral liver lobe was
ligated and its copper content served as pre-perfusion control
(Beuers et al., Hepatology 33, 1206-1216 (2001)). After cannulation
of the bile duct a 20 min sample of bile was collected before the
copper chelators were continuously added to the perfusion medium.
Bile and outflow perfusate were collected in 10 min intervals as
described elsewhere (Beuers et al., log. cit.). D-PA*HCl (20 mg/108
.mu.mol), TETA*2HCl (20 mg/91 .mu.mol), TTM*2NH4 (10 mg/38 .mu.mol)
and MB (40 mg/35 .mu.mol) were each dissolved in 50 ml 0.9% NaCl,
and continuously added to the perfusion medium via a perfusion pump
(Perfusor, Braun, Melsungen) within 2 hours. The molar ratios of
the chelators applied was MB 1:D-PA 3.1:TETA 2.6:TTM 1.1. LDH in
the outflow perfusate was measured every ten minutes as described
(Beuers et al., log. cit.). Control perfusions were done with
Krebs-Ringer bicarbonate solution only.
Histological examination, plasma/serum AST and bilirubin
[0180] Formalin-fixed, paraffin-embedded liver samples were cut
into 4 .mu.m-thick sections and either stained with hematoxylin and
eosin for standard analyses or with Masson trichrome for analysis
of fibrotic tissue. AST activity and bilirubin concentration in
animal plasma or serum were measured with a Reflotron system
(Roche).
Mitochondrial analyses
[0181] Mitochondria were derived either from frozen explanted
livers from WD patients or from freshly prepared rat liver
homogenates as described previously (Zischka et al., Anal Chem 80,
5051-5058 (2008)). Specifically, mitochondria were purified by
differential and density gradient centrifugation using either
Percoll.RTM. or Nycodenz.RTM.. Fresh rat liver mitochondria were
used for respiratory measurements, chelator treatments, analyses of
swelling (MPT), transmembrane potential (.DELTA..sub..PSI.m),
polarization experiments, ATP synthesis and fixed with
glutaraldehyde for subsequent electron microscopy analyses. Stored
frozen mitochondria were used for respiratory complex IV activity
and metal analyses.
[0182] Functional integrity of isolated mitochondria was assessed
by standard respiratory measurements in a Clark-type oxygen
electrode (Oxygraph, Hansatech Instruments) (Zischka et al., log.
cit.). Kit-based assays were used to analyze ATP synthesis (ATP
Bioluminescence Assay Kit, Roche) (Zischka et al., log. cit.).
Mitochondrial swelling was measured by light scattering with an
absorbance reader in 96-well plate formats at 540 nm (Schulz et
al., Biochimica et biophysica acta 1828, 2121-2133 (2013)).
Assessment of .DELTA..sub..PSI.m was followed by Rh123 fluorescence
quenching in a 96-well plate reader (BioTek) (Schulz et al., log.
cit.). Polarisation was measured in DPH and TMA-DPH-dyed
mitochondria (Prendergast et al., log. cit.). In brief,
mitochondria (3 mg/ml) were incubated for 30 minutes at 37.degree.
C. either with DPH or TMA-DPH (50 .mu.M and 20 .mu.M,
respectively). Parallel and perpendicular fluorescence was assessed
in duplicates at ex: 366 nm and em: 425 nm. Polarisation was
calculated (Grebowski et al., Biochim Biophys Acta 1828, 241-248
(2013)) in mPol using the formula
P=(I.sub..parallel.-G
*I.sub..perp.)/(I.sub..parallel.+G*I.sub..perp.); G=0.89.
In vitro treatment of isolated mitochondria with chelators
[0183] Freshly isolated density gradient purified LPP-/-
mitochondria with elevated copper were subjected to chelator
treatments for 30 min with either 2 mM D-PA, TETA, TTM or MB, and
subsequently re-purified by a Nycodenz.RTM.-gradient to separate
copper in solution from copper incorporated into mitochondria. In
validation experiments, mitochondria from control rats (LPP+/-)
were incubated with 1 mM DTT for 5 min at RT and thereafter Cu2+
was added (final concentrations 200-600 .mu.M) for additional 20
min. Copper loaded mitochondria were then re-purified by
Nycodenz.RTM.-gradient centrifugation and subsequently treated with
chelators as above.
Cell culture
[0184] HepG2 cells were kept in MEM with 2% FCS. We found that
Zn-MB is time stable at 37.degree. C. in contrast to metal-free
methanobactin (FIG. 9C). Therefore, Zn-MB, generated by preparing a
20 mM MB solution and adding an equimolar concentration of Zn
solution under pH control, was used in cell culture
experiments.
[0185] Neutral red cell toxicity assay was done as described
elsewhere (Repetto et al., Nat Protoc 3, 1125-1131 (2008)). In
brief, 2.times.104 cells were incubated for 24 hours either with
medium alone (containing 2% FCS, negative control), 0.05 to
[0186] 1 mM Zinc-MB, or 0.25 mM CCCP as mitochondriotoxic positive
control and subsequently analyzed by neutral red.
[0187] For immunofluorescence staining, 2.times.104 cells were
incubated with either medium alone, 500 .mu.M MB or 250 .mu.M CCCP
in black 96-well plates with clear glass bottom. Staining was done
by 1.6 .mu.M Hoechst 33342 (ex 360-400 nm, em 410-480 nm), 300 nM
MitoTracker.RTM. red (ex 620-640 nm, em 650-760 nm), and 1 .mu.M
nonyl acridine orange (NAO, ex 460-490 nm, em 500-550 nm) for 40
minutes at 37.degree. C. After a washing step, fluorescence was
analyzed.
[0188] To determine the cellular de-coppering efficiency of MB,
cells were pretreated with 2% FCS containing medium or 15 .mu.M
copper-histidine for 24 h and subsequently subjected to a 24 h
treatment with 500 .mu.M MB. Thereafter, cells were washed two
times and counted. Copper in 2.5.times.106 cells was determined by
ICP-OES after wet ashing of samples with 65% nitric acid.
[0189] Cellular MB uptake was determined from cell lysates
incubated for 2 or 24 h with MB at different concentrations by a
competitive ELISA using a monoclonal anti-MB antibody.
Generation of HLC from Wilson disease patients
[0190] Urinary epithelial cells were pelleted at 400.times. g for
10 min from freshly donated mid-stream urine (Zhou et al., J Am Soc
Nephrol 22, 1221-1228 (2011)). Cells were cultured in urinary cell
medium (UCM) consisting of Dulbecco's modified Eagle medium/Ham's
F-12 culture medium (DMEM/F12, Lonza) supplemented with 10% fetal
bovine serum (FBS, PAA), 0.1 mM non-essential amino acids (NEAA,
Sigma), 0.1 mM .beta.-mercaptoethanol, 1 mM GlutaMAX (Life
Technologies), and SingleQuot Kit CC-4127 REGM (Lonza). Urinary
epithelial cells were reprogrammed by nucleofection of episomal
expression vectors pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, and pCXLE-hUL
(Addgene) using the Amaxa Basic Nucleofector Kit (Lonza, VPI-1005).
iPS cells (iPSCs) were maintained on Matrigel-coated plates in
mTeSR cell culture medium and dissociated with 1 U/ml dispase (Stem
Cell Technologies) into small clusters and subcultured every 5 to 7
days. WD iPSCs were differentiated into hepatocyte-like cells
(HLCs) by a modification of the method reported previously (Basma
et al., Gastroenterology 136, 990-999 (2009)). 5.times.104 iPSCs
were plated in single cells onto 6-well plates precoated with
Matrigel. The next day, the medium was changed to DMEM/F12 enriched
with 100 ng/ml recombinant Activin-A (Peprotech), 100 ng/ml
fibroblast growth factor-2 (FGF2, Peprotech) plus 50 ng/ml
recombinant human Wnt3a (R&D Systems). Subsequently, medium was
changed according to standard protocols up to day 14 (Basma et al.,
log. cit.). Cells were characterized by flow cytometry and by
qRT-PCR analysis to assess typical markers of hepatocyte
lineage.
[0191] HLCs were incubated at day 14 with Cu-histidin (15 .mu.M)
for 24 h in a 6 well plate. The following day, medium was removed
and changed to OptiMEM containing MB (300 .mu.M). After 24 h
incubation, washed cells were collected, counted and their copper
content assessed.
Methanobactin (MB) antibody generation and competitive ELISA
[0192] Lou/c rats were immunized subcutaneously and
intraperitoneally with a mixture of MB coupled to ovalbumine (50
.mu.g) (Squarix, Marl, Germany), 5 nmol CPG oligonucleotide (Tib
Molbiol, Berlin), 500 .mu.l PBS and 500 .mu.l incomplete Freund's
adjuvant. A boost without adjuvant was given six weeks after the
primary injection. Fusion was performed using standard procedures.
Tissue culture supernatants (TCS) were tested in a solid-phase
immunoassay with MB coupled to BSA or an irrelevant peptide coupled
to BSA coated ELISA plates at a concentration of 4 .mu.g/ml.
Antibodies (mAb) from TCS bound to MB were detected with HRP
conjugated mAbs against the rat IgG isotypes (TIB173 IgG2a, TIB174
IgG2b, TIB170 IgG1 all from ATCC, R-2c IgG2c homemade), thus
avoiding mAbs of IgM class. HRP was visualized with ready to use
TMB (1-Step.TM. Ultra TMB-ELISA, Thermo). 86 hybridomas that
reacted specifically with MB were frozen and the antibody
containing TCS were used for further analysis.
[0193] Competitive ELISA. All 86 TCS were diluted 1:10 with buffer
(PBS, 5% FCS, 0.01% sodium acide). 50 .mu.l of each TCS were
pre-incubated with 50 .mu.l MB solution (1000 ng/ml in buffer) or
buffer overnight. The experiments were run in duplicates. 50 .mu.l
of the pre-incubated sample were added to ELISA plates coated with
MB as used for screening. After 10 min, the plates were washed,
bound antibodies against MB were detected with HRP conjugated mAbs
against the rat IgG isotypes and HRP was visualized with TMB. MAbs
that recognized free MB (no signal when pre-incubated with MB,
positive signal in the buffer control) were further analyzed in a
serial dilution of MB (1000 ng/ml to 2 ng/ml). The four mAbs that
best recognized free MB were established (10B10, 12D9, 18H7, 21G5,
all of rat IgG2a subclass). To increase the sensitivity of the test
system, the TCS of the established hybridomas were titrated on
ELISA plates coated with MB. The titer of 10B10 was far better
(1:1260) than of 12D9 (1:320), 18H7 (1:10) and 21G5 (1:320).
[0194] For the test system 10B10 was used at a dilution of 1:500
and the pre-incubation time reduced to one hour.
Metal content determination
[0195] Copper in liver homogenates, cell lysates and mitochondrial
preparations were analyzed by ICP-OES (Ciros Vision, SPECTRO
Analytical Instruments GmbH) after wet ashing of samples with 65%
nitric acid (Zischka et al., log. cit.).
Miscellaneous
[0196] Electron microscopy of liver tissues and mitochondria was
done as previously described32. For structural analyses of isolated
mitochondria, they have been grouped into type 1: normal structured
mitochondria of the "condensed" type (Hackenbrock, J Cell Biol 37,
345-369 (1968)), type 2: mitochondria with minor alterations like
slightly increased cristae, type 3: mitochondria with massively
increased cristae, and type 4: mitochondria with massive matrix
condensations, matrix vacuolization, detachments of the inner
boundary membrane, and severe cristae deformations. Methanobactin
was isolated from the spent media of Methylosinus trichosporium
OB3b as previously described (Bandow et al., Methods Enzymol 495,
259-269 (2011)). Endotoxin in methanobactin was detected by a
kinetic chromogenic method (Charles River, Ecully, France) and was
on average 4.5 IU/mg. Protein quantification was done by the
Bradford assay (Bradford, Anal Biochem 72, 248-254 (1976)).
Cytochrome C oxidase activity in isolated mitochondria was
determined as described elsewhere (Kiebish et al., J Neurochem 106,
299-312 (2008)).
Copper chelators and chemicals
[0197] D-penicillamine*HCl (D-PA) was a gift from Heyl Pharma
(Berlin), trientine*2 HCl (TETA) was from Sigma (Taufkirchen,
Germany), tetrathiomolybdate*2 NH4 (98% pure) (TTM) was a gift from
KT. Suzuki (Chiba University, Japan). CCCP was from Sigma. DPH and
TMA-DPH were obtained from Life Technologies.
Statistics
[0198] Throughout this study N equals the number of analyzed
animals and n equals the number of technical replicates of
measurements. Data are presented as mean and SD. For Student's
t-test, data were tested unpaired and 2-tailed, except for those
represented in FIG. 3I (unpaired and 1-tailed). Differences were
considered statistically significant when p-values were less than
0.05. P-values mean: *p<0.05, **p<0.01, ***p<0.001.
Example 1: Mitochondrial Impairment is Pathognomonic for Hepatic
Failure in WD Patients and for Liver Damage in LPP-/- Rats
[0199] Mutations causing complete functional loss of ATP7B result
in severe WD phenotypes in humans (Das & Ray, Nat Clin Pract
Neurol 2, 482-493 (2006)). The LPP-/- rat carries an Atp7b mutation
that completely abolishes its hepatic copper transport activity
(Burkhead et al. Biometals 24, 455-466 (2011)). These animals
rapidly progress from a copper-burdened liver to hepatic failure
and death (Zischka, loc. cit.). The diseased livers from untreated
patients with acute onset of WD (who had undergone liver
transplantation) were compared with the livers from LPP-/- rats
with progressive disease states (FIG. 1). In addition, livers from
WD patients that had received unsuccessful D-PA treatment before
transplantation were included in this study (FIG. 6D).
[0200] To compare clinical stages of liver impairment, rats at ages
of 80-100 days were classified, when liver damage becomes apparent,
into three groups: (1) those rats "affected" by elevated liver
copper, with serum AST <200 U/L, bilirubin <0.5 mg/dl, (2)
rats showing "disease onset" with AST >200 U/L, bilirubin
<0.5 mg/dl and, (3) "diseased" rats with AST >200 U/L,
bilirubin >0.5 mg/dl (FIG. 10A).
[0201] Identical tissue damage features were observed in livers of
untreated WD patients and diseased LPP-/- livers (FIG. 1A).
Fibrosis was observed in all WD patient livers, and beginning
fibrosis was found in livers from diseased LPP-/- rats (FIG. 6A).
These characteristics were absent from heterozygous LPP+/- control
livers but steadily progressed in LPP-/- rats (FIG. 6B).
[0202] Another striking analogy between the livers from LPP-/- rats
and WD patients was the structural damage of mitochondria (FIG. 1B
and FIGS. 6C, D). Transparent vacuoles of varying sizes containing
amorphous but also electron-dense material, separated inner and
outer membranes, marked differences in electron densities and
cristae dilations were observed (FIG. 1B and FIGS. 6C, D) depicting
the typical WD mitochondrial phenotype20. Importantly, highly
comparable levels of copper were found in liver homogenate and
mitochondria obtained from diseased LPP-/- rats and untreated WD
patient livers (FIG. 1C). In contrast, lower copper content was
present in the tissue homogenate from the explanted livers and
isolated mitochondria of the D-PA pre-treated WD patients. This
coincided with more heterogeneous impairment of the mitochondrial
structure (FIG. 6D), which probably results from zonal
heterogeneities originating from massive fibrosis within these
livers (FIG. 6D).
Example 2: Increasing Copper Load Impairs the Mitochondrial
Membrane Integrity and Function
[0203] Mitochondrial copper content progressively increases with
disease state in livers from LPP-/- rats (FIG. 1C, FIG. 10A). This
is paralleled by increasingly severe membrane deficits, as
demonstrated directly at the level of freshly isolated mitochondria
(FIG. 2):
[0204] A drastic decrease in structurally normal rat liver
mitochondria (type 1 and 2) in LPP-/- vs. controls and a
corresponding increase in the number of structurally altered
organelles (type 3 and 4, FIG. 2A) was observed.
[0205] Membrane polarization measurements with the fluorophores DPH
and TMA-DPH (Prendergast et al. Biochemistry 20, 7333-7338 (1981))
revealed an alteration of the mitochondrial membrane "fluidity" at
the polar head groups of the membrane lipid-water interface
(TMA-DPH), but not at the membrane inner lipid phase (DPH) (FIG.
2B).
[0206] Upon induction of the mitochondrial permeability transition
(MPT) by either calcium or copper, control mitochondria underwent
large amplitude swelling (Zischka, loc. cit.), which was
significantly reduced in mitochondria from diseased and disease
onset LPP-/- rats (FIG. 2C).
[0207] The capacity of Cys-A to block calcium-induced MPT was
significantly impaired in LPP-/- vs. control mitochondria (FIG.
2D).
[0208] The time stability of the inner mitochondrial transmembrane
potential (.DELTA..PSI.) was jeopardized, and LPP-/- mitochondria
lost their membrane potential at earlier time points compared to
control mitochondria (FIG. 2E).
[0209] LPP-/- mitochondria were found to have an impaired capacity
to produce ATP (FIG. 9F).
Example 3: The Bacterial Peptide Methanobactin Rapidly Depletes
Accumulated Mitochondrial Copper
[0210] Capability of methanobactin (MB) to existing clinically
approved copper chelators D-PA, TETA and the candidate drug TTM to
remove copper from freshly isolated LPP-/- mitochondria were
compared. The MB peptide has an exceptionally high copper affinity
and is produced by methane-oxidizing bacteria when grown in a
copper poor environment (Kim et al., Science 305, 1612-1615
(2004)). In contrast to D-PA and TETA, both MB and TTM rapidly
decreased copper associated with LPP-/- mitochondria (FIG. 3A).
Similar results were obtained with mitochondria from wild-type rats
artificially pre-loaded with copper (FIGS. 7A, B). Furthermore, MB
was found to be significantly less toxic than TTM when assessing
the impairment of the vital copper-dependent mitochondrial
respiratory complex IV (FIGS. 3B, 7C).
[0211] Even a specific MB peptide such as mb-SB2 from Methylocystis
strains SB2, which is structurally and chemically deviating from
other MB peptides (f.e. from mb-OB3b derived from Methylosinus
trichosporium OB3b), acts as a promising copper chelator compared
to existing clinically approved copper chelators such as D-PA. In
three different LPP-/- rats freshly isolated mitochondria
(ATP7B-/-) were incubated 30 minutes with 1 mM cooper chelator
D-PA, mb-OB3b and mb-SB2 and their chelation potency was
investigated compared to the buffer treated control. In all three
LPP-/- rats mitochondria MB peptide mb-SB2 decreased at least as
effective as the MB peptide mb-OB3b derived from Methylosinus
trichosporium OB3b.
Example 4: Methanobactin Efficiently De-Coppers Hepatocytes with
Low Cell Toxicity
[0212] At the cellular level, overnight MB treatments caused a 50%
reduction of copper in HepG2 cells with either basic copper (FIG.
7D) or preloaded with copper amounts that exhibit only mild
toxicity (FIGS. 3C, 7E). Moreover, in an attempt to test the
efficacy of MB on WD patient samples, urinary epithelial cells from
these patients were reprogrammed into induced pluripotent stem
cells (iPSC) and differentiated into hepatocyte-like cells (HLC,
FIGS. 7F-I). Comparable copper depletions upon MB treatment were
found in both copper-preloaded HepG2 and HLCs (FIG. 3C).
[0213] Using a monoclonal antibody specific for MB, MB was found to
be taken up in a dose dependent manner by HepG2 cells (FIG. 3D).
Unwarranted cytotoxic effects of MB were only observed at
millimolar MB concentrations (FIG. 3E). At the mitochondrial level,
non-toxic MB concentrations (500 .mu.M) reduced the mitochondrial
membrane potential only partially (FIG. 3F). Thus, MB efficiently
de-coppers hepatocytes without major toxic side effects.
Example 5: Methanobactin Directs Liver Copper into Bile
[0214] The copper removing efficiency of MB was further validated
at the whole organ level (FIG. 3G-I). During a two-hour perfusion
of LPP-/- livers, tenfold higher amounts of copper were released
into bile, the major physiological excretion route for copper
(Ferenci, Clinical gastroenterology and hepatology: the official
clinical practice journal of the American Gastroenterological
Association 3, 726-733 (2005)), in the presence of MB in comparison
to TTM (FIG. 3G, FIGS. 8A, B). D-PA and TETA did not provoke any
detectable release of copper into the bile (FIG. 3G). However, all
chelators, except TTM, caused an increased presence of copper in
the perfusate (FIG. 3H), which may be linked to TTMs ability to
precipitate copper intra-cellularly (Ogra et al., Toxicology 106,
75-83 (1996)). The release of copper into the perfusate was partly
dependent on the liver disease state, as the liver damage marker
LDH paralleled the copper release curves (FIG. 8C). Noteworthy,
only MB significantly reduced the copper content of the LPP-/-
livers within the two hour perfusion (FIG. 3I).
Example 6: Short-Term Methanobactin Application Reverses Liver
Damage In Vivo
[0215] The efficiency of a short-term MB treatment schedule was
assessed in LPP-/- rats at the age of liver disease onset (85-90
days). Animals received MB (i.p.) either for 3 or 5 days or the
clinically used copper chelators D-PA or TETA, which were orally
administered for 4-days.
[0216] MB application resulted in a strong reduction of
histopathological liver damage markers in LPP-/- livers (FIG. 4A),
in contrast to treatment with D-PA or TETA. The latter two
chelators were unable to avoid the increase of serum AST levels
(indicative of progressive liver damage, FIG. 4B) meaning that
short-term D-PA or TETA treatments were without therapeutic effect.
In contrast, in six out of seven MB treated LPP-/- rats, AST levels
markedly decreased (FIG. 4B) and animals regained body weight (FIG.
10B). Importantly, after 5 days of MB treatment, two LPP-/- animals
with onset disease and one diseased LPP-/- rat were rescued from
liver dysfunction (AST <200 U/L, FIG. 4B, FIG. 10B).
[0217] Concerning drug safety, control LPP+/- rats treated with MB
did not exhibit any signs of toxicity and body weight, liver copper
concentration, serum AST and bilirubin values remained within the
physiological range (N=4, data not shown). Furthermore, MB was
detectable in the serum for only 30 minutes (FIG. 4C).
[0218] MB induced a progressive reduction in total liver copper,
which was even more pronounced in the mitochondrial compartment
(FIG. 4D). No copper reduction was found upon treatment with D-PA
or TETA, neither in whole liver nor in purified mitochondria (FIG.
4D). The mitochondrial de-coppering effect of MB was confirmed by
ultra-structural examinations (FIG. 4E). Severely impaired
mitochondria (type 4, FIG. 2A) were almost absent in isolates from
MB treated LPP-/- animals, but not in isolates from D-PA or TETA
treated animals (FIG. 4E, quantitation in FIG. 9A).
[0219] How long does a short-term MB treatment postpone the onset
of acute liver failure? To address this issue, three LPP-/- rats
were treated with MB for five days and subsequently set on MB drug
holiday. Starting with the MB treatment, zinc enriched food (1000
ppm) (Halestrap, Biochem Soc Trans 38, 841-860 (2010)) was given,
as zinc is a clinically relevant copper maintenance therapy in
WD12. All MB-treated animals showed restoration of normal serum
AST, lasting for at least two weeks, thereafter AST levels rose
again (FIG. 5A). At the time of analysis, one animal was still
healthy and two animals manifested different stages of liver
disease (FIGS. 5A, C). The degree of liver damage correlated with
mitochondrial (but not whole liver) copper levels (FIG. 5B), as
well as with structural (FIG. 5D) and functional defects in
mitochondria (FIG. 9B).
[0220] Intraperitoneal (i.p.) or intravenous (i.v.) MB-application
routes may be alternatively used (FIGS. 10B and C). For i.v.
injections, three LPP-/- rats were catheterized into the femoral
vein. After a three-day recovery period, animals received daily MB
doses on five consecutive days. All animals regained bodyweight
and, in cases with elevated AST or bilirubin, levels returned to
normal (FIG. 10C). Moreover, a profound reduction in copper content
was found at the levels of the whole liver and purified
mitochondria (FIG. 10C).
Example 7: Methanobactin for the Treatment of Acute Liver
Failure
[0221] The capacity of MB was assessed to rescue diseased LPP-/-
rats by an "acute rescue regimen" consisting of two daily MB
injections for one week (i.e. 16 i.p. injections in total, FIG.
10D). Four LPP-/- rats with strongly elevated AST levels were
treated (FIG. 10D). All animals survived, regained weight and
presented with normal serum AST and bilirubin and exceptionally low
copper values at the end of the treatment regimen (FIG. 10D). This
powerful therapeutic effect is best exemplified by the case of
animal no. 3 (FIG. 10D). Diseased LPP-/- rats presenting with
progressive weight loss and bilirubin levels greater than 8 mg/dl
(FIG. 10A) must be considered as moribund as such animals usually
die within few days. In contrast, following the "acute rescue
regimen", animal no. 3 regained 29% in weight, demonstrated a
drastic decrease in AST and bilirubin levels down to normal,
hepatic copper depletion, associated with massive structural and
functional mitochondrial recovery (FIG. 10D, FIG. 9D-F).
Example 8: Repetetive mb Treatment
[0222] Due to the efficiency of the short-term MB treatment, we did
a first test aiming at replacing daily chelation therapy by a
regimen consisting of repetitive treatment cycles interrupted by
longer observation cycles (FIG. 14). Five LPP-/- rats as well as
five age- and sex-matched LPP+/- controls were included. One pair
of rats was sacrificed at experimental days 1, 8, 29, 36 and 85,
respectively. At experimental day one, all animals were healthy,
with the sacrificed LPP-/- rat demonstrating a pronounced liver and
mitochondrial copper load and a slightly impaired mitochondrial
function (87% ATP production capacity) in comparison to its LPP+/--
control (pair 1). The four remaining LPP-/- rats were subjected to
the first treatment cycle consisting of three daily MB injections
(i.p.) for five days. All animals stayed healthy and this resulted
in a 40% reduction in copper load at experimental day eight (pair
2), which increased back to starting levels after additional three
weeks of observation (day 29, pair 3). Upon the second treatment
cycle copper loads decreased again, but now down to 25% of the
starting values, resulting in an unprecedented low copper load in
LPP-/- mitochondria (day 36, pair 4). This decoppering efficiency
of 75% was associated with a subsequent observation period of
further seven weeks during which the remaining LPP-/- rat stayed
healthy. At experimental day 85, liver and mitochondrial copper
loads had risen back to values before beginning of the treatment
(day 1), associated with an impaired mitochondrial function (65%
ATP production capacity) in comparison to its LPP+/- control (pair
5). This corresponds to a doubling of the age when untreated LPP-/-
rats become diseased.
Sequence CWU 1
1
18130PRTMethylosinus sp. 1Met Thr Val Lys Ile Ala Gln Lys Lys Val
Leu Pro Val Ile Gly Arg1 5 10 15Ala Ala Ala Leu Cys Gly Ser Cys Tyr
Pro Cys Ser Cys Met 20 25 30234PRTMethylosinus sp. 2Met Ala Ile Lys
Ile Ala Lys Lys Glu Val Leu Pro Val Val Gly Arg1 5 10 15Leu Gly Ala
Met Cys Ser Ser Cys Pro Met Cys His Cys Gly Pro Leu 20 25 30Cys
Pro332PRTMethylocystis sp. 3Met Ala Ile Lys Ile Ser Lys Lys Glu Val
Leu Pro Val Val Gly Arg1 5 10 15Leu Gly Ala Met Cys Ser Ser Cys Pro
Met Cys Gly Pro Leu Cys Pro 20 25 30432PRTMethylosinus sp. 4Met Ala
Ile Lys Ile Ala Lys Lys Glu Val Leu Pro Val Val Gly Arg1 5 10 15Leu
Gly Ala Met Cys Ser Ser Cys Pro Met Cys Gly Pro Leu Cys Pro 20 25
30531PRTMethylosinus sp. 5Met Thr Ile Lys Val Val Lys Lys Glu Ile
Leu Pro Val Ile Gly Arg1 5 10 15Val Gln Ala Met Cys Ala Cys Asn Pro
Pro Trp Cys Gly Thr Cys 20 25 30635PRTMethylocystis sp. 6Met Ala
Ile Lys Ile Val Lys Lys Glu Ile Leu Pro Val Ile Gly Arg1 5 10 15Val
Gln Ala Phe Cys Ser Ser Asp Ser Gly Gly Gly Gln Ile Gly Cys 20 25
30Gly Pro Ala 35730PRTMethylocystis sp. 7Met Thr Ile Arg Ile Ala
Lys Arg Ile Thr Leu Asn Val Ile Gly Arg1 5 10 15Ala Ser Ala Arg Cys
Ala Ser Thr Cys Ala Ala Thr Asn Gly 20 25 30830PRTMethylocystis sp.
8Met Thr Ile Arg Ile Ala Lys Arg Ile Thr Leu Asn Val Ile Gly Arg1 5
10 15Ala Ser Ala Arg Cys Ala Ser Thr Cys Ala Ala Thr Asn Gly 20 25
30930PRTMethylocystis sp. 9Met Thr Ile Arg Ile Ala Lys Arg Ile Thr
Leu Asn Val Ile Gly Arg1 5 10 15Ala Ser Ala Met Cys Ala Ser Thr Cys
Ala Ala Thr Asn Gly 20 25 301027PRTMethylocystis sp. 10Met Thr Ile
Lys Ile Val Lys Arg Thr Ala Leu Ala Val Asn Gly Arg1 5 10 15Ala Gly
Ala Asp Cys Gly Thr Ala Cys Trp Ala 20 251127PRTMethylocystis sp.
11Met Ala Ile Asn Ile Val Lys Arg Thr Thr Leu Val Val Asn Gly Arg1
5 10 15Thr Gly Ala Asp Cys Gly Thr Ala Cys Trp Gly 20
251227PRTMethylosinus sp. 12Met Ala Ile Asn Ile Val Lys Arg Thr Thr
Leu Val Val Asn Gly Arg1 5 10 15Ser Gly Ala Asp Cys Gly Thr Ala Cys
Trp Gly 20 251329PRTTistrella mobilis 13Met Ser Ile Lys Ile Ser Ala
Arg Lys Ala Leu Gln Ile Ala Gly Arg1 5 10 15Ala Gly Ala Arg Cys Ala
Thr Ile Cys Ala Val Ala Gly 20 251430PRTCupriavidus basiliensis
14Met Thr Ile Lys Ile Ser Lys Lys Glu Ala Ile Glu Val Arg Gly Arg1
5 10 15Ser Gly Ala Cys Cys Gly Ser Cys Cys Ala Ala Ile Gly Ala 20
25 301531PRTPseudomonas extremaustralis 15Met Ser Ile Lys Ile Ala
Lys Lys His Thr Leu Gln Ile Ala Gly Arg1 5 10 15Ala Gly Ala Cys Cys
Ala Ser Cys Cys Ala Pro Leu Gly Val Asn 20 25 301631PRTAzospirillum
sp. 16Met Thr Ile Lys Ile Ala Lys Lys Gln Thr Leu Ser Val Ala Gly
Arg1 5 10 15Ala Gly Ala Cys Cys Gly Ser Cys Cys Ala Pro Val Gly Val
Asn 20 25 301731PRTComamonas composti 17Met Lys Ile Lys Val Thr Lys
Lys Thr Thr Met Thr Val Ala Gly Arg1 5 10 15Ala Gly Ala Cys Cys Ala
Ser Cys Cys Ala Pro Val Gly Val Asn 20 25 301835PRTMethylocystis
sp. 18Met Ala Ile Lys Ile Val Lys Lys Glu Ile Leu Pro Val Ile Gly
Arg1 5 10 15Val Gln Ala Phe Cys Ser Ser Cys Ser Gly Gly Gly Gln Cys
Gly Cys 20 25 30Gly Pro Ala 35
* * * * *