Note: Descriptions are shown in the official language in which they were submitted.
CA 02493199 2005-O1-20
WO 2004/019936 PCT/EP2003100?556
PEROXYNITRITE REARRANGEMENT CATALYSTS FOR TREATMENT OR
PROPHYLAXIS OF DISEASES THAT ARE CAUSED BY PEROXYNITRITE-
MEDIATED REACTIONS
The invention relates to the use of metal-containing complexes that catalyze
the
rearrangement of peroxynitrite for the production of pharmaceutical agents for
treating
diseases.
As early as 1990, peroxynitrite was described by Beckman et al. (Beckman et
al.,
1990, Proc. Natl. Acad. Sci. USA. 87, 1620-1624) as a toxic metabolite, which
is
produced by the diffusion-controlled reaction between nitrogen monoxide (NO,
nitric
oxide) and superoxide anion (OZ-). Peroxynitrite is involved in a number of
inflammatory
processes that play an important role in diseases, such as, for example,
Alzheimer's
dementia, multiple sclerosis, and amyotrophic lateral sclerosis, and are made
responsible
for cellular degeneration and the induction of apoptosis.
Peroxynitrite reacts with a number of proteins by amino acid radicals being
oxidized or nitrated. Nitrotyrosine radicals are increasingly found in the
tissue of patients
who are suffering from multiple sclerosis, since peroxynitrite ensures the
nitration of
tyrosine radicals of the filaments of motor neurons. A neuronal dysfunction
(Estevez et
al., 1999, Science 286, 2498-2500) results by the thus disrupted contraction
of the
filaments. A cause of the vasoconstriction that is impaired after a stroke
consists in the
oxidation of the lipid radicals of the cell membrane, induced by
peroxynitrite, in which
CA 02493199 2005-O1-20
2
damage to the endothelium and edema resulting therefrom - and the formation of
neutrophils - result.
A pharmacological intervention to prevent the actions mediated by
peroxynitrite
can take place on the part of the starting substances (NO, and OZ ) or on the
part of the
product.
An approach on the part of the product peroxynitrite was first described by
Salvemini et al. (Salvemini et al., 1998, Proc. Natl. Acad. Sci. USA., 95,
2659-2663). In
this approach, peroxynitrite is rearranged by means of a catalyst into
harmless end
products. It is possible to convert large amounts of peroxynitrite with only
small
concentrations of catalyst. An advantage of this approach is based on the fact
that it
cannot result in the formation of disadvantageous decomposition products, such
as, for
example, the reaction oxygen species, and that it results in eliminating the
inhibition of
the superoxide-dismutase (SOD) by peroxynitrite. This treatment method with
novel
compounds consequently has a two-fold advantage in the treatment of diseases.
'Thus, on
the one hand, the rate of the conversion of peroxynitrite is accelerated, and,
on the other
hand, the SOD is protected relative to inactivation by peroxynitrite.
As possible conversion catalysts, metal-containing complexes are known to date
(WO 95/31197, US 6,245,758, WO 98/04132, US 5,872,124, WO 00/75144, WO
01 /26655, US 6,372,727). The metallopoiphyrins that are described by
Salvemini et al.
show protective action in inflammation models (Salvemini et al., 1998, Proc.
Natl. Acad.
Sci. USA. 95, 2659-2663 and British J. Pharmacol., 1999, 127, 685-692). The
same class
of compounds was described as effective by Cuzzocrea et al. in an intestinal
artery
occlusion model (Cuzzocrea et al., 2000, FASEB, J. 14 (9), 1061-1072 and
Cuzzocrea et
AMENDED
SHEET
CA 02493199 2005-O1-20
3
al., 2001, Pharmacology Rev. 53, 135-159). Cross et al. demonstrated the
effectiveness
of these substances in an MS model ("experimental autoimmune
encephalomyelitis" _
EAE) in mice, (Cross et al., 2000, J. Neuroimmunology 107, 21-28). Mackensen
et al.
showed for the first time the effectiveness of a manganese-containing
porphyrin in a focal
ischemia model, the focal MCAO (middle cerebral artery occlusion) (Mackensen
et al.,
2001, J. Neurosci. 21, 4582-4592).
To date, little is known on the side effects, such as toxicity and in vivo
availability, as well as the blood-cerebrospinal permeability of these known
rearrangement catalysts.
For the treatment and prophylaxis of diseases that have their cause in the
reactions
that are mediated by peroxynitrite, the urgent problem is to produce well-
tolerated,
chemically stable substances (catalysts) that are available in vivo to
increase the
rearrangement of peroxynitrite in harmless products. These substances that are
effective
in vivo can be used to develop medications for treating diseases.
This invention solves the problem by producing porphyrin complexes that are
used as peroxynitrite rearrangement catalysts. These porphyrins are
distinguished by their
good in vivo availability as well as by their chemical stability. They have
already been
used as agents to diagnose tumors, and their use for necrosis and infarction
imaging was
already disclosed in W099/62512 and in WO 00105235. Their use in photodynamic
therapy and in MRI diagnosis is claimed in W099/62512.
In this invention, it was possible to show by means of NMR and UV/VIS
spectroscopy that the porphyrins of WO 00/05235 according to the invention
catalyze the
rearrangement of peroxynitrite in harmless end products, namely nitrate and
nitrite. By
means of a model for cell damage, it was possible to show that the porphyrins
according
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SHEET
CA 02493199 2005-O1-20
4
to the invention are protective and protect the cells from peroxynitrite
damage, induced
by the peroxynitrite donor SIN-1. These porphyrins are already very well
characterized,
and it is known that they have no side effects, good water solubility and a
good in-vivo
availability.
The use of porphyrin complexes, which have, on the one hand, a peroxynitrite-
rearranging property, and, on the other hand, diagnostic properties, makes
possible a
specific treatment of the diseases that are caused by peroxynitrite and their
diagnosis with
use of imaging processes, such as, e.g., MRT.
This invention provides pharmaceutical agents for this specific treatment and
relates to a porphyrin complex that consists of a ligand of general formula I
R~
R'
~3
as well as at least one ion of an element of atomic numbers 20-32, 37-39, 42-
51 or 57-83,
in which
M stands for a paramagnetic ion,
R~ stands for a hydrogen atom, for a straight-chain C~-C6 alkyl radical, a
CA 02493199 2005-O1-20
C~-C~2-aralkyl radical or for a group OR' , in which
R' is a hydrogen atom or a C~-C3-alkyl radical,
R2 stands for R3, a group -CO-Z or a group -(NH)o-(A)q-NH-D, in which
Z is a group -OL, with L in the meaning of an inorganic or organic
cation or a C~-C4-alkyl radical,
A means a phenylenoxy group or a C~-C~2-alkylene group or a C7-C~2
aralkylene group that is interrupted by one or more oxygen atoms,
o and q, independently of one another, mean the number 0 or l, and
D means a hydrogen atom or a group -CO-A-(COOL)o-(H)m, with m
equal to 0 or l, and provided that the sum of m and o is equal to l,
R3 stands for a group -(C=Q)(NR4)o-(A)q-(NRS)-K,
in which Q stands for an oxygen atom or for two hydrogen atoms,
4
R means a group -(A)q-H, and
K means a complexing agent of general formula (IIa), (IIb), (IIc),
(IId) or (IIe), whereby RS stands for the case that K is a
complexing agent of Formula (IIa) and has the same meaning as
R4, and RS stands for the case that K is a complexing agent of
Formula (IIb), (IIc), (IId) or (IIe) and has the same meaning as D,
provided that a direct oxygen-nitrogen bond is not allowed,
and K stands for a complexing agent of general formula (Ila), (IIb), (IIc),
(IId), (IIe) or (IIf)
(IIa)
'~~--CO~ ~ ~COOL~
N~ N
3 'COOL2
COOL4 COOL
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6
OOL2
'~~ A' ~'N
H
(Ilb),
L300C--\ ~~~COOL'
N N N
LcOOL4
~cOOL2
R6 ~CO~
(llc),
rim 2
~OL3
"",~ X A
H
4
COOL
(I!d),
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7
6
COOL
~'',~.. X - A2 NH N
~N
4
~~ N
OOL4
(Ile),
rnni 2
L' 00 OOL3
~'v' X- A2 '~ N
OOL4 ( I If)
in which
q has the above-indicated meaning,
A' has the meaning that is indicated for A,
R6 stands for a hydrogen atom, a straight-chain or branched C~-C~-alkyl
group, a phenyl or benzyl group,
AZ stands for a phenylene-, -CH2-NHCO-CHZ-CH (CHZCOOH) -C6H4-(3-,
-C6H4-O-(CHZ)o_5-(3, -C6H4-(OCHZCHZ)o_~-N(CHZCOOH)-CH2-(3 or a
C~-C~2-alkylene- or C~-C,2-alkylene group that is optionally substituted by
one or more oxygen atoms, I to 3 -NHCO groups or 1 to 3 -CONH groups
and/or substituted with 1 to 3 -(CHZ)o-sCOOH groups, whereby ~3 stands
COOL2
CA 02493199 2005-O1-20
for the binding site to X,
stands for a -CO- or NHCS group, and
L', L2, L3 and L4, independently of one another, stand for a hydrogen atom or
a
metal ion equivalent of an element of the above-mentioned atomic
number, provided that at least two of these substituents stand fox metal ion
equivalents and that other anions are present to compensate for optionally
present charges in the metalloporphyrin, and in which free carboxylic acid
groups that are not required for complexing can also be present as salts with
physiologically compatible inorganic and/or organic cations or as esters or as
amides,
and that, moreover, also increase the rearrangement rate of peroxynitrite in
harmless products and thus can be used for the production of a
pharmaceutical agent for treatment and prophylaxis of radically-mediated
cell damage.
It was possible to show by means of NMR and UV/VIS spectroscopy that the
compounds according to the invention catalyze the rearrangement of
peroxynitrite in
harmless end products, namely nitrate and nitrite. Peroxynitrite is a strong
oxidant that is
produced by the reaction of nitrogen oxide (NO) and superoxide-anion (02-). It
was
possible to show that NO is generated in many cells, such as, for example, in
macrophages, in neutrophil cells, hepatocytes and endothelial cells. The
direct reaction of
NO and 02- results in the formation of the peroxynitrite ion, which quickly
dissolves into
oxidizing intermediate compounds under physiological conditions. These
intermediate
oxidation stages are responsible for the damage to the biological targets.
CA 02493199 2005-O1-20
9
The results of this damage can be associated with pathological consequences,
including the oxidation and nitration of proteins, lipids and DNA.
Peroxynitrite can pass
through cell membranes at a significantly higher speed than other oxidants,
and
peroxynitrite can quickly penetrate the interior of the cell even in the
presence of a
biological membrane. Peroxynitrite is known for the nitration of tyrosine
radicals in
proteins, and it oxidizes sulfhydryl radicals, methionines and macromolecules,
such as,
for example, metal enzymes, DNA and lipids.
Because of its high reactivity, peroxynitrite was brought into contact with
many
diseases. The invention relates to the use of the compounds according to the
invention
for the production of a pharmaceutical agent for treatment and prophylaxis of
radically-
mediated cell damage. These include neurodegenerative diseases, inflammatory
diseases,
autoimmune diseases, and cardiovascular diseases.
For example, there can be mentioned:
Cerebral ischemia, ischemic reperfusion disease, hypoxia and other
neurodegenerative diseases that are associated with inflammations, such as
multiple
sclerosis, ALS (amyotrophic lateral sclerosis) and comparable sclerotic
diseases,
Parkinson's disease, Huntington's disease, Korksakoff s disease, epilepsy,
vomiting,
sleep disturbances, schizophrenia, depression, stress, pain, migraine,
hypoglycemia,
dementia, such as, e.g., Alzheimer's disease, HIV dementia and presenile
dementia.
They are also suitable for treating diseases of the cardiovascular system,
such as
arteriosclerosis, and for treating autoimmune and/or inflammatory diseases,
such as
hypotension, ARDS (adult respiratory distress syndrome), sepsis or septic
shock,
rheumatoid arthritis, osteoarthritis, insulin-dependent diabetes mellitus
(IDDM),
CA 02493199 2005-O1-20
inflammatory disease of the pelvis/intestine (bowel disease), meningitis,
glomerulonephritis, acute and chronic liver diseases, diseases by rejection
(for example
allogenic heart, kidney or liver transplants) or inflammatory skin diseases
such as
psoriasis, etc.
The porphyrin complexes according to the invention contain as a paramagnetic
ion in the porphyrin skeleton the iron(III), manganese(III), copper(II),
cobalt(III),
chromium(III), nickel(II) or vanadyl(II) ion, whereby the first three ions
mentioned are
preferred.
If one of the ions that is bonded in the porphyrin is present in a higher
oxidation
stage than +2, the excess charges) is (are) compensated for by, e.g., anions
of organic or
inorganic acids, preferably by acetate, chloride, sulfate, nitrate, tartrate,
succinate and
maleate ions or by the negative charges that are present in RZ and/or R3.
The carboxyl groups that are not required for the complexing of the metal ions
can optionally be present as esters, as amides, or as salts of inorganic or
organic bases.
Suitable ester radicals are those with 1 to 6 C atoms, preferably ethyl ester;
suitable
inorganic canons are, for example, the lithium ion and the potassium ion, and
especially
the sodium ion. Suitable canons of organic bases are those of primary,
secondary or
tertiary amines, such as, for example, ethanolamine, diethanolamine,
morpholine,
glucamine, N,N-dimethylglucamine, in particular the meglumine.
As complexing agent radical K, preferably derivatives of diethylenetriamine-
pentaacetic acid and 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid can
be
mentioned, which are bonded via a linker to the respective porphyrin.
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The production of the complex compounds of general formula I is earned out
according to methods that are known in the literature (see, e.g., DE 4232925
for II a and
II b; see, e.g., DE 19507822, DE 19580858 and DE 19507819 for III c; and see,
e.g., US-
5,053,503, WO 96/02669, WO 96/01655, EP 0430863, EP 255471, US-5,277,895, EP
0232751, and US-4,885,363 for II d, II a and II f).
The production of the compound according to the invention is already described
in WO 00/17205.
The compounds in which R2 and R3 stand for CONHNHK groups are preferred.
The synthesis of the 3, 3'-(7, 12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-
diyl)di(propanohydrazide) that is required as an educt in this connection is
described in Z.
Physiol Chem. 241, 209 ( I 936).
The introduction of the desired metals (e.g., Mn) in the porphyrins is earned
out
according to methods that are known in the literature (e.g., The Porphyrins,
ed. D.
Dolphin, Academic Press, New York 1980, Vol. V, 459; DE 4232925), whereby
essentially the following can be mentioned:
a) The substitution of the pyrrolic NHs (by heating the metal-free ligand
with the corresponding metal salt, preferably the acetate, optionally
with the addition of acid-buffering agents, such as, e.g., sodium acetate,
in a polar solvent), or
b) The "recomplexing," in which a metal that is already complexed by a
ligand is displaced by the desired metal.
As a solvent, primarily polar solvents, such as, e.g., methanol, glacial
acetic acid,
dimethylformamide, chloroform and water, are suitable.
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12
The introduction of paramagnetic metal M into the porphyrin system can be
carried
out before or after the linkage of complexing agent radical K. As a result, an
especially
flexible procedure for the synthesis of the compounds according to the
invention is made
possible.
The chelation of radical K is carried out in a way that is known in the
literature (see,
e.g., DE 34 OI 052) by the metal oxide or metal salt (e.g., the nitrate,
acetate, carbonate,
chloride or sulfate) of the respectively desired metal being suspended or
dissolved in polar
solvents such as water or aqueous alcohols and being reacted with the
corresponding
amount of the complexing ligand. If desired, acidic hydrogen atoms or acid
groups that are
present can be substituted by cations of inorganic and/or organic bases or
amino acids.
The neutralization is carried out in this case with the aid of inorganic
bases, such as,
e.g., alkali hydroxides or alkaline-earth hydroxides, -carbonates or-
bicarbonates and/or
organic bases such as, i.a., primary, secondary and tertiary amines, such as,
e.g.,
ethanolamine, morpholine, glucamine, N-methylglucamine and N,N-
dimethylglucamine, as
well as basic amino acids, such as, e.g., lysine, arginine and ornithine, or
amides of
originally neutral or acidic amino acids.
For the production of the neutral complex compounds, enough of the desired
bases
can be added to, for example, the acidic complex salts in aqueous solution or
suspension
such that the neutral point is reached. The solution that is obtained can then
be evaporated
to the dry state in a vacuum. It is frequently advantageous to precipitate the
neutral salts that
are formed by adding water-miscible solvents, such as, for example, lower
alcohols (e.g.,
methanol, ethanol, isopropanol), lower ketones (e.g., acetone), polar ethers
(e.g.,
tetrahydrofuran, dioxane, 1,2-dimethoxyethane) and thus to obtain easily
isolated and
CA 02493199 2005-O1-20
13
readily purified crystaIlizates. It has proven especially advantageous to add
the desired base
as early as during the complexing of the reaction mixture and thus to save a
process step.
If the acidic complex compounds contain several free acid groups, it is often
suitable
to produce neutral mixed salts that contain both inorganic and organic canons
as
counterions.
This can happen, for example, by the complexing ligands being reacted in
aqueous
suspension or solution with the oxide or salt of the element that yields the
central ion and
half the amount of an organic base that is required for neutralization, the
formed complex
salt being isolated, optionally purified and then mixed with the necessary
amount of
inorganic base for complete neutralization. The sequence in which the base is
added can
also be reversed.
Another possibility for resulting in neutral complex compounds consists in
converting the remaining acid groups in the complex completely or partially
into esters.
This can happen by subsequent reaction in the finished complex (e.g., by
exhaustive
reaction of the free carboxy groups with dimethyl sulfate).
The production of the pharmaceutical agents according to the invention is also
carried out in a way that is known in the art by the complex compounds
according to the
invention - optionally with the addition of the additives that are commonly
used in
galenicals - being suspended or dissolved in aqueous medium and then the
suspension or
solution optionally being sterilized. Suitable additives are, for example,
physiologically
harmless buffers (such as, e.g., tromethamine), small additions of complexing
agents (such
as, e.g., diethylenetriaminepentaacetic acid), or, if necessary, electrolytes,
such as, e.g.,
sodium chloride or, if necessary, antioxidants, such as, e.g., ascorbic acid.
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14
In principle, it is also possible to produce the pharmaceutical agents
according to the
invention even without isolating the complex salts. In each case, special care
must be used
to perform the chelation such that the salts and salt solutions according to
the invention are
virtually free of uncomplexed metal ions that have a toxic effect.
This can be ensured, for example, with the aid of color indicators, such as
xylenol
orange, by control titrations during the production process. The invention
therefore also
relates to the process fox the production of complex compounds and salts
thereof. As a final
precaution, there remains purification of the isolated complex salt.
To use the compounds according to the invention as pharmaceutical agents, the
latter
are brought into the form of a pharmaceutical preparation that in addition to
the active
ingredient for the enteral or parenteral administration contain suitable
pharmaceutical,
organic or inorganic inert carrier materials, such as, for example, water,
gelatin, gum arabic,
lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene
glycols, etc. The
pharmaceutical preparations can be present in solid form, for example as
tablets, coated
tablets, suppositories, or capsules, or in liquid form, for example as
solutions, suspensions or
emulsions. Moreover, they optionally contain adjuvants, such as preservatives,
stabilizers,
wetting agents or emulsifiers; salts for changing the osmotic pressure, or
buffers.
These pharmaceutical preparations are also the subject of this invention.
For parenteral use, in particular injection solutions or suspensions, in
particular
aqueous solutions of the active compounds in polyhydroxyethoxylated castor
oil, are
suitable.
If suspensions or solutions of the agents according to the invention in water
or in
physiological salt solution are desired for enteral administration or other
proposes, they are
CA 02493199 2005-O1-20
mixed with one or more adjuvant(s) that are commonly used in galenicals (e.g.,
methyl
cellulose, lactose, mannitol) andJor surfactants) (e.g., lecithins, Tweeri ,
Myrj~ and/or
flavoring substances for taste correction (e.g., ethereal oils).
As carrier systems, surface-active adjuvants such as salts of bile acids or
animal or
vegetable phospholipids, but also mixtures thereof as well as liposomes or
components
thereof can also be used.
For oral administration, in particular tablets, coated tablets, or capsules
with talc
and/or hydrocarbon vehicles or binders, such as, for example, lactose, corn or
potato starch,
are suitable. The application can also be carned out in liquid form, such as,
for example, as
juice, to which optionally a sweetener is added.
The enteral, parenteral and oral administrations are also subjects of this
invention.
The dosage of the active ingredients can vary depending on the method of
administration, age and weight of the patient, type and severity of the
disease to be treated
and similar factors. The daily dose is 0.5-1000 mg, preferably 50-200 mg,
whereby the dose
can be given as a single dose to be administered once or divided into 2 or
more daily doses.
This invention also relates to the use of the porphyrin complexes according to
the
invention according to Formula (I) for the treatment and prophylaxis of
diseases that are
caused by the peroxynitrite-mediated reactions and that are weakened and/or
treated by the
increase in the conversion rate of peroxynitrite.
This invention relates in particular to the use of the porphyrin complexes of
general
formula (I) according to the invention for the treatment and prophylaxis of
diseases that
include neurodegenerative diseases, inflammatory diseases, autoimmune
diseases, and
cardiovascular diseases. For example, there can be mentioned:
CA 02493199 2005-O1-20
16
Cerebral ischemia, ischemic reperfusion disease, hypoxia and other
neurodegenerative diseases that are associated with inflammations, such
as multiple sclerosis, amyotrophic lateral sclerosis and comparable
sclerotic diseases, Parkinson's disease, Huntington's chorea,
Korksakoff's syndrome, epilepsy, vomiting, sleep disturbances,
schizophrenia, depression, migraine, hypoglycemia, dementia, such as,
e.g., Alzheimer's disease, HIV dementia and presenile dementia.
They are also suitable for treating diseases of the cardiovascular system
and for treating autoimmune andlor inflammatory diseases such as
hypotension, ARDS (adult respiratory distress syndrome), sepsis or
septic shock, rheumatoid arthritis, osteoarthritis, insulin-dependent
diabetes mellitus (IDDM), inflammatory disease of the pelvis/intestine
(bowel disease), meningitis, glomerulonephritis, acute and chronic liver
diseases, diseases by rejection (for example allogenic heart, kidney or
liver transplants) or inflammatory skin diseases such as psoriasis, etc.
Based on their profile of action, the compounds according to the
invention are very well suited for rearranging the peroxynitrite in
harmless products.
The subject matter of this invention is also the use of compounds of general
formula (I), characterized in that M stands for an Fe3*, Mn3+, Cu2+, Co3+,
VOZ+, Cr3+ or
Niz+-ion, and that are especially effective.
In addition, the subject matter of this invention is the use of porphyrin
complex
compounds of general formula I, characterized in that RZ and R3 in each case
stand for a
CA 02493199 2005-O1-20
17
-CONHNHK, -CONH(CH2)2NHK, -CONH(CHZ)3NHK, -CONH(CHZ)4NHK, or
-CONH(CHZ)z0(CHZ)ZNHK group.
The subject matter of this invention is also the use of porphyrin complex
compounds of general fomula (I), characterized in that RZ and R3 in each case
stand for a
-CONHNHK.
These compounds are quite especially effective if K is a complexing agent of
general formula (IIa):
~-CO-\N~N COOL
3'COOL2
COOL4 COOL
Additional subjects of this invention are in particular porphyrin complex
compounds according to Formula (I), namely
{ mu-[ { I 6, I 6'-[Chloromanganese(III)-7,12-diethyl-3,8, I 3,17-
tetramethylporphyrin-2,18-diyl]-bis[3,6,9-tris(carboxymethyl)-11,14-dioxo-
3,6,9,12,13-
pentaazahexadecanoato] } (8-)] } digadolinato(2-), -disodium,
{ mu[ { 16, I 6'-[Chloroiron(III)-7,12-diethyl-3, 8,13,17-tetramethylporphyrin-
2,18-
diyl]-bis[3,6,9-tris(carboxymethyl)-11,14-dioxo-3, 6, 9, 12, 13-pentaazahexa-
decanoato] } (8-)] ] -digadolinato(2-), -disodium,
{mu[{ 16,16'-[copper(II)-7,12-diethyl-3,8,13,17-tetramethylporphyrin-2,18-
diyl]-
bis[3,6,9-tris(carboxymethyl)-I 1,14-dioxo-3, 6, 9, 12,13-
pentaazahexadecanoato]}(8-)]}-
digadolinato(2-), -disodium.
CA 02493199 2005-O1-20
Ig
The good water solubility of the agents according to the invention allows the
production of highly-concentrated solutions, so as to keep the volume burden
of the
circulatory system within reasonable limits and to compensate for the dilution
by bodily
fluids. In addition, the agents according to the invention show not only a
high stability in
vitro, but also a surprisingly high stability in vivo, so that a release or an
exchange of the
ions, which are inherently toxic and not covalently bonded in the complexes,
can be
disregarded within the time that it takes for the contrast media to be
completely excreted
again.
Surprisingly enough, the complexes according to the invention show a
significantly higher relaxivity compared to the previously known, structurally
similar
compounds. Since the relaxivity can be regarded as a yardstick for the
contrast medium
action of a compound, a comparable, positive signal effect is possible even at
a low dose
with use of the complexes according to the invention in the area of NMR
diagnosis. This
significantly increases the safety margin, for which the product of relaxivity
and
compatibility can be considered as a guide value.
In addition, this invention relates to the use of the porphyrin complexes of
general
formula I according to claim I of the invention for diagnosis of diseases that
comprise the
group of the following diseases: ischemic reperfusion diseases, such as, e.g.,
stroke, head
trauma and myocardial ischemia, sepsis, chronic or acute inflammation (such
as, e.g.,
arthritis or inflammatory intestinal disease), adult respiratory stress
syndrome, cancer,
bronchio-pulmonary dysplasia, cardiovascular diseases, diabetes, multiple
sclerosis,
Parkinson's disease, familial amyotrophic lateral sclerosis and colitis and
special
neuronal diseases.
CA 02493199 2005-O1-20
19
Description of the Figures:
Figure 1 shows a ~4NMR spectrum.
Figure 2 shows the time-dependent degradation of peroxynitrite with and
without
a porphyrin catalyst, which was administered in a 100x deficit.
CA 02493199 2005-O1-20
Examples
1. Study of the Decomposition of Peroxynitrite with NMR-Spectroscopic Methods
For the study, 3 samples are prepared, whereby one of the samples contains the
original aqueous peroxynitrite solution without additives, and the others
contain this
solution in the same amount but with defined additions of a reference
substance or the
substance to be studied. From each sample, a 14-N spectrum with the same
acquisition
and processing parameters is recorded. The differences of the integrals of the
nitrate
signal between the treated and the untreated peroxynitrite solution indicates
the increase
in the nitrate owing to rearrangement of the peroxynitrite. Comparisons of the
data
between the substance to be studied and the reference compound allow a
quantification of
this process (Figure 1).
2. Measurement of the Kinetics of the Conversion of Peroxynitrite to Nitrate
by
Means of UV-Spectrometry
Used are: A UV-spectrometer
A stopped-flow device with a flanged cuvette
Laboratory devices for volumetric works
Reagents (buffers, peroxynitrite solution, catalysts)
The content of peroxynitrite is determined from the concentrated peroxynitrite
solution. A molar extinction coefficient of s = 1670 is taken as the baseline.
The
solution is diluted with water such that an absorption of about 1.6 at the
observation
wavelength of 301 nm is reached. The pH of the thus produced storage solution
is not to
fall below I 1.
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Corresponding to the set concentration of the peroxynitrite, a solution of the
catalyst to be studied in the phosphate buffer is produced such that taking
into
consideration the given ratios of the stopped-flow device, the desired amounts
of catalyst
solution and peroxynitrite can be merged to bring about the reaction. The
buffer of the
catalyst solution must have sufficient capacity to be able to set and to hold
the still strong
alkaline peroxynitrite solution at the desired pH. The catalysts are added in
a 100x
deficit.
The metering sprayers of the stopped-flow unit are filled with the two
solutions;
and the cuvette that is located in the UV-spectrometer is filled therefrom.
The absorption
values are simultaneously measured at 301 nm. Because of the rearrangement of
the
peroxynitrite in the nitrate, absorption is decreased, assuming a stable value
after some
length of time. At this time, the rearrangement is terminated, and the data
registration is
brought to a halt.
The measurement data are analyzed, and the peroxynitrite concentration can be
calculated from the resulting kinetic characteristics of the curve. These data
are used to
characterize the catalyst that is to be studied. To take into consideration
the self
decomposition of the peroxynitrite, first the decomposition behavior of the
peroxynitrite
solution in any preparation of peroxynitrite that is used is determined in
adding the buffer
without catalyst content and whose characteristics are set in relation to
those that are
obtained from a measurement with catalyst.
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Relative speed constant - Spontaneous decomposition of the peroxynitrite that
is used at the described pH of the catalyzed
decomposition.
(Figure 2).
3. SIN-1 Damage Assay with Neuronal Primary Cultures from the Cerebellum of
Neonatal Rats
For in-vitro testing of substances for neuroprotection with respect to damage
that
is induced by peroxynitrite, a primary culture of cells from the cerebellum of
neonatal
rats is applied. The measurement of cell death or survival of neurons in this
culture is
carried out indirectly by measurement of the reaction of the dye Alamar blue
in its
reduced fluorescent form. For injury, the peroxynitrite donor SIN-1 (3-
morpholino-
sydnonimine) is used.
To obtain the cells, Wistar rats (P8) are killed by decapitation, the
cerebella are
obtained, the meninges from the cerebellum are removed (HBSS (GIBCO, 14025-
050)
4°C), crushed and transferred into a 1 S ml Falcon tube, the
supernatant is suctioned off,
and then the cerebella are trypsinized by means of adding 500 pl of trypsin-
EDTA
solution (GIBCO #2530-054)/cerebellum. After an incubation (20 minutes,
37°C), the
trypsinized cerebella are washed 3x with 10 ml of HBSS. A trituration by
adding 500 pl
of 0.05% DNAseI (BOEHRINGER MANNHEIM, #14953000) per cerebellum follows.
By means of a 5 ml pipette, then with a fire-polished Pasteur pipette and
finally (if
necessary) with an elongated, fire-polished Pasteur pipette, the cells are
isolated and
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mixed with 10 ml of complete medium (100 m1 of neurobasaI (GIBCO #21103-049),
1
ml of B27 supplement (GIBCO #17504-044), 0.4 ml of Pen/Strep (10,000
IU/ml/10000
UG/ml) (GIBCO #15140-106), 0.8 ml of KCl-stock solution (MERCK, 1.04936.0500),
and 1 ml of L-glutamine (100x - 200 mmol) (GIBCO #15140-106). The isolated
cells are
then centrifuged off ( 10 minutes at 600 rpm), washed I x with complete medium
and
resuspended with 20 ml of complete medium, counted and diluted to 2 x 106/ml.
Per hole
of a 96-hole microtiter plate, 100 ~1 of complete medium is introduced and
mixed with
100 pl of cell suspension (=divl = day 1 in vitro). The microtiter plates are
coated
beforehand as follows: 50 pl of poly-L-lysine (MW ?0-l OSkD) (SIGMA #P-6282)
is
applied per hole, and the plates are then incubated in an incubator for about
90 minutes.
Before the cells are flattened out, the solution is suctioned off again and
washed 2x with
HBSS or with sterile bidistilled water. 24 hours after flattening out, the
cells are
damaged by adding SIN-I. Test substances are applied 1 hour before SIN-1 is
added
(individual concentration of 10 or 30 pM, or as a concentration series,
CALBIOCHEM
567028). The measurement of the cell function is carned out on div2 (day2 in
vitro) with
Alamar blue (10 pl/well) (BIOSOURCE INT., DAL 1100). After a 3-hour
incubation,
the measurement in the fluorescence reader follows (Victor, Wallac Company,
Extinction
544 nm/emission 590 nm). ICSO values are calculated with the Excel Plug-in
XLfit.
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The results from Examples 2 and 3 are indicated in the following table.
Example Complex Speed ConstantCell Toxicity
Assay
ED50 [pM]
1 3.75 >30
O
p
N
~ NFe
~
+ v' ~ i
Na Na+
O N O O N O O_
h~N~N ~N O
O
3+
3+
Gd
Gd
N
0 01 ENO o
ooo0
2 1.61 9.6
~ N ~~ v
O IV nN_/
'
O
v
~ i
O N O Oa-N O O
?~~N~ N ~ N~d
O
Na O Na
3+
a+
Gd
Gd
N
00
o~ o oo_o ~o
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Example Complex Speed ConstantCell Toxicity
Assay
ED50 (pM)
Fe(III)TMPy~ 8.05 2.2
i
P N
I
w
[ ci - J5
\y
_ N
~~ .
_
Fe31 ~ N ,
N
