U.S. patent application number 12/863061 was filed with the patent office on 2011-02-24 for tetrahydropyridoethers for treatment of amd.
Invention is credited to Ulrich Schraermeyer.
Application Number | 20110046377 12/863061 |
Document ID | / |
Family ID | 40097475 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110046377 |
Kind Code |
A1 |
Schraermeyer; Ulrich |
February 24, 2011 |
TETRAHYDROPYRIDOETHERS FOR TREATMENT OF AMD
Abstract
A medication comprising tetrahydropyridoethers for use in the
treatment of AMD.
Inventors: |
Schraermeyer; Ulrich;
(Hechingen, DE) |
Correspondence
Address: |
HENRY M FEIEREISEN, LLC;HENRY M FEIEREISEN
708 THIRD AVENUE, SUITE 1501
NEW YORK
NY
10017
US
|
Family ID: |
40097475 |
Appl. No.: |
12/863061 |
Filed: |
January 16, 2009 |
PCT Filed: |
January 16, 2009 |
PCT NO: |
PCT/EP09/00248 |
371 Date: |
July 15, 2010 |
Current U.S.
Class: |
546/82 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 31/4375 20130101; A61K 31/50 20130101; A61K 31/437 20130101;
A61P 27/00 20180101 |
Class at
Publication: |
546/82 |
International
Class: |
C07D 471/14 20060101
C07D471/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 16, 2008 |
EP |
08000761.0 |
Jun 12, 2008 |
EP |
08010697.4 |
Claims
1. A medication comprising tetrahydropyridoethers for use in the
treatment of AMD.
2. A medication comprising tetrahydropyridoethers for use in the
treatment of dry AMD.
3. A medication comprising a compound according to the following
formula I for use in the treatment of AMD or dry AMD ##STR00005##
in which R1 is methyl or hydroxymethyl, one of the substituents R2a
and R2b is hydrogen and the other is hydroxy, methoxy, ethoxy,
isopropoxy, methoxyethoxy or ethoxypropoxy, one of the substituents
R3a and R3b is hydrogen and the other is hydroxy, methoxy, ethoxy,
isopropoxy, methoxyethoxy or methoxypropoxy, where R2a or R2b on
the one hand and R3a or R3b on the other hand are not
simultaneously hydroxy, and its salts.
4. The medication for use in the treatment of AMD or dry AMD,
comprising a compound of claim 3, in which R1 is methyl, one of the
substituents R2a and R2b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, one of the
substituents R3a and R3b is hydrogen and the other is hydroxy, and
its salts.
5. The medication for use in the treatment of AMD or dry AMD
according to claim 3, in which R1 is methyl, one of the
substituents R2a and R2b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, one of the
substituents R3a and R3b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, and its
salts.
6. The medication for use in the treatment of AMD or dry AMD
according to claim 3, in which R3b is hydrogen.
7. The medication for use in the treatment of AMD or dry AMD
according to claim 3, in which R2a and R3b are hydrogen.
8. The medication for use in the treatment of AMD or dry AMD
according to claim 3, in which R2a is hydrogen and R2b is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, and its
salts.
9. The medication for use in the treatment of AMD or dry AMD
according to claim 3, comprising a compound according to the
following formula I* ##STR00006## in which R1 is methyl or
hydroxymethyl, one of the substituents R2a and R2b is hydrogen and
the other is hydroxy, methoxy, ethoxy, isopropoxy, methoxyethoxy or
ethoxypropoxy, one of the substituents R3a and R3b is hydrogen and
the other is hydroxy, methoxy, ethoxy, isopropoxy, methoxyethoxy or
methoxypropoxy, where R2a or R2b on the one hand and R3a or R3b on
the other hand are not simultaneously hydroxy, and its salts.
10. The medication for use in the treatment of AMD or dry AMD
according to claim 9, in which R1 is methyl, one of the
substituents R2a and R2b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, one of the
substituents R3a and R3b is hydrogen and the other is hydroxy, and
its salts.
11. The medication for use in the treatment of AMD or dry AMD
according to claim 9, in which R1 is methyl, one of the
substituents R2a and R2b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, one of the
substituents R3a and R3b is hydrogen and the other is methoxy,
ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy, and its
salts.
12. The medication for use in the treatment of AMD or dry AMD
according to claim 9, in which R3b is hydrogen.
13. The medication for use in the treatment of AMD or dry AMD
according to claim 9, in which R2a and R3b are hydrogen.
14. A medication comprising a compound according to the following
formula II* for use in the treatment of AMD or dry AMD ##STR00007##
In which one of the substituents Ra and Rb is hydrogen and the
other is methoxy, ethoxy, isopropoxy, methoxyethoxy or
methoxypropoxy, and its salts.
15. The medication for use in the treatment of AMD or dry AMD,
comprising a compound of claim 14, in which Ra is hydrogen and Rb
is methoxy, ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy,
and its salts.
16. The medication for use in the treatment of AMD or dry AMD
according to claim 3, which is
(7R,8R,9R)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,10-t-
etrahydro-imidazo[1,2-h][1,7]naphthyridine and its salts or
(7R,8R,9R)-2,3-Dimethyl-7-ethoxy-8-hydroxy-9-phenyl-7,8,9,10-tetrahydro-i-
midazo[1,2-h][1,7]naphthyridine and its pharmacologically
acceptable salts for the treatment of AMD or dry AMD.
Description
[0001] The invention relates to tetrahydropyridoethers for the
treatment of AMD and claims the priority of the European Patent
Application 08 000 761.0 of Jan. 16, 2008.
[0002] Age-related macular degeneration (AMD) is the main cause of
blindness in the western world (Bird A C, Bressler N M, Bressler S
B, Chisholm I H, Coscas G, Davis M D, de J P, Klaver C C, Klein B
E, Klein R (1995) International classification and grading system
for age-related maculopathy and age-related macular degeneration:
Surv. Ophthalmol 39: 367-374). About 30 million people suffer from
age-related macula degeneration (AMD), which leads to a loss of
central vision.
[0003] The macula is the most important part of the retina. The
retinal pigment endothelium (RPE) is essential for retinal
function. In healthy eyes, undisturbed transport of metabolites
takes place between photoreceptors and the RPE-choroid.
Accumulation of material between Bruch's membrane and the RPE
inhibits the transport of metabolites. Years of daily phagocytosis
of the shed photoreceptor tips by RPE are thought eventually to
take their toll in some individuals.
[0004] Over time, lipofuscin accumulates in the aging RPE until, in
some cases, the cells are virtually engorged with this material and
function is almost certainly compromised. It is generally accepted
that this aging process is a causative factor in age-related
macular degeneration.
[0005] Melanin
[0006] Optical measurements of the pigments of the RPE and choroid
have been made in human autopsy eyes varying in age between 2 weeks
and 90 years old. The choroidal melanin content increased from the
periphery to the posterior pole. The RPE melanin concentration
decreased from the periphery to the posterior pole with an increase
in the macula. The amount of RPE Lipofuscin increased from the
periphery to the posterior pole with a consistent dip at the fovea.
In humans there is an inverse relationship between RPE lipofuscin
concentration and RPE melanin concentration.
[0007] A biochemical examination has been carried out on the
lipofuscin content, lysosomal enzyme activities and melanin level
in the retina and choroid of normal human eyes. The melanin level
was two to three times higher in the macular RPE and choroid than
in other areas. Blue-light-induced photoreactivity of melanosomes
increases with age, perhaps providing a source of reactive oxygen
species and leading to depletion of vital cellular reductants,
which, together with lipofuscin, may contribute to cellular
dysfunction (Rozanowska M, Korytowsky W, Rozanowsky B, Skumatz C,
Boulton M G, Burke J M, Sarna T Photoreactivity of aged human RPE
melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis
Sci 2002, 43, 2088-96).
[0008] Lipofuscin
[0009] Lipofuscin is a pigment that is formed in tissues with high
oxidative stress (heart, liver, brain, eye) (Terman A, Brunk U T
(1998) Lipofuscin: Mechanisms of formation and increase with age.
APMIS 106: 265-276) Lipofuscin, also called age pigment, is a
brown-yellow, electron-dense, autofluorescent material that
accumulates progressively over time in lysosomes of postmitotic
cells, such as neurons and cardiac myocytes and the RPE. The exact
mechanisms behind this accumulation are still unclear. It can be
detected histologically by its autofluorescence properties. The
origin of lipofuscin in the RPE is still under debate (Kennedy C J,
Rakoczy P E, Constable I J (1995) Lipofuscin of the retinal pigment
epithelium: a review. Eye 9: 763-771). Numerous studies indicate
that the formation of lipofuscin is due to the oxidative alteration
of macromolecules by oxygen-derived free radicals generated in
reactions catalyzed by redox-active iron of low molecular weight.
Two principal explanations for the increase of lipofuscin with age
have been suggested. The first one is based on the notion that
lipofuscin is not totally eliminated (either by degradation or
exocytosis) even at a young age, and, thus, accumulates in
postmitotic cells as a function of time. Since oxidative reactions
are obligatory for life, they would act as age-independent
enhancers of lipofuscin accumulation, as well as of many other
manifestations of senescence. The second explanation is that the
increase of lipofuscin is an effect of aging, caused by an
age-related enhancement of autophagocytosis, a decline in
intralysosomal degradation, and/or a decrease in exocytosis. No
reports state that lipofuscin can be degraded or exocytosed by RPE
cells. In the eye, lipofuscin accumulates with age, especially in
the RPE, and occupies a considerable part of the cell volume in
elderly persons. Lipofuscin content, expressed as fluorescence
intensity, in the macular retinal pigment epithelium (RPE) and
choroid was two to three times higher than in other areas, and
increased with aging.
[0010] Interestingly, there is an association of melanin and
lipofuscin in the RPE. By use of enzyme cytochemistry, fluorescence
microscopy, and lipid extraction, two types of melanin-containing
complex granules have been identified: melanin with a cortex of
lipofuscin (melanolipofuscin and melanin with a cortex of
non-lipid, enzyme reactive material (melanolysosomes).
[0011] Lipofuscin and aged melanin in the RPE can generate oxygen
radicals, and both are believed to be involved in making the RPE
dysfunctional. The more lipofuscin the RPE at the margins of the
geographic atrophy contains, the quicker the atrophy will progress
(Holz et al. (2007) Am J Ophthalmol 143; 4639; Schmitz-Valckenberg
et al 2006; IOVS 47:2648).
[0012] AMD
[0013] This correlation is well accepted in ophthalmology. If the
progressing atrophy (AMD) reaches the macula, the patients become
legally blind. Two forms exist: Wet AMD is characterized by
neovascularization whereas dry AMD leads to geographic atrophy of
the RPE and retina. Macular degeneration in both forms is
associated with an accumulation of lipofuscin and melano-lipofuscin
(Feeney L (1978) Lipofuscin and melanin of human retinal pigment
epithelium. Fluorescence, enzyme cytochemical and ultrastructural
studies. Invest. Ophthalmol. Vis. Sci. 17: 583-600), an increase in
large deposits between the RPE cell layer and the Bruch's membrane
(called drusen).
[0014] A high cost anti-VEGF therapy (Ranibizumab) has been
developed against wet AMD.
[0015] 80 to 85% of the AMD patients have dry AMD, for which no
treatment modality currently exists.
[0016] It is consequently an object of the invention to provide a
compound for the treatment of AMD, especially for dry AMD.
[0017] It has now been found that tetrahydropyridoethers
especially, Soraprazan (INN Name)
(7R,8R,9R)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,10-t-
etrahydro-imidazo[1,2-h][1,7]naphthyridine and its salts and
related compounds remove lipofuscin from RPE cells and can
therefore serve as active ingredient in the treatment of AMD
degeneration, especially of dry AMD.
[0018] Until this observation, it was believed that the RPE cells
could not eliminate their lipofuscin during life.
[0019] The compounds, including Soraprazan, used for treatment
according to the invention have been described in WO 00/17200
(tetrahydropyridoethers) and EP 1 115 725 B1 that are herewith
incorporated by reference. EP 1 115 725 B1 especially describes
preferred compounds and methods of preparation including starting
compounds described e.g. in EP-A-0 299 470 or Kaminski et. al., J.
Med. Chem. 1985, 28, 876-892. The compounds according to the
invention can be prepared, for example starting from N-protected
8-amino-imidazo[1,2-a]pyridines in an enantioselective synthesis as
described in EP 1 115 725. The full process with different
variations and examples is incorporated by reference including the
examples of the final products 1-8 and starting compounds A1-D.
[0020] The invention furthermore relates to medications which
contain one or more compounds described in EP 1 115 725 and/or
their pharmacologically tolerable salts.
[0021] These compounds and examples for their preparations are
described as follows:
[0022] The invention relates to compounds of the formula I
##STR00001## [0023] in which [0024] R1 is methyl or hydroxymethyl,
[0025] one of the substituents R2a and R2b is hydrogen and the
other is hydroxy, methoxy, ethoxy, isopropoxy, methoxyethoxy or
methoxypropoxy, [0026] one of the substituents R3a and R3b is
hydrogen and the other is hydroxy, methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0027] where R2a or R2b on the one
hand and R3a or R3b on the other hand are not simultaneously
hydroxy, and their salts.
[0028] Suitable salts of compounds of the formula I are especially
all acid addition salts. Particular mention may be made of the
pharmacologically tolerable salts of the inorganic and organic
acids customarily used in pharmacy. Those suitable are
water-soluble and water-insoluble acid addition salts with acids
such as, for example, hydrochloric acid, hydrobromic acid,
phosphoric acid, nitric acid, sulfuric acid, acetic acid, citric
acid, D-gluconic acid, benzoic acid, 2-(4-hydroxybenzoyl)benzoic
acid, butyric acid, sulfosalicylic acid, maleic acid, lauric acid,
malic acid, fumaric acid, succinic acid, oxalic acid, tartaric
acid, embonic acid, stearic acid, toluenesulfonic acid,
methanesulfonic acid or 3-hydroxy-2-naphthoic acid, where the acids
are employed in salt preparation--depending on whether a mono- or
polybasic acid is concerned and depending on which salt is
desired--in an equimolar quantitative ratio or one differing
therefrom.
[0029] Pharmacologically intolerable salts which can be initially
obtained as process products, for example in the preparation of the
compounds according to the invention on an industrial scale, are
converted into pharmacologically tolerable salts by processes known
to the person skilled in the art.
[0030] According to expert's knowledge the compounds of the
invention as well as their salts may contain, e. g. when isolated
in crystalline form, varying amounts of solvents. Included within
the scope of the invention are therefore all solvates and in
particular all hydrates of the compounds of formula I as well as
all solvates and in particular all hydrates of the salts of the
compounds of formula I.
[0031] The compounds of the formula I have three chiral centers.
The invention relates to all eight conceivable stereoisomers in any
desired mixing ratio with one another, including the pure
enantiomers, which are a preferred subject of the invention.
[0032] In a preferred embodiment of the invention compounds are
used of the formula I*
##STR00002## [0033] in which [0034] R1 is methyl or hydroxymethyl,
[0035] one of the substituents R2a and R2b is hydrogen and the
other is hydroxy, methoxy, ethoxy, isopropoxy, methoxyethoxy or
methoxypropoxy, [0036] one of the substituents R3a and R3b is
hydrogen and the other is hydroxy, methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0037] where R2a or R2b on the one
hand and R3a or R3b on the other hand are not simultaneously
hydroxy, [0038] and their salts.
[0039] An embodiment (embodiment a) of the invention are compounds
of the formula I*, [0040] in which [0041] R1 is methyl, [0042] one
of the substituents R2a and R2b is hydrogen and the other is
methoxy, ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy,
[0043] one of the substituents R3a and R3b is hydrogen and the
other is hydroxy, [0044] and their salts.
[0045] A further embodiment (embodiment b) of the invention are
compounds of the formula I*, [0046] in which [0047] R1 is methyl,
[0048] one of the substituents R2a and R2b is hydrogen and the
other is hydroxy, [0049] one of the substituents R3a and R3b is
hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0050] and their salts.
[0051] A further embodiment (embodiment c) of the invention are
compounds of the formula I*, [0052] in which [0053] R1 is methyl,
[0054] one of the substituents R2a and R2b is hydrogen and the
other is methoxy, ethoxy, isopropoxy, methoxyethoxy or
methoxypropoxy, [0055] one of the substituents R3a and R3b is
hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0056] and their salts.
[0057] A further embodiment (embodiment d) of the invention are
compounds of the formula I*, [0058] in which [0059] R1 is
hydroxymethyl, [0060] one of the substituents R2a and R2b is
hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0061] one of the substituents R3a
and R3b is hydrogen and the other is hydroxy, [0062] and their
salts.
[0063] A further embodiment (embodiment e) of the invention are
compounds of the formula I*, [0064] in which [0065] R1 is
hydroxymethyl, [0066] one of the substituents R2a and R2b is
hydrogen and the other is hydroxy, [0067] one of the substituents
R3a and R3b is hydrogen and the other is methoxy, ethoxy,
isopropoxy, methoxyethoxy or methoxypropoxy, [0068] and their
salts.
[0069] A further embodiment (embodiment f) of the invention are
compounds of the formula I*, [0070] in which [0071] R1 is
hydroxymethyl, [0072] one of the substituents R2a and R2b is
hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0073] one of the substituents R3a
and R3b is hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0074] and their salts.
[0075] Preferred compounds of the embodiments a to f are those, in
which R3b is hydrogen.
[0076] Particularly preferred compounds of the embodiments a to f
are those, in which R2a and R3b are hydrogen.
[0077] Preferred compounds within the scope of the invention are
those of embodiment a, which can be characterized by the formula
I**
##STR00003## [0078] in which [0079] one of the substituents Ra and
Rb is hydrogen and the other is methoxy, ethoxy, isopropoxy,
methoxyethoxy or methoxypropoxy, [0080] and their salts.
[0081] Particularly preferred compounds of embodiment a are those
of formula I**, in which [0082] Ra is hydrogen and [0083] Rb is
methoxy, ethoxy, isopropoxy, methoxyethoxy or methoxypropoxy,
[0084] and their salts.
[0085] With the aid of the general formula I*, the following
exemplary preferred compounds according to the invention may
actually be mentioned by means of the substituent meanings for R1,
R2a, R2b, R3a and R3b in the following Table 1 (Tab. 1):
TABLE-US-00001 TABLE 1 R1 R2a R2b R3a R3b CH.sub.3 H OCH.sub.3 OH H
CH.sub.3 H OC.sub.2H.sub.5 OH H CH.sub.3 H OCH(CH.sub.3).sub.2 OH H
CH.sub.3 H OCH.sub.2CH.sub.2OCH.sub.3 OH H CH.sub.3 H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 OH H CH.sub.3 H OH OCH.sub.3 H
CH.sub.3 H OH OC.sub.2H.sub.5 H CH.sub.3 H OH OCH(CH.sub.3).sub.2 H
CH.sub.3 H OH OCH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 H OH
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 H OCH.sub.3 OCH.sub.3
H CH.sub.3 H OC.sub.2H.sub.5 OC.sub.2H.sub.5 H CH.sub.3 H
OCH(CH.sub.3).sub.2 OCH(CH.sub.3).sub.2 H CH.sub.3 H
OCH.sub.2CH.sub.2OCH.sub.3 OCH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.2OH H OCH.sub.3 OH H
CH.sub.2OH H OC.sub.2H.sub.5 OH H CH.sub.2OH H OCH(CH.sub.3).sub.2
OH H CH.sub.2OH H OCH.sub.2CH.sub.2OCH.sub.3 OH H CH.sub.2OH H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 OH H CH.sub.2OH H OH OCH.sub.3 H
CH.sub.2OH H OH OC.sub.2H.sub.5 H CH.sub.2OH H OH
OCH(CH.sub.3).sub.2 H CH.sub.2OH H OH OCH.sub.2CH.sub.2OCH.sub.3 H
CH.sub.2OH H OH OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.2OH H
OCH.sub.3 OCH.sub.3 H CH.sub.2OH H OC.sub.2H.sub.5 OC.sub.2H.sub.5
H CH.sub.2OH H OCH(CH.sub.3).sub.2 OCH(CH.sub.3).sub.2 H CH.sub.2OH
H OCH.sub.2CH.sub.2OCH.sub.3 OCH.sub.2CH.sub.2OCH.sub.3 H
CH.sub.2OH H OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 OCH.sub.3 H OH H
CH.sub.3 OC.sub.2H.sub.5 H OH H CH.sub.3 OCH(CH.sub.3).sub.2 H OH H
CH.sub.3 OCH.sub.2CH.sub.2OCH.sub.3 H OH H CH.sub.3
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H OH H CH.sub.3 OH H OCH.sub.3 H
CH.sub.3 OH H OC.sub.2H.sub.5 H CH.sub.3 OH H OCH(CH.sub.3).sub.2 H
CH.sub.3 OH H OCH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 OH H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3 OCH.sub.3 H OCH.sub.3
H CH.sub.3 OC.sub.2H.sub.5 H OC.sub.2H.sub.5 H CH.sub.3
OCH(CH.sub.3).sub.2 H OCH(CH.sub.3).sub.2 H CH.sub.3
OCH.sub.2CH.sub.2OCH.sub.3 H OCH.sub.2CH.sub.2OCH.sub.3 H CH.sub.3
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.2OH OCH.sub.3 H OH H
CH.sub.2OH OC.sub.2H.sub.5 H OH H CH.sub.2OH OCH(CH.sub.3).sub.2 H
OH H CH.sub.2OH OCH.sub.2CH.sub.2OCH.sub.3 H OH H CH.sub.2OH
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H OH H CH.sub.2OH OH H OCH.sub.3
H CH.sub.2OH OH H OC.sub.2H.sub.5 H CH.sub.2OH OH H
OCH(CH.sub.3).sub.2 H CH.sub.2OH OH H OCH.sub.2CH.sub.2OCH.sub.3 H
CH.sub.2OH OH H OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H CH.sub.2OH
OCH.sub.3 H OCH.sub.3 H CH.sub.2OH OC.sub.2H.sub.5 H
OC.sub.2H.sub.5 H CH.sub.2OH OCH(CH.sub.3).sub.2 H
OCH(CH.sub.3).sub.2 H CH.sub.2OH OCH.sub.2CH.sub.2OCH.sub.3 H
OCH.sub.2CH.sub.2OCH.sub.3 H CH.sub.2OH
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H
OCH.sub.2CH.sub.2CH.sub.2OCH.sub.3 H
[0086] And the salts of these compounds.
[0087] The compounds according to the invention can be prepared as
described by way of example in the following examples, or using
analogous process steps starting from appropriate starting
compounds (see, for example, EP-A-0 299 470 or Kaminski et al., J.
Med. Chem. 1985, 28, 876-892). The starting compounds are known or
can be prepared analogously to the known compounds. The compounds
according to the invention can be prepared for example starting
from N-protected 8-amino-imidazo[1,2-a]pyridines according to the
following reaction scheme:
##STR00004##
[0088] The above scheme represents an example of an
enantioselective synthesis. The N-protected (Piv represents a
customary protective group, preferably the pivaloyl group),
8-aminoimidazo[1,2-a]pyridine deprotonated in the 7-position is
reacted with an enantiomerically pure dioxolane. This initially
leads to a condensation product which can be cyclized under
strongly acidic conditions with removal of the protecting groups.
The subsequent reduction of the keto group using sodium borohydride
leads in over 90% enantiomeric purity to the 7,8-trans-diol
indicated. The subsequent etherification which is carried out
according to known processes, e. g. as described in the Examples,
leads to the final products of formula I* in which R2a and R3b are
hydrogen. The corresponding 7,8-cis-compound is obtained from the
mother liquor, which is left after separating off the
7,8-trans-compound, by chromatographic purification.
[0089] The substances according to the invention are isolated and
purified in a manner known per se, for example, by distilling off
the solvent in vacuo and recrystallizing the residue obtained from
a suitable solvent or subjecting it to one of the customary
purification methods, such as, for example, column chromatography
on suitable support material.
[0090] Salts are obtained by dissolving the free compound in a
suitable solvent, e. g. in a chlorinated hydrocarbon, such as
dichloromethane or chloroform, or a low molecular weight aliphatic
alcohol (ethanol, isopropanol) which contains the desired acid, or
to which the desired acid is subsequently added. The salts are
obtained by filtering, reprecipitating, precipitating with a
nonsolvent for the addition salt or by evaporating the solvent.
Salts obtained can be converted by alkalization or by acidification
into the free compounds, which in turn can be converted into salts.
In this way, pharmacologically intolerable salts can be converted
into pharmacologically tolerable salts.
[0091] The pure enantiomers, in particular the pure enantiomers of
the formula I*, to which the invention preferably relates, can be
obtained in a manner familiar to the person skilled in the art, for
example by enantioselective synthesis (see, for example, the
Scheme), by chromatographic separation on chiral separating
columns, by derivatization with chiral auxiliary reagents,
subsequent separation of diastereomers and removal of the chiral
auxiliary group, by salt formation with chiral acids, subsequent
separation of the salts and liberation of the desired compound from
the salt, or by (fractional) crystallization from a suitable
solvent. Trans-products obtained (with R2a and R3b=hydrogen) can be
converted (at least partly) to the corresponding cis-products (with
R2b and R3b=hydrogen) by standing under acidic conditions (e. g. 2
equivalents of acid, such as sulfuric acid) in the corresponding
alcohol R2a-OH. Likewise, cis-products obtained can be converted to
the corresponding trans-products. The cis- and trans-products are
separated e. g. by chromatography or by crystallization.
[0092] The following examples serve to illustrate the invention
further without restricting it. Likewise, further compounds of the
formula I whose preparation is not described explicitly can be
prepared analogously or in a manner familiar to the person skilled
in the art using customary process techniques. The abbreviation min
stands for minute(s), h for hour(s) and ee for enantiomeric
excess.
EXAMPLES
[0093] Final Products
1A.
(7R,8R,9R)-2,3-Dimethyl-8-hydroxy-7-methoxy-9-phenyl-7,8,9,10-tetrahyd-
ro-imidazo[1,2-h][1,7]naphthyridine
[0094] Method a
[0095] 20 g (65 mmol) of
(7R,8R,9R)-2,3-dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidaz-
o[1,2-h][1,7]naphthyridine are dissolved in methanol (350 ml). 13.5
g of sulfuric acid are added and the solution is stirred for 48 h
at 50 C. After cooling the reaction mixture is poured into 250 ml
of water. The pH is adjusted by aqueous saturated sodium hydrogen
carbonate solution to neutral pH. The precipitate is collected and
purified on silica gel (eluent: diethylether). 2.5 g of the title
compound are obtained as colourless crystals of melting point
164-165.degree. C. (2-propanol).
[0096] Method b
[0097] 10 g (32.5 mmol) of
(7R,8R,9R)-2,3-dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidaz-
o[1,2-h][1,7]naphthyridine are dissolved in 200 ml of dry
dimethylformamide. 1.9 g of commercially available sodium hydride
in paraffin (80%) are added in small portions at room temperature.
After 1 h 9.1 g (65 mmol) of methyl iodide, dissolved in 4 ml of
dimethylformamide, are added and the mixture is stirred for an
additional hour. The reaction mixture is poured into cold water. 20
ml of a saturated aqueous ammonium chloride solution is added; the
yellow precipitate is collected and discarded. The filtrate is
extracted several times with ethyl acetate, the combined organic
phases are washed several times with water and the solvent is
evaporated in vacuo. The solid residue is purified on silica gel
(diethylether).
[0098] 2 g of the title compound are obtained as colourless
crystals of melting point 164-165.degree. C. (2-propanol).
1B.
(7S,8S,9S)-2,3-Dimethyl-8-hydroxy-7-methoxy-9-phenyl-7,8,9,10-tetrahyd-
ro-imidazo[1,2-h][1,7]naphthyridine
[0099] The title compound of melting point 161-162.degree. C. is
obtained similarly to the procedure described in Example 1, Method
a, using
(7S,8S,9S)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo-
[1,2h][1,7]naphthyridine as starting material.
2A.
(7S,8R,9R)-2,3-Dimethyl-8-hydroxy-7-methoxy-9-phenyl-7,8,9,10-tetrahyd-
ro-imidazo[1,2-h][1,7]naphthyridine
[0100] 6 g of the title compound are obtained as colourless powder
of melting point 108-110.degree. C. after purification on silica
gel according to Example 1A, Method a, starting from
(7S,8R,9R)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidaz-
o[1,2-h][1,7]naphthyridine.
2B.
(7R,8S,9S)-2,3-Dimethyl-8-hydroxy-7-methoxy-9-phenyl-7,8,9,10-tetrahyd-
ro-imidazo[1,2-h][1,7]naphthyridine
[0101] The title compound of melting point 171-172.degree. C. is
obtained from the mother liquor of Example 1B after purification on
silica gel (eluent: diethyl ether).
3.
(7R,8R,9R)-2,3-Dimethyl-7-ethoxy-8-hydroxy-9-phenyl-7,8,9,10-tetrahydro-
-imidazo[1,7]naphthyridine
[0102] 500 mg of the title compound are obtained by reaction of
(7R,8R,9R)-2,3-dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidaz-
o[1,2-h][1,7]naphthyridine with ethanol and sulfuric acid according
to Example 1, Method a, after purification on silica gel (eluent:
diethylether). Melting point: 188-190.degree. C.
4.
(7S,8R,9R)-2,3-Dimethyl-7-ethoxy-8-hydroxy-9-phenyl-7,8,9,10-tetrahydro-
-imidazo[1,7]naphthyridine
[0103] 800 mg of the title compound of melting point
135-137.degree. C. are obtained as a solid by further purification
of the mother liquor of Example 3 on silica gel.
5A.
(7R,8R,9R)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,1-
0-tetrahydro-imidazo[1,2-h][1,7]naphthyridine
[0104] Method a
[0105] 5 g of the title compound of melting point 130-131.degree.
C. are obtained by reaction of 20 g
(7R,8R,9R)-2,3-dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidaz-
o[1,2-h][1,7]naphthyridine with 2-methoxy-ethanol according to
Example 1, Method a.
[0106] Method b
[0107] To a solution of 100 g of
(7R,8R,9R)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo-
[1,2h][1,7]naphthyridine in 1 L of 2-ethoxyethanol, 64 g of
concentrated. sulfuric acid are added slowly at room temperature
under an argon atmosphere. The rate of addition is such that the
temperature of the mixture does not exceed 35.degree. C. After
further 15 hours of stirring at room temperature the greenish
solution is poured into a mixture of 1 kg of crushed ice and 800 ml
of dichloromethane. The pH of the stirred mixture is adjusted to
7.5 by addition of a 10 M aqueous sodium hydroxide solution, the
organic layer is separated off, the aqueous layer is extracted
three times with dichloromethane (200 ml each), the dichloromethane
layers are washed collectively with 500 ml of water (six times) and
are then dried over sodium sulfate. After complete evaporation of
the solvent under reduced pressure the remaining oily residue is
treated with 450 ml of acetone to yield 75 g off-white crystals
consisting of a 1:1 mixture of the title compound and its
(7S,8R,9R)-epimer. The mixture is separated by preparative HPLC
using methanol as eluent. 28 g of the title compound of melting
point 128-129.degree. C. are obtained after recrystallization from
ethyl acetate.
5B.
(7S,8S,9S)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,1-
0-tetrahydro-imidazo[1,2-h][1,7]naphthyridine
[0108] The title compound of melting point 130-131.degree. C. is
obtained similarly to the procedure described in Example 5A, Method
a, using
(7S,8S,9S)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo-
[1,2h][1,7]naphthyridine as starting material.
6A.
(7S,8R,9R)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,1-
0-tetrahydro-imidazo[1,2-h][1,7]naphthyridine
[0109] 7.8 g of the title compound of melting point 131-132.degree.
C. are obtained as a solid from the mother liquor of Example 5A
after purification on silica gel (eluent: diethyl ether).
6B.
(7R,8S,9S)-2,3-Dimethyl-8-hydroxy-7-(2-methoxyethoxy)-9-phenyl-7,8,9,1-
0-tetrahydro-imidazo[1,2-h][1,7]naphthyridine
[0110] The title compound of melting point 131-132.degree. C. is
obtained from the mother liquor of Example 5B after purification on
silica gel (eluent: diethyl ether).
7.
(7S,8R,9R)-2,3-Dimethyl-8-hydroxy-9-phenyl-7-(2-propoxy)-7,8,9,10-tetra-
hydro-imidazo[1,2-h][1,7]naphthyridine
[0111] 1 g of the title compound of melting point 168-9 C is
obtained by reaction of 3 g of
(7R,8R,9R)-2,3-di-methyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imida-
zo[1,2-h][1,7]naphthyridine with 2-propanol according to Example 1,
Method a.
8.
(7R,8R,9R)-2,3-Dimethyl-7,8-dimethoxy-9-phenyl-7,8,9,10-tetrahydro-imid-
azo[1,2-h][1,7]naphthyridine
[0112] 8 g of the title compound of melting point 155-156.degree.
C. are obtained by reaction of 10 g of (7R,8R,
9R)-2,3-dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imidazo[1,2-h-
][1,7]naphthyridine with 1.9 g of sodium hydride (80%) and 9.1 g of
methyl iodide according to Example 1, Method b.
Starting Compounds
A1.
2,3-Dimethyl-7-[(2R,3S)-2,3-O-isopropylidene-3-phenylpropan-1-on-1-yl]-
-8-pivaloylamino-imidazo[1,2-a]pyridine
[0113] 60 g (0. 245 mol) of
2,3-dimethyl-8-pivaloylaminoimidazo[1,2-a]pyridine are dissolved in
1.5 L of anhydrous diethyl ether with exclusion of moisture and
under an argon atmosphere and cooled to -75.degree. C. By means of
a flex needle, 408 ml (0.612 mol) of tert-butyllithium solution
(1.5 M in n-pentane) are added dropwise such that the temperature
does not exceed -65.degree. C. (30 min). A red suspension is
formed. After addition is complete, the suspension is stirred at
-75.degree. C. for further 30 min. 1/3 of a solution of 145 g of
methyl (2R,3S)-2,3-O-isopropylidene-3-phenylpropionate (ee: 99.05%,
Daicel Chiralcel HPLC) in 150 ml of dry THF is then slowly added
dropwise at a temperature below -65.degree. C. during the course of
30 min. The residual quantity is then briskly added (5 min), a
temperature rise to -60.degree. C. taking place. After addition is
complete the cooling bath is removed. On reaching an internal
temperature of -30.degree. C., 20 ml of methanol are added and at
an internal temperature of 0.degree. C. 200 ml of distilled water
are added. The aqueous phase is separated off in a separating
funnel, the organic phase is washed five times with 100 ml of
distilled water each time, then the organic phase is extracted
three times with 10% strength sulfuric acid (200 ml, 50 ml, 50 ml).
The sulfuric acid phases are combined, treated with 200 ml of
dichloromethane and adjusted to pH 2.3 with 10N sodium hydroxide
solution and with ice cooling and vigorous stirring. The organic
layer is separated off. The aqueous phase is extracted with 30 ml
of dichloromethane. The combined dichloromethane phases are washed
twice with a little distilled water. The organic layer is then
dried over anhydrous sodium sulfate and the solvent is completely
stripped off in vacuo. A brown oil is obtained which is treated
with 50 ml of diethyl ether. After seeding, crystals are formed
which are filtered off after standing overnight and washed with
diethyl ether. After drying in vacuo, 57.7 g (52.5%, ee>99%,
Daicel Chiralcel HPLC) of the title compound of melting point
76-80.degree. C. are obtained as a pale yellow powder.
A2.
2,3-Dimethyl-7-[(2S,3R)-2,3-O-isopropylidene-3-phenylpropan-1-on-1-yl]-
-8-pivaloylamino-imidazo[1,2-a]pyridine
[0114] The title compound (ee: 98.3%, Daicel Chiralcel HPLC) is
obtained similarly to the procedure described in example A1 by
using methyl (2S,3R)-2,3-O-isopropylidene-3-phenylpropionate (ee:
98%, Daicel Chiralcel HPLC) as acylating agent.
B1.
(8R,9R)-2,3-Dimethyl-8-hydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo[1,2-
-h][1,7]naphthyridin-7-one
[0115] 10.8 g (24 mmol) of
2,3-dimethyl-7-[(2R,3S)-2,3-O-isopropylidene-3-phenylpropan-1-on-1-yl]-8--
pivaloylaminoimidazo[1,2-a]pyridine (ee>95%, Daicel Chiralcel
HPLC) are introduced into 50 ml of 70% strength sulfuric acid with
ice cooling during the course of 4 min. A suspension is formed in
the course of this, which turns into an orange solution after 30
min. After addition is complete, the ice bath is removed and the
mixture is stirred on at room temperature. The reaction solution is
added after 50 h to ice water and dichloromethane is added, then
the mixture is adjusted to pH 8 using 6N sodium hydroxide solution
and saturated sodium hydrogen-carbonate solution. The organic phase
is separated off. The aqueous phase is extracted twice with
dichloromethane. The organic phases are combined and washed with a
little distilled water. The organic layer is then dried over
anhydrous sodium sulfate, filtered and concentrated on a vacuum
rotary evaporator. The concentrated residue is chromatographed on
silica gel (eluent: dichloromethane/methanol 100/1). The main
fraction is concentrated and treated with ethyl acetate, and the
title compound crystallizes in the course of this as a yellow
solid. This precipitate is filtered off with suction and dried to
constant weight in a vacuum drying oven at 50.degree. C. 4.22 g
(57%, ee>95%, Daicel Chiralcel HPLC) of the title compound of
melting point 231-234.degree. C. are obtained.
B2.
(8S,9S)-2,3-Dimethyl-8-hydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo[1,2-
-h][1,7]naphthyridin-7-one
[0116] The title compound (ee: 94.0%, Daicel Chiralcel HPLC) is
obtained according to the procedure described in example B1
starting from
2,3-dimethyl-7-[(2S,3R)-2,3-O-isopropylidene-3-phenylpropan-1-on-1-yl]-8--
pivaloylaminoimidazo[1,2-a]pyridine.
C1.
(7R,8R,9R)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydroimid-
azo[1,2-h][1,7]-naphthyridine
[0117] 6 g (19.52 mmol) of
(8R,9R)-2,3-dimethyl-8-hydroxy-9-phenyl-7,8,9,10-tetra-hydroimidazo-[1,2--
h][1,7]naphthyridin-7-one (ee>90%, Daicel Chiralcel HPLC) are
suspended in 60 ml of methanol and cooled to -5 to 0.degree. C. in
a methanol-ice bath. At this temperature, sodium borohydride (0.81
g, 21.47 mmol) is added by spatula during the course of 0.5 h
(evolution of gas). After addition is complete, the mixture is
stirred for a further 10 min, and then concentrated in a vacuum
rotary evaporator at a bath temperature of 40.degree. C. The oily
residue obtained is taken up in distilled water and extracted three
times with chloroform. The organic phases are combined and washed
with a little water, then dried using anhydrous sodium sulfate and
filtered. The filtrate is concentrated on a vacuum rotary
evaporator and co-evaporated with acetone; the title compound
crystallizes out in the course of this. The precipitate is filtered
off, washed with acetone and dried to constant weight at 50.degree.
C. in a vacuum drying oven. 5.15 g (85.3%, ee>90%, Daicel
Chiralcel HPLC) of the title compound are obtained as a colorless
crystallizate of melting point 206-9.degree. C.
C2.
(7S,8S,9S)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7s8,9f1o-tetrahydroimid-
azo[1,2-h][1,7]naphthyridine
[0118] The title compound of mp 207-208.degree. C. (ee: 98.7%,
Daicel Chiralcel HPLC) is obtained according to the procedure
described in example C1 using
(8S,9S)-2,3-dimethyl-8-hydroxy-9-phenyl-7,8,9,10-tetrahydroimidazo[1,2-h]-
[1,7]naphthyridin-7-one as starting material.
D.
(7S,8R,9R)-2,3-Dimethyl-7,8-dihydroxy-9-phenyl-7,8,9,10-tetrahydro-imid-
izo[1,2-h][1,7]naphthyridine
[0119] 2 g of the mother liquor of Example C1 are chromatographed
on silica gel (eluent: ethyl acetate/methanol 19/1) to give 0.35 g
of the title compound as an oil which crystallizes upon addition of
ethyl acetate. Melting point: 199-200.degree. C. (ethyl
acetate).
[0120] The medication according to the invention is prepared by
processes known per se, which are familiar to the person skilled in
the art. As medication, the pharmacologically active compounds
according to the invention are employed either as such, or
preferably in combination with suitable pharmaceutical auxiliaries
or excipients in the form of intraocular devices, where the active
compound content is advantageously and where, by the appropriate
choice of the auxiliaries and excipients, a pharmaceutical
administration form exactly suited to the active compound and/or to
the desired onset of action can be achieved.
[0121] The person skilled in the art is familiar, on the basis of
his expert knowledge, with auxiliaries or excipients which are
suitable for the desired pharmaceutical formulations.
[0122] The active compounds are preferably administered orally,
topically, intravitreally, subretinally or periocularly. It has
proven advantageous to administer the active compound (s) in a dose
from 10-50 ng/ml. Favourably a dosage of about 10 to about 50 mg/kg
body weight, in particular about 10 to about 40 mg/kg, more
preferably of about 10 to about 36 mg/kg body weight is
administered to the patient. The optimal dose and manner of
administration of the active compounds necessary in each case can
easily be determined by any person skilled in the art on the basis
of his expert knowledge.
[0123] If the compounds according to the invention and/or their
salts are to be employed for the treatment of the above mentioned
diseases, the pharmaceutical preparations can also contain one or
more pharmacologically active constituents of other pharmaceutical
groups.
[0124] Soraprazan was administrated in oral application of 6, 12
and 24 mg/kg/day for 52 weeks in the Cynomolgus monkey.
[0125] Some monkeys from the control and the high-dose group were
subjected to a recovery period of 3 months.
[0126] Conventional histopathology revealed no alterations after
treatment with 6 and 12 mg/kg/day. Three out of 12 monkeys treated
with 24 mg/kg/day, including 1 animal with a funduscopic
abnormality, showed migration of individual macrophages either
beneath the RPE (1 animal), and/or into the subretinal space (3
animals). Two of these 3 monkeys had depigmentation of RPE cells
although the photoreceptors facing the depigmented RPE stayed
healthy. These RPE cells had released melanin as well as lipofuscin
granules to secondary cells that had migrated between Bruch's
membrane and the RPE cell layer or into the subretinal space.
Therefore this shows that it is possible to stop the progression of
lipofuscin accumulation in conditions where there is a risk of
getting dry AMD.
[0127] As lipofuscin can be easily detected in the fundus, the
invention would also allow prevention of the disease, as detection
can already be done at an early stage of the disease
development.
DESCRIPTION OF EXAMPLES
[0128] In the present example the effect of Soraprazan on
pigmentation of the retinal pigment epithelium in the Cynomolgus
monkey (4 years of age) after oral application of 24 mg/kg/day for
52 weeks is shown.
[0129] Methods
[0130] Right eyes from the following animals were subjected to
transmission electron microscopy:
TABLE-US-00002 No. of animals investigated Soraprazan by
transmision electron (mg/kg/day) microscopy Group 52 weeks m/f 1 0
3/4 2 24 4/3
[0131] Sampling, Fixation, Embedding
[0132] Right eyes were removed carefully. A circular slit was cut
at the limbus in order to immerse the inner eye with the fixation
fluid (5% Glutaraldehyd in 100 .mu.mol Cacodylat buffer). From
these eyes, specimens (1 mm.sup.3 in diameter) from the macula and
from the mid-area were cut out, were postfixed in OsO.sub.4,
treated with uranylacetate, dehydrated and embedded in Epon
resin.
[0133] Sectioning, Evaluation
[0134] Semithin sections (0.7 .mu.m) were prepared from Epon resin
blocks. They were stained with toluidin blue and evaluated under a
light microscope. Subsequently, ultrathin sections (50 nm) were cut
from Epon resin blocks, contrasted and evaluated in a transmission
electron microscope.
[0135] Ultrastructure of RPE and Photoreceptor Outer Segments in
Control Animals
[0136] The RPE cells of the Cynomolgus monkeys contain many
microvilli at the apical cell surface. Spindle-shaped melanin
granules are located in normal RPE within these microvilli. The
spindle shaped melanosomes are 1.6 .mu.m long and 0.5-0.7 .mu.m
thick. The central parts of the RPE cells contain predominately
round melanosomes and lipofuscin granules with a diameter between
0.7-1.2 .mu.m in most cases. Also many mixed type granules
(melanolipofuscin) containing both melanin and lipofuscin are
present in the central parts of the RPE cells. The outer segments
of cones contain irregular disk membranes and homogenous material,
whereas the outer segments of the rods contain more regularly
shaped and highly ordered disk membranes.
[0137] Treatment-Related Findings in RPE Cells and Photoreceptor
Cells
[0138] Ultrastructural alterations in the retina compared to
untreated monkeys could not be detected in any of the treated
monkeys (Table 1).
TABLE-US-00003 TABLE 1 Summary of ultrastructural findings
Lipofuscin removal from RPE RPE cell N = normal, morphology ++ =
(except moderate pigment Structure of Animal +++ = granules)
photoreceptors Group No. complete N = normal N = normal 1 1 N N N
(0 mg/kg/day) 2 N N N 3 N N N 4 N N N 5 N N N 6 N N N 7 N N N 2 8
+++ N N (24 mg/kg/day) 9 +++ N N 10 ++ N N 11 ++ N N 12 ++ N N 13
++ N N 14 +++ N N
[0139] Lysosomes in RPE cells exhibited a regular morphology.
Accumulation of secondary lysosomes which degrade the shed tips of
the outer segments were not found in any RPE cells investigated in
this study. RPE cells did not divide, nor did they show any signs
related to cell death.
[0140] Tight junctions between RPE cells appeared normal in all
groups. Separation of RPE cells or enlargement of intercellular
clefts between RPE cells was not observed in any eye from this
study.
Example 1
[0141] The most prominent alteration was loss of melanin and
lipofuscin in RPE cells in the eyes of 3 out of 7 monkeys from
animals treated with 24 mg/kg/day (Table 1). Different stages of
degradation of spindle shaped melanosomes within the apical
microvilli were observed.
[0142] These spindle shaped melanosomes became 0.4-0.2 .mu.m thin
and then separated into bead-like structures, before dividing up
into separate individual small granules with diameters between
0.2-0.5 .mu.m. Finally the spindle shaped granules disappear
completely from the microvilli. In these animals there were many
areas with a diameter up to 2 micrometers in which the RPE was more
or less completely free of melanin and lipofuscin granules. Such
areas were also observed below the macula. In addition, fusion of
melanosomes in large lysosomes was observed in all 7 monkeys that
were investigated from group 2.
Example 2
[0143] In the vicinity of depigmented RPE cells, macrophage-like
cells were frequently present. Staining with CD 68 antibodies
showed that these cells were macrophages. They were located in most
cases between Bruch's membrane and RPE. They were also seen within
Bruch's membrane. These macrophages were filled with lipofuscin
granules and melanosomes as well as melanolipofuscin granules and
therefore were highly pigmented. Pigment granules within these
macrophages were often collected in lysosomes. These findings show
that the RPE cells can release their pigment granules.
Example 3
[0144] Section was illuminated under the fluorescence microscope
with 360 nm wavelength light. Lipofuscin granules were detected by
the emission of gold-yellow light with 540 .mu.m wavelength. In RPE
cells that were depigmented by bright light examination, the
lipofuscin granules were completely or almost completely absent.
Macrophages between the RPE and Bruch's membrane, however,
contained many lipofuscin granules. The majority of these granules
were smaller than those in RPE cells of untreated animals. These
findings show that the RPE cells can release their pigment
granules.
Example 4
[0145] The number of lipofuscin granules that were smaller than 0.4
.mu.m in diameter was counted in the cytoplasm of ultrathin
sections of RPE cells from untreated and treated monkeys. In
addition the same counts were performed in macrophages located
between RPE and Bruch's membrane of treated monkeys. The results
were 4.9.+-.0.6/50 .mu.m.sup.2, 0.6.+-.0.2/50 .mu.m.sup.2;
13.6.+-.0.9/50 .mu.m.sup.2. Lipofuscin granules larger than 1 .mu.m
in diameter were absent in RPE cells of treated animals. In
contrast many of them were present in RPE cells of untreated
monkeys. These findings show that the RPE cells of treated monkeys
can eliminate the lipofuscin granules.
Example 5
[0146] In order to investigate whether trace element concentration
is altered by drug administration, X-ray microanalysis of
melanosomes within RPE cells from untreated and drug treated
monkeys was performed. In addition melanosomes within macrophages
were also analysed.
[0147] An increase of the Na, P and Ca concentration and a
statistically insignificant decrease of Fe was found in RPE
melanosomes after treatment with Soraprazan. These results show
that pigment granules are chemically modified by Soraprazan
treatment. This may be the reason why the pigment granules are
extruded from the RPE cells.
[0148] No morphological changes in the neuroretina were observed at
the ultrastructural level after treatment with Soraprazan 24
mg/kg/day for 52 weeks.
[0149] The present study shows for the first time that RPE cells of
the adult monkey can eliminate lipofuscin and degrade melanin. Both
findings were induced by drug administration. These findings are
extremely unusual and surprising, because until this finding it was
believed that the RPE cells could not eliminate their lipofuscin
during life.
[0150] Therefore, with the present invention it is possible to
prevent the progression of lipofuscin accumulation or to remove
lipofuscin in patients at risk of getting AMD, especially dry
AMD.
[0151] As lipofuscin can be easily detected in the fundus, this new
treatment method can already be applied at an early stage of the
disease development of dry and wet AMD.
Example 6
[0152] Retinal pigment epithelium (RPE) cells from human donor eyes
were cultured and exposed permanently to either vehicle solution,
solution of Soraprazan or solution of
7R,8R,9R)-2,3-Dimethyl-7-ethoxy-8-hydroxy-9-phenyl-7,8,9,10-tetrahydro-im-
idazo-[1,2-h][1,7]-naphthyridin (called substance 1) at a
concentration of 50 .mu.g/ml or 0.25 mM. At several time points,
digital images were taken and analysed with respect to the portion
of lipofuscin and pigmentation in general. As the results did
strongly depend on the random choice of microscopic field and the
variations of the data were very high, the values of four
consecutive days of five independent experiments were averaged. The
results for the lipofuscin content in the RPE cells are shown in
the diagram (FIG. 18).
[0153] Retinal pigment epithelium (RPE) cells from human donor eyes
were cultured and exposed permanently to either vehicle solution,
solution of Soraprazan or solution of substance 1 (concentration 50
.mu.g/ml or 0.25 mM). At several time points, digital images were
taken and analysed with respect to the portion of lipofuscin and
pigmentation in general.
[0154] The degree of lipofuscin content and total pigmentation
decreases slightly in the control samples, which can be explained
by a weak division of RPE cells and thus dilution of pigment. In
the cell cultures treated with Soraprazan, lipofuscin and pigment
content are decreased compared to the control. In the RPE cells
treated with substance 1, there was an even more clear and
significant decrease in both lipofuscin content and
pigmentation.
[0155] Then the treatment-dependent ability of the cells to
phagocytose, which is a crucial function of the RPE, was checked.
For this purpose, RPE cell cultures from human donor eyes were
exposed to vehicle or substance 1. After three weeks, fluorescent
latex beads were added to the cell cultures for four hours. The
cells were then washed to remove non-phagocytosed beads and fixed.
Fluorescent images of the cells were taken and analysed for the
contents of lipofuscin and the number of phagocytosed beads. The
results shown in FIG. 19 are presented as the relationship between
lipofuscin content and phagocytosed bead number in both controls
and substance-1-treated cells.
[0156] It can clearly be seen that the RPE cells contain much more
lipofuscin under control conditions than under the influence of
substance 1. In addition, it is obvious that RPE cells of a high
lipofuscin content do not phagocytose many latex beads, in most
cases not a single one. In contrast, many cells with a small
portion of lipofuscin have phagocytosed a significantly higher
number of latex beads.
[0157] As a summary of the in vitro experiments, it can be
concluded that the human RPE cells get rid of their lipofuscin when
they are treated with Soraprazan or substance 1, and that loss of
lipofuscin is associated with an enhanced ability of
phagocytosis.
Example 7
[0158] The effects of lipofuscin in vivo, i.e. in the living eye of
experimental rats after an intravitreal injection of the compounds
were investigated. Vehicle solution or solutions of either
substance 1 or Soraprazan were injected intravitreally in half-year
old Wistar rats. The final concentration in the vitreous was the
same as in the cell cultures, i.e. 50 .mu.g/ml or 0.25 mM. In order
to avoid mutual interactions between the two eyes of an animal,
both eyes of an animal were treated the same way, with three
animals (i.e. six eyes) per group.
[0159] Two kinds of evaluation were performed in these
animals--electroretinography (ERG) for functional testing, and
counting of lipofuscin particles to check whether the
administration of the compounds leads to a decrease of lipofuscin
contents in the RPE.
[0160] Electroretinography:
[0161] Before the injection, electroretinograms were measured to
obtain base line values. Additional ERG measurements were performed
one, two and three weeks after the intravitreal injections. Some
results are shown in the diagrams below.
[0162] The ERG amplitudes obtained one week after the injection
were smaller than the base line values, in most cases
significantly. Such a decrease is a direct consequence of the
injection procedure and has been observed also in other studies
were intravitreal injection has been performed, and the extent of
the decrease depended on the kind of injected solution and the kind
of electroretinographic parameter.
[0163] In the diagrams (FIG. 20), changes of the amplitudes of
a-waves and b-waves are shown, recorded at the highest intensity of
light stimulation. After an injection of vehicle solution
(containing 20 vol % DMSO), a decrease of amplitudes is observed.
If Soraprazan or substance 1 are injected, the decrease of the
amplitudes is even more pronounced. During the following time, a
certain recovery of the amplitudes can be observed. Amplitudes
obtained in animals treated with vehicle or substance 1 solutions
did recover almost completely three weeks after the injection,
whereas the values obtained in Soraprazan-treated animals remained
significantly lower than the base line. Such a behaviour was also
seen in the photopic b-waves, i.e. the cone-driven response of the
post-receptoral systems.
[0164] The amplitudes of the scotopic oscillatory potentials did
not show a recovery after the initial decrease in all three groups,
and the same is true for the 30-Hz Flicker response. There is
obviously a permanent damage by the injection that cannot be
repaired, probably by the DMSO that is present in the injected
solution. The kind of damage suggests that inner neurones and/or
the communication between them may be disturbed.
[0165] As a summary, the used compounds interfere with the function
of the photoreceptors and post-receptoral systems. Nevertheless,
disturbance of retinal function was almost reversible compared to
vehicle-injected eyes if substance 1 had been used. Less side
effects on retinal function can be expected by reduction of DMSO
content in the injected solution and an optimised injection
routine.
[0166] Lipofuscin Content:
[0167] The eyes were isolated after three weeks, fixed in formalin
and embedded in paraffin. Paraffin sections were made, and digital
fluorescence images were evaluated. The number of fluorescent
lipofuscin particles per 50 .mu.m RPE layer length was counted. The
results are shown in the diagram (FIG. 21).
[0168] In the vehicle-treated eyes, 21.0.+-.7.8 lipofuscin
particles were found per 50 .mu.m. In contrast, only 15.0.+-.8.3
particles were found in Soraprazan-treated eyes, and 9.8.+-.6.1
particles in eyes treated with substance 1. The difference between
these values was significant. Consequently, even one single
injection of the compounds leads to a clear decrease in the
lipofuscin content in the RPE.
Example 8
[0169] Heavily pigmented human donor RPE cells (passage1) were
treated with 50 .mu.g/ml substance1 or 30 .mu.g/ml Soraprazan and
cultured for 28 days. Cells without treatment were used as
controls. Cells were fixed for electron microscopy in 2%
glutaraldehyde and embedded in EPON. Semithin and ultrathin
sections were cut.
[0170] Ultrastructurally, treated cells contained big clusters of
pigment-like granules (FIG. 22), covered by a limiting membrane.
Individual lipofuscin, melanin or melanolipofuscin granules were
missing, but were present in the controls. These clusters contained
unusual small melanin granules embedded into a lipofuscin-like
electron opaque matrix. The total amount of normal appearing
pigment granules was largely reduced in these cells. In treated
cells many small electron lucent and opaque granules were present.
Clusters and the electron lucent granules could only infrequently
be observed in the controls.
[0171] In the semithin sections, the clusters could be observed as
well. Here, the pigmented cells bearing one or more clusters were
counted. Degradational clusters were detected in 90.5%+/-21.3 of
cells treated with substance 1 and in 80.1%+/-22.6 of cells treated
with Soraprazan, but only in 16.8%+/-21.6 of untreated cells
(p<0.0001).
[0172] Thus, the pigments undergo degradation in substance treated
human RPE cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0173] FIG. 1:
[0174] Lipofuscin fluorescence as a function of age at 7.degree.
temporal to the fovea (top) and at the fovea (bottom). The solid
lines are linear regression lines for ages 20 to 70 years
(P<0.0001). The interrupted lines are linear regression lines
for ages 70 to 80 years (P<0.12). From Delori F C, Goger D G,
Dorey C K Age-related accumulation and spatial distribution of
lipofuscin in RPE of normal subjects. Invest Ophthalmol Vis Sci.
2001; 42:1855-66.
[0175] FIG. 2:
[0176] RPE from a 72 year old women contains few melanosomes, but
many lipofuscin or melanolipofuscin granules
[0177] FIG. 3:
[0178] FIG. 3, in contrast to FIG. 2 shows that RPE from young
individuals contains many melanosomes
[0179] FIG. 4:
[0180] Several macrophages have migrated between Bruch's membrane
and RPE just below the macula of a monkey after treatment with
Soraprazan. Whereas the RPE is nearly free of lipofuscin, the
macrophages are highly pigmented (see also FIG. 5 for more
details). The photoreceptors appear healthy.
[0181] FIG. 5:
[0182] Several macrophages have migrated between Bruch's membrane
and RPE just below the macula of a monkey after treatment with
Soraprazan. Whereas the RPE is nearly free of Lipofuscin, the
macrophages are highly pigmented. The photoreceptors appear
healthy.
[0183] FIG. 6:
[0184] The normal pigmented RPE of an untreated monkey is shown
(top). After treatment with Soraprazan the RPE is nearly free of
lipofuscin (arrow), which is now localised within the macrophage
(bottom).
[0185] FIG. 7:
[0186] A macrophage is localised between Bruch's membrane and the
RPE of a monkey after treatment with Soraprazan, as shown in an
electron micrograph. The RPE is nearly free of lipofuscin, which is
now localisedI within the macrophage (FIG. 9) below. Rod outer
segments appear normal.
[0187] FIG. 8:
[0188] Ultrathin section from a monkey treated with Soraprazan
reveals a trilayer of cells in the parafovea shown in a semithin
section in FIG. 6. Blood vessels are not present in this layer. The
morphology of the choriocapillaris and Bruch's membrane is normal.
The cells are separated by an extracellular matrix. The outer
segments of the photoreceptors are completely normal. The RPE has
lost melanin and lipofuscin granules.
[0189] FIG. 9:
[0190] Ultrathin section from a monkey treated with Soraprazan
shows small lipofuscin granules within a macrophage localised
between Bruch's membrane and the RPE. Such lipofuscin granules
within macrophages were measured and counted and compared to those
within the RPE cells of untreated monkeys (see FIG. 15).
[0191] FIG. 10:
[0192] The arrows show macrophages located between Bruch's membrane
and the RPE which had taken up lipofuscin granules from RPE cells.
The lipofuscin granules are identified by their golden-yellow
autofluorescence in a light micrograph. The RPE cells are nearly
free of lipofuscin granules. The rod outer and inner segments of
the photoreceptors appear normal.
[0193] FIG. 11:
[0194] The arrows show macrophages located between Bruch's membrane
and the RPE which had taken up lipofuscin granules from RPE cells.
The lipofuscin granules are identified by their golden-yellow
autofluorescence in a light micrograph. The RPE cells are nearly
free of lipofuscin granules. The rod outer and inner segments of
the photoreceptors appear normal.
[0195] FIG. 12:
[0196] Paraffin section from a monkey treated with Soraprazan. A
macrophage (red) identified by immunostaining with CD 68 antibodies
has migrated between Bruch's membrane and RPE.
[0197] FIG. 13:
[0198] Ultrathin section from a monkey treated with Soraprazan
shows small lipofuscin granules within a macrophage localised
between Bruch's membrane and the RPE.
[0199] FIG. 14:
[0200] Ultrathin section of RPE cells of monkey not treated
Soraprazan (Control).
[0201] FIG. 15:
[0202] The lipofuscin granules within macrophages of FIG. 13 were
measured and counted and compared to those of FIG. 14. The number
of small lipofuscin granules is significantly enhanced in
macrophages indicating degradation after Soraprazan treatment.
[0203] FIG. 16:
[0204] The concentration of C, Na, P,S, Ca and Fe in RPE
melanosomes or in melanosomes from macrophages after drug treatment
are presented in atom % as detected by EDX. These findings were
compared to the concentrations without treatment. A significant
increase of Ca, Na and P was found in RPE melanosomes after
treatment. (p=p-values from Student's T-test; n=number of
measurements). The animal was treated with 24 mg
Soraprazan/kg/day.
[0205] FIG. 17:
[0206] The table shows that lipofuscin depigmentation in the RPE
was observed in monkeys treated with 24 mg Soraprazan/kg/day. The
morphology of the photoreceptors as judged by the ultrastructure of
the outer segments was normal. This indicates that the function of
the RPE was not altered, although some mild changes were seen in
the microvilli and basal labyrinth.
[0207] FIG. 18:
[0208] Lipofuscin and melanin is reduced in cultured human RPE
cells after 26 days of treatment with Soraprazan and substance
1.
[0209] FIG. 19:
[0210] The results shown in this diagram are presented as the
relationship between lipofuscin content and phagocytosed bead
number in both controls and substance-1-treated human RPE cells.
Treated and depigmented cells phagocytosed more beads than
untreated cells.
[0211] FIG. 20:
[0212] Soraprazan and substance 1 were injected into the vitreous
of Wistar rats. The amplitudes of a-waves (photoreceptors) and
b-waves (retinal neurons) are shown by electroretinography (ERG),
recorded at the highest intensity of light stimulation. After an
injection of vehicle solution (containing 20 vol % DMSO), a
decrease of amplitudes is observed. If Soraprazan or substance 1 is
injected, the decrease of the amplitudes is even more pronounced.
During the following time, a certain recovery of the amplitudes can
be observed. Amplitudes obtained in animals treated with vehicle or
substance 1 solutions did recover almost completely three weeks
after the injection, whereas the values obtained in
Soraprazan-treated animals remained significantly lower than the
base line.
[0213] FIG. 21:
[0214] Soraprazan and substance 1 were injected into the vitreous
of Wistar rats. The eyes were isolated after three weeks, fixed in
formalin and embedded in paraffin. Paraffin sections were made, and
digital fluorescence images were evaluated. The number of
fluorescent lipofuscin particles per 50 .mu.m RPE layer length was
counted.
[0215] FIG. 22:
[0216] Human RPE cells treated with substance 1 contained clusters
of pigment-like granules, covered by a limiting membrane as shown
in an electron micrograph. These clusters contained unusual small
melanin granules embedded into a lipofuscin-like electron opaque
matrix indicating lipofuscin and melanin degradation.
* * * * *