Note: Descriptions are shown in the official language in which they were submitted.
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ANTIOXIDANT METALLO~ORGANIC TREATMENT OF INFLAMMATION
Field of the Invention
Our present invention relates to a method of treatment of
mammalian subjects for inflammatory conditions and, more
particularly, arthritis and like active-oxygen or superoxide
based conditions.
Background of the Invention
It has been recognized for some time that inflammation in
mammalian species can be traced at least in part to active
oxygen species, including superoxide, and radicals associated
therewith at the inflammatory site. Considerable r~search has
been undertaken to measure and detect oxygen radicals, to
establish the mechanisms whereby enzymes such as superoxide
dismutase is effective in countering oxygen radical toxicity
and even in the development and use of copper amine oxidases in
preventing tissue damage and even in promoting damaged-tissue
recovery.
However, the compounds which have been developed
heretofore for active-oxygen or superoxide antagonism and
destruction n Vivo have not proved as effective as desired or
were characterized by side reactions or could not be made in
commercially significant quantities at reasonable cost.
Ob~ects of the Invention
It is, therefore, the principal ob~ect of our invention to
provide an improved m~thod of treatment of mammalian subjects
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for inflammatory conditions whereby the aforedescribed
drawbacks are obviated.
Another object of our invention is to provide an improved
method of treating a mammalian subject for a condition
resultiny from active oxygen or superoxide toxicity which may
result in acute or chronic inflammation or any other disorder.
Still another object is to provide a method of treatm~nt
which is nontoxic and has no measurable side-effects but which
is particularly effective in the treatment of arthritic
conditions cr the lika.
Summary of the Invention
These objects and others which will become apparent
hereinafter are attained, in accordance with the present
invention in a method of treating active-oxygen and superoxide
associated inflammator~ conditions in mammalian subjects which
comprises administering an eEfective and nontoxic dose for an
inflammation ameliorating period to the subject of at least one
metallo-organic complex capable of in vivo superoxide
anti-oxidant effects and wherein the metal of the complex is
selected from the group which consists of cobalt and iron.
Preferably the method is used in the treatment of acute or
chronic arthritis in a dosage of 0.1 to 250 mg/kg of body
weight, but in all cases at most 50% of the LD50 value of the
compound, when the complex is administered orally as is
pre~erred. However the complex or a combination of the
complexes can be administered subcutaneously or even topically
in a suitable vehicle, e.g. physiological saline in the case of
s.c. administration an~ dimethylsulfoxide (DMSO) in the case of
a topical administration, although ointments, salves or like
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conventional vehicles may be employed.
For oral administration, the complex or mixture of
complexes may be prepared in suitable dosage forms. For example
it may be prepared a5 dragees, as capsules, as tablets, as an
elixir or other oral dosage form.
The dose may he administered one to six times daily,
depending upon the severity of the inflammatory condition,
preferably under medical supervision so that the dosage can be
reduced or the number of daily administrations limited as the
inflammatory condition subsides.
The compounds of the invention may also have prophylactic
properties in preventing the spread of arthritic inflammation
and has been found to be effective in reducing the sevPrity of
the actual condition which develops in subjects who are prone
to such inflammatory states. The compounds may also be
effective in preventing postischemic heart damage and for
geriatric applications other than as antiarthritics.
We have found that the anti-inflammatory effect is
exceptionally pronounced with low-toxicity metallo-organic
complexes selected from the group which consists of:
[Co~2,3,g,10-tetra~low~r-alkyl}-1,4,8,11-tetraazacyclotetradeca-
1,3,8,10-tetraene}Cl2~Cl,
a 1,1-dimethylfexrocenium or other alkyl ferrocenium salt,
a cobalt(III)-bis(acetyl or propio acetone)-ethylenediimine
complex, and
[Co{2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca--
1(17),2,11,13,15-pentaene}C12]Cl H20.
In the case of the ferrocenium derivatives, we have
discovered that the key to the effect is the ferrocenium ion,
so that any physiologically compatible or pharmaceutically
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effective salt thereof can be employed. Furthermore, the methyl
groups arQ important only in the sense that two methylene
structures -CH2- moieties are attached to the ferrocenium
moiety. The methyls can thus be substituted by Cl to C5
-alkyl, alkenyl or alkoxyO Best results are obtained with
1,1-dimethylferrocenium salts.
In the case of the (Co{2,3,9,10-tetra{lower-alkyl}-1,4,8,-
11-tetraazacyclotetradeca-1,3,8,10-tetraene}C12)Cl, we have
~ound that the lower alkyl can be Cl to C6-alkyl but that
best results are obtained when the tCo~2,3,9,10-tetra{lower-
-alkyl}-1,4,8,11-tetraazacyclotetradeca-1,3,8,10-tetraene}C12)
Cl is Co{2,3,9,10-tetramethyl-1,4,8,11-tetraazacyclotetradeca--
1,3,8,10-tetraene)C12)Cl.
Preferably the cobalt(III)-bis(acetyl or propio acetone)-
e~thylenediimine complex is a cobalt(III)-bis(acetylacetone)-
-ethylenediimine complex, and more specifically the
[Co(bis~acetylacetone}-ethylenediimine)(NH3)2]+ Cl-.
We have found that the complexes specifically identified
above can be used together with hitherto-known anti-inflammator-
ies with propionic acid side chains, especially indomethacine
to further alleviate the suffering of arthritic inflammation.
Preparation o~ the Compounds
[Co(Tim)C12]Cl - Compound 8)
Tim = 2,3,9,10 tetramethyl 1,4,8,11-tetraazcyclotetradeca-
-1,3,3,10-tetraene.
The procedure of Bush et al ~Inorg. Chem., 1972, 11, 2893)
; was employed to prepare [Co(Tim~C12]PF6.
A saturated solution of tetramethylammonium chloride in
68~
acetone was added slowly to a saturated solution of
[Co(Tim)C12]PF6 in acetone until the solution became
cloudy. Cooling afforded light green crystals of compound 8.
Anal. Calcd. for C14H24N4CoC13: C, 40.63; H, 5.8;
N, 13:54. Found: C, 40.57; H, 5.67; N, 13.15.
l,l-Dimethylferrocenium Salts (PF6- Cl-~
- 4~ 1_4-~ -- Compound 19)
The procedure of Wahl et al. (J. Phys. Chem., 1975, 79,
2049-52) was employed.
A solution of dimethylferrocene (0.65 g. 3 mmol) in
concentrated sulfuric acid (8 ml) was stirred at room temp. for
10 min. and then poured into 60 ml. of cold, dist. water. The
insoluble material was removed by ~iltration, the deep-blue
; filtrate treated with 0.82 g (5 mmol) of ammonium
hexafluorophosphate, stirred for 1/2 hr. and cooled. The
separated solid (compound 19) was collected, washed with ethyl
ether and dried under vacuum for two days.
Anal. Calcd. for C12H~4FePF6: C, 40.14; H, 3.93.
Found: C, 39.98; H, 4.23.
Cobalt(III~ Bis(acetylacetone)-ethylenediimine Complex
~Compound 23)
[C(BAE)(NH3)2]cl
~his complex was prepared by a modification of the
procedure described by G. Costa et al., J. Organometal. Chem.,
966, 6, 1~1 - 187.
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Bis(acetylacetone)-ethylenediimine (BAE, 2.24 g, 10 mmol)
was dissolved in methanol (100 ml) and treated with cobalt
chloride hexahydrate (2.38 g, 10 mmol). The brown solution was
stirred at room temperature for 1 1/2 days in order to allow
oxidation of the cobalt (no precipitate formed), treated with 7
ml of conc. ag. ammonia solution, and then heated under reflux
for 2 hrs. The yellow-brown solid that separated upon standing
was recrystd. from ethanol/water (compound 23).
Anal. Calcd. for C12H242N4C~l C, 41.09; H,
6.90. Found: C, 40.81: ~, 7.00.
[Co(CR)C12]Cl H20 (Compound 39~
CR= 2,12-dimethyl-3,7,11,17-tetraazabicyclo[11.3.1]
heptadeca-1(17),2,11,13,15-pentaene
The Co(II) complex, [Co(CR)C12], was prepared and
oxidized by the procedure described by Poon et al. for the
analogous perchlorate (J. Chem. Soc. Dalton, 1977, 1247-1251).
The complex ~compound 39) was recrystallized from acetone.
Anal. Calcd. for : C16H240N4CoC13: C, 42035; H,
5.33; N, 12.35; Cl, 23.45. Found: C, 41.35; H, 5.69; N,
12.07; Cl, 23.64.
In vivo_Inflammatory Studies
The drugs used are prspared just before performing the ex-
periment. Drugs are dissolved at a concentration of 10 2M (or
2 x 10 2M or 4 x 10 2M) in pyrogen free sterile saline. The
dissolved drug is then filtered through a sterile and
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pyrogen-free 0.2 micron filter (acrodisc, Gelman).
Procedure
8-12 CD-l female mice (Charles river), age 2-5 months are
numbered, weighed and distrihuted to 2-3 cages, 3-4 mice in
each cage. 0.2 ml of pyrogen-free saline or drug are injected
subcutaneously in a randomized order.
Thirty minutes after injecting the drugs or the saline,
tha right paw of each mouse is injected with 25 microliters of
1% carrageenin (vi6carin type, Marine Colloids) in pyrogen-free
saline or with 5 microliters (0.227 U) of xanthineoxidase
(Sigma).
1.5 hr to 2 hrs after injecting the inflammatory stimulus
to the right paw, both paws of the animal are amputated at the
knee joint and weighed. The uninjected left paw serves as an
internal control for the degree oE swelling of the right paw in
each animal.
In some experiments the surface temperature of the right
and left paws were recorded also before the amputation was
performed.
Calculations
The difference in mg between the weight of the right and
left paw in control animals (injected with saline) rapresents
lOO~o Of the acute inflammatory response. Concomitantly, the
difference between paws of the drug treated animals is
calculated and compared to control.
TAB~E 1: Effect of several pruducts on carrageenin paw oedema
in mice measured by paw weight.
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Paw Wei~ht
Product No.of total route of average % % inhib-
expts. mice admin. mg/kg activity ition-
saline - - *S.CO - 100% 0
23 4 14 *S.C. 2563.1% 36.9%
8 4 15 *S.C. 29.873.2% 26.8%
39 5 18 *S.C. 35.764.7% 35.3%
19 1 ~ *S.C. 23.588.2% 11.8%
19 3 11 *S.C. 58.954 % 46 %
*subcutaneously
TABLE 2: Effect o~ several products on xanthine oxidase paw
oedema measured by temperature reduction
Product No.of total route of average % % inhib-
expts. mice admin. mg/kg activity ition-
saline ~ - *S.C. -100 % 0
23 2 6 *S.C. 22.731 % 69 %
8 3 10 *S.C. 25.133.2% 66.8%
19 1 4 *S.C. ~6.869.5% 30.5%
19 3 12 *S.C. 91.549.6% 50.4%
-
*subcutaneously
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TABLE 3: LD50 and ED50 of various drugs on xanthine
oxidase and carrageenin paw oedema in mice
stimulus :xanthine carrageenin
oxidase
measurement :temp. _weight mg_
DrugLD50 ED50 ED50
mq/kq _ mq/kq m~/k~
23 75 <38 25
19 >>875 93 65
39 85 - 43
8 172 ~24 not
determined
Orally_and S.C.Administerable Com~ositions
Example 1
The composition o~ tablets is as ~ollows:
,
active ingredient (one or more
of compounds 8, }9, 23~or 39) 25~0 mg.
corn starch 97.0 mg.
polyvinyl pyrrolidone 175.0 mg~
magnesium stearate 3.0 mg.
300.0 mg.
The active ingredient and th~ corn starch are wetted by an
_ g _
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aqueous polyvinyl pyrrolidone solution of approx. 15% w/v/,
followed by granulation, and drying of the wet granules at
about 40-45C. The dried granulate is thoroughly mixed with
magnesium stearate, and the mixture so obtained is further
processed by a tabl~t machine, equipped with an appropriate
pressing tool, to give tablets of 300 mg. weight containing 25
mg. of active ingredient. One manufacturing lot includes 1000
tablets.
Example 2
Dragees of the following composition are preparedo
active ingredient (one or more
of compounds 8, 19, 23 or 39) 50.0 mg.
lactose 94.0 mg.
polyvinyl pyrrolidone 154.0 mg.
magnesium stearate 2.0 mg.
300 mg.
Granulate~ are preparPd according to Example 1, and from
them dragee kernels of 150 mg. weight are pressed. The dragee
kernels are coated with a layer containing sugar and talc
followed by coloring with an approved food colorant and
polishing with bees wax.
Ex~amplP 3
25 mg. of active ingredient (one or more of compounds 8,
19, 23 or 39) are dissolved in 1000 ml. of distilled water. The
solution is filled into 500 ampoules. In this way ampoules
containing 2 ml. of a solution containing 25 mg./ml. of active
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agent each are obtained. The contents of an ampoule are
injected subcutaneously.
Example_4
Gelatin capsules of the following composition are
prepared:
active ingredient (one or more
of compounds 8, 19, 23 or 39~ 25.0 mg.
maize starch 122.0 mg.
colloidal silica 3.0 mq.
150.0 mg.
The ingredients are homogenized, and the homogenate is
filled into hard gelatine capsules. 1000 capsules of 150 mg.
~filling) weight each, containing 25.0 mg. of active ingredient
per capsule, make a lot~
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The formulae of some of the compounds referred
to in the specification are the following:
~8) / CH2
Cl
~ CO ~
l Cl 1 3~ C1
CH2 ~CH2
CH2
CH
#19) ~ 3
~ Lc~ IPP6
~,
.
~l3
#23) 3 \ CH3 ~
~ C - N / I ~ ~ > ~ Cl
:~ / \ NH3 ~
:: ~ H3C CH2 _~ CH2 CH3
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L H3C ¦ ~ 3
:
:: :
~: :
` :
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