WO1999015160A2

WO1999015160A2 - Use of 6,7-substituted 2-aminotetralines suitable for preparing pharmaceutical composition for the therapeutic treatment of inflammatory and/or autoimmune pathologies

Info

Publication number: WO1999015160A2
Application number: PCT/IT1998/000250
Authority: WO
Inventors: Piero Foresta; Vito Ruggiero
Original assignee: Sigma Tau Industrie Farmaceutiche Riunite SpA
Current assignee: Sigma Tau Industrie Farmaceutiche Riunite SpA
Priority date: 1997-09-22
Filing date: 1998-09-18
Publication date: 1999-04-01
Anticipated expiration: 2000-03-22
Also published as: DK1017377T3; DE69802828T2; AU9366198A; DE69802828D1; ITRM970569A1; IT1294932B1; NZ503391A; JP2001517618A; EP1017377A2; CN1276721A; AU735369B2; US6242497B1; BR9812489A; KR20010030670A; ATE209910T1; WO1999015160A3; ES2167942T3; EP1017377B1; PT1017377E; MXPA00002774A

Abstract

The use of 6,7-substituted 2-aminotetralines is disclosed for preparing pharmaceutical compositions for the therapeutic treatment of inflammatory and/or autoimmune pathologies induced by inflammatory cytokines.

Description

Use of 6,7-substituted 2-aminotetralines suitable for preparing a

pharmaceutical composition for the therapeutic treatment of

inflammatory and/ or autoimmune pathologies.

The present invention relates to the use of 6,7-substituted-2-

aminotetralines and their pharmacologically acceptable salts suitable

for the prophylactic and therapeutic treatment of septic shock and

for the treatment of inflammatory and/ or autoimmune pathologies

which will be better defined here below, in which the

aetiopathogenetic role of inflammatory cytokines is well established.

6,7-Substituted-2-aminotetralines, which are active in the

treatment of septic shock, are well known. EP-A-0730861, which is

incorporated herein for reference purposes, describes a class of such

6,7-substituted-2-aminotetralines and particularly the compound

(R,S)-2-amino-6-fluoro-7-methoxytetraline (ST 626).

The 6,7-substituted-2-aminotetralines for the use according to

the present invention are also known compounds but for completely

different therapeutic applications. In fact, in J Chem Soc (1997),

288-93 they are described as possessing bronchodilatory activity; in

Tetrahedron let, 22/38, 3707- 10, 1981 they are described as

compounds endowed with dopaminergic activity.

It is clear that there is no relationship between the known bronchodilatory and dopaminergic activity of such aminotetralines

and their therapeutic activity in septic shock and in inflammatory

and/ or autoimmune pathologies in which the aetiopathogenetic role

of the inflammatory cytokines is well established.

The inflammatory and/ or autoimmune pathologies to be

treated with the compounds of the invention described herein are, for

example, rheumatoid arthritis, pancreatitis, inflammatory bowel

disease, systemic lupus erythematosus, glomerulo-nephritis and

encephalomyelitis.

Hereinafter, reference will be made only to septic shock, it

being understood that the other pathologies due to inflammatory

cytokines can also be effectively treated according the invention.

Septic shock is an extremely severe clinical syndrome which

may set in as a result of infections mainly caused either by gram-

negative or gram-positive bacteria, by protozoa or by viruses, and

characterised by leukocytosis, fever, tachycardia, hypotension and

renal, respiratory, cardiac and hepatic insufficiency. It should be

stressed, however, that the severity of septic shock is independent of

the type of micro-organism responsible for the syndrome (Parrillo

J.E., Pathogenetic mechanisms of septic shock, N Engl J Med,

328: 1471-1477, 1993) but is related to the individual inflammatory response to the antigen responsible for the toxic insult. Despite the

significant improvement in antibiotic therapy and in intervention

protocols in intensive care units over the past few years, shock

remains one of the major causes of morbidity and mortality in

hospitalised patients. It is estimated that in the USA it is responsible

for approximately 100,000 deaths/year (Glauser M.P., Zanetti G.,

Baumgartner J.D. and Cohen J., Septic shock: pathogenesis, Lancet,

338:732-736, 1991).

The most decisive and characteristic feature of septic shock is

the body's reaction to products deriving from lysis or from microbial

metabolism.

The first of these substances to be identified and the one most

used in experimental research is lipopolysaccharide (LPS), a

constituent of the gram-negative bacterial wall chemically consisting

in a polysaccharide portion which varies according to the bacterial

species and a lipid portion (lipid A) which is constant, and present in

the blood of septicaemic subjects in the form of micelles. If

administered to animals, LPS is capable of reproducing all the

cardio circulatory and neurological symptoms encountered in shock

(Olson C, Salzer W.L., McCall C.E., Biochemical, physiological and

clinical aspects of endotoxaemia, Molec Aspects Med, 10: 511-629, 1988). It is therefore identifiable as the prime mover in the chain of

events, which leads to the triggering of the clinical symptoms via

activation of the intrinsic and extrinsic pathways of the coagulative

cascade and the . secretion of cytokines of mainly macrophage-

monocyte origin, such as, for instance, TNF, IL- 1 and IL-6.

The increasing importance this syndrome has come to take on

over the past few years, its severity and the inadequate therapeutic

measures currently available make the rapid discovery of therapeutic

agents capable of effectively combating the progression of the disease

a highly desirable goal.

It has now been found that a class of known 6, 7 -substituted

2-aminotetralines exhibits potent activity in the prevention and

therapeutic treatment of the above- mentioned pathologies.

6,7-Substituted-2-aminotetralines according to the invention

can occur both as free bases with general formula (I):

(I) and as pharmacologically acceptable salts with general formula (II):

(II)

wherein:

R is methoxy or hydroxy and

X^~ is the monovalent anion of a pharmacologically acceptable acid.

Among the 6,7-substituted-2-aminotetralines with general formula (I)

or (II) the following compounds are particularly preferred wherein:

R = methoxy: (R,S)-2-amino-6,7-dimethoxytetraline hydrochloride

(hereinafter: ST 1213)

R = OH: (R,S)-2-a_mino-6,7-dihydroxytetraline hydrochloride

(hereinafter: ST 1236).

What is meant by pharmacologically acceptable salts of 6,7-

substituted-2-aminotetralines with formula (I) are any of its salts

with an acid that does not give rise to unwanted toxic or side effects. Such acids are well known to pharmacologists and to experts in

pharmacy and pharmaceutical technology.

Non-limiting examples of such salts are chloride, bromide,

orotate, acid aspartate, acid citrate, acid phosphate, fumarate and

acid fumarate, lactate, maleate and acid maleate, acid oxalate, acid

sulphate, glucose phosphate, tartrate and acid tartrate. FDA-

approved salts are listed in Int J Pharm 33 (1986), 201-217, which is

incorporated herein for reference purposes.

The methodological approach most widely employed for the

purposes of assessing the possible protective effect of a substance in

septic shock, in preclinical investigation, is the use of experimental

models of intoxication with a toxic substance (exo- or endotoxin)

injected directly into the laboratory animal or released in massive

amounts by the infecting cells with which the animal is inoculated.

Here below are given the results obtained with the according

to the invention, (R,S)-2-amino-6,7-dimethoxytetraline hydrochloride

(ST 1213) and (R,S)-2-amino-6,7-dihydroxytetraline hydrochloride

(ST 1236), in comparison with the reference compound (R,S)-2-

amino-6-fluoro-7-methoxytetraline hydrochloride (ST 626). As

mentioned above, the compound ST 626 is the compound known to

be most closely related structurally to the compounds for use as per the invention and also known to be endowed with the same

pharmacological activity.

These results demonstrate the preventive and therapeutic

activity of the compounds according to the invention (ST 1213 and

ST 1236) in comparison with the known compound ST 626 and also

provide indications as to one of the possible mechanisms of action

responsible for the favourable pharmacological profile of the

compounds: a drastic reduction in blood levels of inflammatory

cytokines (TNF, IL- lbeta, IL-6, IFN-γ levels, and in serum nitric

oxide (NOx) levels.

EVALUATION OF THE EFFECTS OF ST 1213 AND ST 1236

IN ANIMAL MODELS OF SEPTIC SHOCK.

Male BALB/C mice (C. River) aged approx. 6 weeks were

utilised (10 animals per experimental group). The animals, housed in

cages at a constant temperature 22± 2°C and 50+15% relative

humidity with 12 hours of light (7am-7pm) and 12 hours of darkness

(7 pm-7 am), had unrestricted access to food and drinking water.

The compounds tested were ST1213, ST 1236 and ST 626

(reference compound). The pH of the compound solutions was

corrected, as necessary, with NaOH 0.1 N (maintaining the solution

cold and under stirring) to obtain values no lower than pH 5.5. The substances utilised were: LPS (Escherichia coli serotype

026:B6), batch 73570 JB (Difco), LPS (Salmonella typhosa), batch

81H4018 (Sigma), SEB (Staphylococcus aureus), batch 144H4024

(Sigma), and D-galactosamine batch 031EE002485 (Merck).

Lethality induced by E. coli and S. typhosa LPS

Animals were treated with LPS from E. coli or S. typhosa. Prior

to use the endotoxin was first dissolved in sterile saline and then

injected mtraperitoneally (i.p.) in a volume of 200 μL at doses of

10.0-12.5 mg/kg (E. coli) and 23.0-27.0 mg/kg (S. typhosa),

corresponding to approximately the LDβo.

The compounds tested were administered intravenously (i.v.)

in a volume of 200 μL of sterile saline, corresponding to

approximately 1/ 10 of the respective LDsos, 30 min before and again

5 min after treatment with LPS, or 5 and 30 min after the endotoxic

challenge.

Lethality induced by E. coli LPS in mice sensitised with

D-galactosamine

Animals were sensitised with D-galactosamine (1000 mg/kg

i.p.) and, at the same time, treated with E. coli LPS (0.30 mg/kg i.p.)

in a total volume of 200 μL. The dose of LPS utilised corresponded

approximately to the endotoxin LDβo in the animals sensitised with D-galactosamine.

The compounds tested were administered intravenously (i.v.)

in a volume of 200 μL of sterile saline at the dose corresponding

approximately to 1/ 10 of the respective LD50S, 30 min before and 5

min after, or 5 and 30 min after the LPS challenge.

Lethality induced by SEB (Staphylococcus aureus) in mice

sensitised with D-galactosamine

Animals were sensitised with D-galactosamine (1000- 1500

mg/kg i.p.) and, at the same time, treated with the enterotoxin SEB

(3 mg/kg i.p.) in a total volume of 200 μL. The dose of SEB utilised

corresponds to approximately the LDso and was determined in a

preliminary experiment.

The compounds tested were administered intravenously (i.v.)

in a volume of 200 μL of sterile saline at the dose corresponding

approximately to 1/ 10 of the respective LDsos, 30 min before and 5

min. after, or 5 min. and 30 min. after the SEB challenge.

The animals were observed for 10 days in all experiments,

taking daily note of any deaths.

The statistical significance of the protective effect of the study

compounds was evaluated using the one-tailed Fisher's exact test. RESULTS

Lethality induced by E. coli LPS

The results obtained with this experimental model of shock

with E. coli LPS are reported in Table 1 (A and B). The compound

ST1213 significantly reduces the lethality induced by LPS (p<0.001),

when it is administered 30 min before and 5 min after the LPS

challenge (Tab. 1A). Such protection is also obtained in the post-

challenge treatment protocol, though the degree of statistical

significance is lower (p<0.05) (Tab. IB).

Table 1 Protective effect of ST 1213 i.v. administration on lethality induced in mice by injection of E. coli LPS. Pre- and post- challenge treatment (-30 and +5 min) (A) or post-challenge only (+5 and +30 min) (B).

a = Increase in survival (%) of treated animals compared to LPS control, b = Statistical significance calculated using one-tailed Fisher's exact test.

a = Increase in survival (%) of treated animals compared to LPS control. b = Statistical significance calculated using one-tailed Fisher's exact test.

Lethality induced by S. typhosa LPS

In this experimental model of endotoxic shock with S. typhosa

LPS, the compounds ST 1213 and ST 1236 significantly reduce

lethality when administered pre- and post-challenge, p<0.001 and p<

0.05, respectively (Tab. 2).

ST 1213, when administered in a second post-challenge

treatment protocol, retains its protective efficacy, though to a lesser

extent (Tab. 3).

Table 2 Protective effect of ST 1213 and ST 1236 i.v.

administration on lethality induced in mice by injection of

S. typhosa LPS. Pre- and post-challenge treatment (-30

and +5 min).

Table 3 Protective effect of ST 1213 i.v. administration on lethality

induced in mice by injection of S. typhosa LPS. Post-

challenge treatment (+5 and +30 min).

Lethality induced by E. coli LPS in mice sensitised with

D-galactosamine

Animals were sensitised with D-galactosamine and at the

same time treated with E. coli LPS. The compound ST 1236

significantly reduces the lethality (ρ< 0.001) (Tab. 4).

Table 4 Protective effect of ST 1236 i.v. administration on lethality

induced by injection of E. coli LPS in mice sensitised with

D-galactosamine. Pre- and post- challenge treatment (-30

and +5 min).

a = Increase in survival (%) of treated animals compared to LPS + D-

GalN control, b = Statistical significance calculated using one-tailed Fisher's exact test.

Lethality induced by enterotoxin SEB (Staphylococcus aureus) in

mice sensitised with D-galactosamine

The results obtained with this experimental model of shock

with enterotoxin SEB in mice sensitised with D-galactosamine are

reported in Tables 5 and 6.

The compounds ST 1213 and ST 1236 reduce the lethality in

comparison with the control (increases in survival = 46% and 90%,

respectively) when administered 30 min before and 5 min after the challenge (Tab. 5). Following the post-challenge treatment the

protective effect remains, but fails to prove statistically significant

(Tab. 6).

Table 5 Protective effect of ST 1213 and ST 1236 i.v. administration

on lethality induced by injection of LPS from enterotoxin

SEB in mice sensitised with D-galactosamine. Pre- and

post-challenge treatment (-30 and +5 min).

a = Increase in survival (%) of treated animals compared to LPS control, b = Statistical significance calculated using one-tailed Fisher's exact test. Table 6 Protective effect of ST 1213 and ST 1236 i.v. administration

on lethality induced by injection of LPS from enterotoxin

SEB in mice sensitised with D-galactosamine. Post-

challenge treatment (-30 and +5 min).

EVALUATION OF THE EFFECT OF ST 1213 AND ST 1236 ON

SERUM TNF (TUMOR NECROSIS FACTOR) LEVELS INDUCED BY

LPS IN RAT WHOLE BLOOD CULTURE

Cultures of whole blood cells stimulated by LPS have been

utilised in recent years as an experimental model, which, though

presenting a number of limitations, mimics the physiopathological

condition of endotoxaemia, a situation in which the lipo-

polysaccharide of gram-negative bacteria is released into the bloodstream, thus coming into contact with the immune system

cells. This experimental model, in fact, has recently been adopted for

the evaluation of potential inhibitors of the release of TNF and IL- 1

(GC Rice et al., Shock, 4:254-266, 1994. AJH Gearing et al., Nature,

370:555-557, 1994. K Tschaikowsky, Biochim Biophys Acta,

1222: 1 13- 121 , 1994. A Haziot et al., J Immunol, 152:5868-5876,

1994).

Male Wistar rats (C. River) weighing 175-200 g were utilised in

these experiments.

The animals, housed in cages at a constant temperature of

22± 2°C and 50+ 15% relative humidity with 12 hours of light (7 am-7

pm), had unrestricted access to food and drinking water.

The compounds tested were ST 1213 and ST 1236.

The endotoxin utilised was LPS from Salmonella typhosa,

batch 81 H4018 (Sigma) .

Heparinised blood samples (0.450 mL/test tube) were taken

from Wistar rats sacrificed by decapitation. Volumes of 0.025 mL

(20x solution) of the compounds tested (final concentration 0.050

mM) dissolved in sterile saline were added to the test tubes

containing the blood samples. 0.025 mL (20x solution) of LPS from

Salmonella typhosa were added (final LPS concentration = 1 μg/mL) to the test tubes incubated for 1 h at 37°C in a 5% C02 humidified

atmosphere. The test tubes were incubated in the same conditions

for 4 h and then centrifuged for 5 min at 10,000 rpm and the

supernatants were stored frozen at -80°C pending TNF assay.

TNF biological activity was determined in RPMI medium added

with 1% FCS. For the TNF assay serial dilutions of the samples (50

μL) containing TNF (TNF standard, culture supernatants, serum,

biological fluids, etc.) were made directly in the 96-well Primaria

microtitre plates. Actinomycin D-mannitol (50 μL) at a final

concentration of 4 μg/mL, prepared in RPMI medium added with 1%

FCS, was added to the wells. This inhibitor enhances cell sensitivity

to TNF.

100 μL of a standardised suspension of 4x10⁵ cells/mL of

L929 (murine fibrosarcoma sensitive to the toxic action of TNF) was

added to each well. Appropriate controls, i.e. the Actinomycin D

control (cells + Actinomycin but without TNF) and the cell control

(cells in the presence of culture medium alone), were also prepared.

After further incubation for 18 h at 37°C with 5% C02, the

cells were stained with a freshly prepared solution of 1 mg/mL XTT

(sodium 3'-[l-[(phenylamino)-carbonyl]-3,4-tetrazolium]-bis(4-

methoxy-6-nitro)benzene-sulphonic acid hydrate) and 125 μM PMS (phenazine methosulphate) according to the method described here

below.

The PMS mother solution is 100 mM (stable for about 20 days

at +4°C in the dark) and is prepared by dissolving PMS in PBS,

followed by brief sonication to dissolve the PMS completely. The 100

mM PMS solution is then diluted 1 :800 in XTT, thus obtaining a final

PMS concentration of 125 μM in XTT 1 mg/ml. The XTT-PMS

staining mixture must be filtered prior to use.

On completing the 18 h incubation, cells were stained by

adding 50 μL of the XTT-PMS staining solution to each well, thus

obtaining a final volume of 250 μL with final XTT and PMS

concentrations of 0.2 mg/mL and 25 μM, respectively. A "blank",

consisting of wells containing 200 μL of culture medium + 50 μL of

XTT-PMS staining solution, was also prepared.

The microtitre plates were incubated for 2-2.5 hours at 37°C

with 5% C02 (total incubation time = about 20 hours).

The absorbance values of each sample were measured with a

microtitre plate reader using a 450 nm readout wavelength and a

620 nm reference wavelength (the colorimetric microtiter plate reader

was programmed to automatically deduct the "blank" absorbance

value from the sample value). The TNF titre was calculated as described here below. By

definition, 1 unit of biological activity is given by the semimaximal

value (=50%) of the Actinomycin-D absorbance.

Sample dilutions give rise to an absorbance value curve whose

linear portion is described by the equation y=ax+b. After inserting

the a and b values (obtained by computerised linear regression

analysis) and after substituting for y the semimaximal absorbance

value (corresponding to 1 biological unit) of the Actinomycin-D

control, the equation is resolved for x, which represents the

reciprocal of the sample dilutions. The value obtained gives the TNF

titre expressed in U/mL.

Data were analysed using the two-tailed Student's t-test.

RESULTS

The results obtained with this experimental model and

reported in Table 7 show that the compounds tested reduce, though

to different extents, TNF production by the blood culture stimulated

with LPS. In fact, the compounds ST 1236 and ST 1213 induce 59%

and 31% reductions, respectively, in TNF levels. Table 7 Effect of compounds ST 1213 and ST 1236 on TNF

production induced in rat blood cultures (n°=5) stimulated

with S. typhosa LPS (1 μg/mL). The compounds were

tested at a concentration of 50 μM. The experimental

conditions were those described in Materials and Methods.

* Statistical significance evaluated using two-tailed Student's t test.

EVALUATION OF THE EFFECT OF ST 1213 AND ST 1236 ON

SERUM TNF LEVELS INDUCED BY LPS IN MICE

Male BALB/c mice (C. River), aged approx. 6 weeks were

utilised (10 animals per experimental group).

The animals, housed in cages at a constant temperature of

22+2°C and 50+15% relative humidity with 12 hours of light (7 ara-7

pm) and 12 hours of darkness (7 pm-7 am), had unrestricted access

to food and drinking water.

The compounds tested were ST1213, ST 1236 and ST 626

(reference compound). The pH of the compound solutions was corrected, as necessary, with NaOH 0.1 N (maintaining the solution

cold and under stirring) to obtain values no lower than pH 5.5.

The substances utilised were: LPS (from E. coli serotype

026:B6, batch 73570 JB (Difco), LPS (Salmonella typhosa), batch

81H4018 (Sigma), D-galactosamine, batch 031EE002485 (Merck).

Lethality induced by E. coli and S. typhosa LPS

The experimental conditions were exactly the same as those

previously described.

Lethality induced by E. coli LPS in mice sensitised with D-

galactosamine

The experimental conditions were exactly the same as those

previously described.

In both the experimental models the blood samples were taken

90 min after the challenge (TNF serum peak level).

Ether- anaesthetised mice were bled by retro-orbital sinus

puncture. Blood samples were incubated at room temperature for 2

h and the serum thus obtained was centrifuged for 20 min at 3000

rpm and stored frozen at -80°C pending TNF assay.

TNF biological activity was determined in RPMI medium

containing 1% FCS.

50 μL/well serial dilutions of the sample containing TNF were added directly to the Primaria microtitre plate. The experimental

conditions utilised were the same as those previously described.

Data were analysed using the one-tailed Student's t-test.

RESULTS

Lethality induced by E. coli and S. typhosa LPS

The results obtained in these experimental models show that

the compound ST 1213 significantly reduces TNF levels induced by

E. coli LPS (p<0.01) (Tab. 8) and produces an even more substantial

reduction in serum TNF levels in the experiment with Salmonella

typhosa LPS (p<0.0001) (Tab. 9).

Table 8 Effect of ST 1213 administration (6 mg/kg i.v.) on serum

TNF levels in E. coli LPS septic shock model in mice. Pre-

and post-challenge treatment (-30 and +5 min).

Table 9 Effect of ST 1213 administration (6 mg/kg i.v.) on serum

TNF levels in S. typhosa LPS septic shock model in mice.

Pre- and post- challenge treatment (-30 and +5 min).

Lethality induced by E. coli LPS in mice sensitised with D-

galactosamine

The results obtained with this experimental model of TNF

production induced by E. coli LPS in animals sensitised with D-

galactosamine are reported in Table 10.

The compound ST 1236 significantly reduces the release of

TNF (p<0.0001) induced by LPS in mice sensitised with D-

galactosamine. Table 10 Effect of ST 1236 administration (19 mg/kg i.v.) on serum

TNF levels induced by E. coli LPS in mice sensitised

with D-galactosamine. Pre- and post-challenge treatment

(-30 and +5 min).

EVALUATION OF THE EFFECT OF ST 1213 ON SERUM

INTERLEUKIN-1 BETA (IL-lβ), INTERLEUKIN-6 (IL-6) AND

INTERFERON-GAMMA (IFN-γ, LEVELS INDUCED BY E. coli or

SEB ENTEROTOXIN IN MICE

Male BALB/c mice (C. River), aged approximately 6 weeks,

were utilised (10 animals per experimental group). The animals.

housed in cages at a constant temperature of 22+2°C and 50+ 15

relative humidity, with 12 hours of light (7 am-7 pm) and 12 hours of

darkness (7 pm-7 am) had unrestricted access to food and drinking

water.

The compound tested was ST 1213. The substances utilised were: LPS from E. coli 026:B6, batch

73570 JB (Difco), SEB (Staphylococcus aureus), batch 144H4024

(Sigma), and D-galactosamine, batch 031EE002485 (Merck).

Lethality induced by E. coli LPS

The experimental conditions were exactly the same as those

previously described.

Lethality induced by SEB from S. aureus in mice sensitised with

D-galactosamine

The experimental conditions were exactly the same as those

previously described.

In both models, blood samples were taken 2 h post-challenge

for IL-6, 4 h post-challenge for IL- lβ, and 6 h post-challenge for IFN-

γ-

Ether- anaesthetised mice were bled by retro-orbital sinus

puncture. Blood samples were incubated at room temperature for 2

h and the serum thus obtained was centrifuged for 20 min at 3000

rpm and stored frozen at -80°C until assayed.

Biological assays were carried out according to the procedures

indicated in the assay kits utilised. In particular, the following were

used: - Mouse IL-lβ Immunoassay (MLBOO, R&D Systems)

- Mouse IL-6 EIA Kit (8-6706, PerSeptive Diagnostics)

- Mouse IFN-γ EIA Kit (8-6716, PerSeptive Diagnostics).

Data were analysed using the one-tailed Student's t-test.

RESULTS

Lethality induced by E. coli LPS

The results obtained with this experimental model are

reported in Table 11.

The compound ST 1213 significantly reduces (ρ<0.0001) the

production of IFN-gamma, but does not reduce the levels of the other

two cytokines analysed .

Table 11 Effect of ST 1213 administration (6 mg/kg i.v.) on serum levels of IL- lβ. IL-6 and IFN-γ in mice stimulated with E. coli LPS. Pre- and post-challenge treatment (-30 and +5 min).

Lethality induced by S. aureus SEB in mice sensitised with

D-galactosamine

The results obtained with this experimental model are

reported in Table 12.

The compound ST 1213 significantly reduces serum IL- l β and

IL-6 levels but not serum IFN-γ levels.

Table 12 Effect of ST 1213 administration (6 mg/kg i.v.) on serum

levels of IL- l β, IL-6 and IFN-γ in mice stimulated with S.

aureus SEB. Pre- and post-challenge treatment (-30 and

+5 min).

EVALUATION OF THE EFFECT OF ST 1213 ON SERUM NITRIC

OXIDE (NOx) INDUCED BY E. coli LPS IN MICE

Male BALB/c mice (C. River), aged approx. 6-7 weeks, were

utilised (6-9 animals per experimental group) . The animals, housed in cages at a constant temperature of

22±2°C and 50±15% relative humidity with 12 hours of light (7 am-7

pm) and 12 hours of darkness (7 pm-7 am), had unrestricted access

to food and drinking water.

The compound tested was ST 1213.

The endotoxin utilised was LPS from E. coli 026:B6, batch

73570, Difco, previously dissolved in sterile saline, which was

injected mtraperitoneally at the dose of 5 mg/kg.

Compounds ST 1213 and ST 626 (reference compound) were

administered i.v. at the dose of 6 mg/kg, corresponding to

approximately 1 / 10 of the LD50, +5 min and +30 min after the LPS

challenge.

Ether-anaesthetised mice were bled by retro-orbital sinus

puncture, taking blood samples 20 h after the LPS challenge, when

NOx reached its serum peak levels in mice.

The blood, put into heparinised test tubes, was centrifuged for

10 min at 2200 rpm and stored frozen at -80 C pending NOx assay.

Before testing the samples were diluted 1 :3 with distilled

water and then centrifuged for 90 min at 4700 g on Ultrafree-MC,

10,000 NMWL Millipore filters (Cat. No. UFC3LGC00).

The sample assay for NOx was carried out using the recently marketed assay kit manufactured by Cabru (Nitrate/ Nitrite assay

kit, Cat. No. 780001).

Data were analysed using the two-tailed Student's t-test.

RESULTS

The results obtained with this experimental model are

reported in Table 13.

The compound ST 1213 significantly reduces NOx levels (42%

reduction) induced by E. coli LPS in BALB/c mice, when

administered post-challenge.

In the same experimental model, the reference compound ST

626 does not significantly reduce serum levels of NOx (21%

reduction) .

Table 13 Effect of ST 1213 administration (6 mg/kg i.v.). Treatment 5 and 30 min after the i.p. LPS challenge (5 mg/kg).

a = Decrease (%) in NOx levels of treated animals compared to the control group, b = Statistical significance evaluated using the two-tailed Student's t-test.

Claims

1. Use of 6,7-substituted 2-aminotetralines with general formula (I)

(I)

or their pharmacologically acceptable salts with general formula

(II)

(II)

wherein:

R is methoxy or hydroxy and

X is the monovalent anion of a pharmacologically acceptable acid suitable for preparing a pharmaceutical composition for the

therapeutic treatment of inflammatory and/ or autoimmune

pathologies induced by inflammatory cytokines.

2. Use according to claim 1 , in which the pharmaceutical

composition is suitable for the prophylactic and the therapeutic

treatment of septic shock.

3. Use according to claim 1 , in which the pharmaceutical

composition is suitable for the therapeutic treatment of

rheumatoid arthritis, pancreatitis, inflammatory bowel disease,

systematic lupus erythematosus, glomerulonephritis and

encephalomy elitis .

4. Use according to claims 1-3, in which the monovalent anion of

the pharmacologically acceptable acid is selected from among

chloride, bromide, orotate, acid aspartate, acid citrate, acid

phosphate, fumarate and acid fumarate, lactate, maleate and

acid maleate, acid oxalate, acid sulphate, glucose phosphate,

tartrate and acid tartrate.

5. Use according to claims 1-3, in which the 6,7-substituted 2-

aminotetraline is (R,S)-2-amino-6,7-dimethoxytetraline hydro-

chloride.

6. Use according to claims 1-3, in which the 6,7-substituted 2-

aminotetraline is (R,S)-2-amino-6,7-dihydroxytetraline hydro¬

chloride.