WO2006117624A2

WO2006117624A2 - Kynurenic acid and salts thereof for the prevention and treatment of shock

Info

Publication number: WO2006117624A2
Application number: PCT/IB2006/001059
Authority: WO
Inventors: Flavio Moroni; Andrea Cozzi
Original assignee: Universita'degli Studi Di Firenze
Priority date: 2005-04-29
Filing date: 2006-04-28
Publication date: 2006-11-09
Also published as: ITFI20050083A1; WO2006117624A3

Abstract

The use in therapy of kynurenic acid and pharmaceutically acceptable salts there for preparing drugs effective in the prevention and treatment of shock, in particular septic shock, is described.

Description

KYNURENIC ACID AND SALTS THEREOF FOR THE PREVENTION AND TREATMENT OF SHOCK Field of the invention

The present invention relates to the field of compounds useful for the prevention and treatment of shock, in particular septic shock. Prior art

Shock is a very serious pathological condition characterised by considerable damage to the major organs and tissues due to serious changes in cardiovascular function. Often, the state of shock rapidly becomes irreversible, resulting in the death of affected patients.

The pathogenetic process that culminates in irreversible shock can be triggered by various pathological situations (traumas, haemorrhages, cardiac pathologies, serious infections) and appears to be due to the action on the heart and circulation of several toxic molecules able to modify cardiovascular homeostatic processes. For example, lipopolysaccharides (LPS) present in the outer capsule of various microorganisms are toxins regarded as responsible for septic shock and other types of shock. These molecules, when injected into experimental animals, result in a pathological response similar to that observed with shock. Monocyte-macrophage cells play a particularly important role in pathogenic processes that give rise to shock. LPSs are regarded as capable of stimulating the CD 14 receptor on said cells, thus initiating a series of events characterised by the introduction into the circulation of massive amounts of phlogistic mediators such as cytokines, tumor necrosis factor α (TNF-α), platelet activating factor (PAF), kinins, complement factors, arachidonic acid metabolites, nitric oxide (NO). These mediators interact with various targets and tend to amplify phlogistic processes with substantial effects on both blood circulation and the heart. In particular macrophage activation, associated with the expression of endothelial cell adhesion receptors, results in the deterioration of microcirculation and blood supply to the tissues, with adhesion of white blood cells to capillary walls, their marginalization and their increased migration into tissues. In the presence of LPS and TNF-α, NO production increases in both macrophages and endothelial cells, with consequent further lowering of arterial pressure. Indeed, cardiac function can also be considerably reduced by factors released from centres with inadequate blood supply thus worsening the clinical situation.

In support of these concepts, non-expression of the enzyme iNOS (inducible nitric oxide synthetase), and therefore reduction in NO production, has been shown to significantly reduce mortality induced by administering high doses of LPS. It has also been demonstrated that by blocking macrophage activation and marginalization processes with adhesion receptor monoclonal antibodies, mortality in shock models can be reduced. As measures proposed for reducing mortality in states of shock, the use of iNOS inhibitors and molecules able to antagonize the effect of LPS or the function of adhesion receptors have been considered. In any case, a significant number of clinical studies have shown that these procedures do not improve the prognosis of this serious pathological situation and are not utilized in clinical practice.

On the other hand, the role of tryptophan metabolites in cerebral function is known. At least two of said metabolites, quinolinic acid and kynurenic acid, are able to interact, in a functionally opposing manner, with NMDA glutamate (N- methyl-D-aspartate) receptors and are present in the CNS, where among other things they tend to increase with ageing and during various pathological processes. (Moroni, F. et al., Brain Research 295, 352-355 (1984); Moroni, F. et al., Neuroscience Letters 47, 51-55 (1984); Moroni, F et al., Neuroscience Letters 94, 145-150 (1988); Moroni, F. et al., Journal of Neurochemistry 51 , 177-181 (1988); Moroni, F. et al., Journal of Neurochemistry 57, 1630-1635 (1991)). Molecules able to increase the plasma and cerebral content of kynurenic acid have also been shown to cause sedation, to increase convulsion threshold and to reduce post-ischemic brain damage (Carpenedo, R. et al., Neuroscience 61 , 237- 244 (1994); Carpenedo, R. et al., Journal of Neurochemistry 82, 1465-1471 (2002); Chiarugi, A. et al., Neuroscience 102, 687-695 (2000); Cozzi, A. et al., Journal of Cerebral Blood Flow and Metabolism 19, 771-777 (1999)). It has also been noted that kynurenic acid can significantly reduce synaptic concentrations of the inhibitory neurotransmitter glutamate, probably by acting on particular receptors that have yet to be identified at a molecular level (Carpenedo, R. et al., 2001 , cit; Carpenedo, R. et al., 2002, cit). Summary of the invention

It has now surprisingly been found that kynurenic acid is strongly active in preventing and treating shock, as demonstrated herein on suitable animal models of this pathology. The present inventors have also shown the specific ability of kynurenic acid to inhibit macrophage activation, which represents an important contribution to anti-shock activity. A further encountered effect is the reduction in overproduction of nitrites in blood; this latter typically accompanies the state of shock and causes vasodilation. The reduction of nitrites by kynurenic acid treatment returns vessel tonicity to physiological conditions, thus effectively counteracting the various forms of shock. Brief description of the figures

Figure 1: concentration of nitrites in control animals, in animals treated with LPS and in animals treated with LPS plus kynurenic acid (200 mg/kg s.c, three times). Figure 2: IDO activity in pure cultures of human macrophages exposed to LPS (1 μg/ml for 24 hours) in the presence and absence of kynurenic acid (30-100 nM). Detailed description of the invention

The present invention concerns the use of kynurenic acid and its pharmaceutically acceptable salts in therapy; in particular the invention relates to the use of kynurenic acid and its salts for the preparation of drugs useful in the prevention and treatment of shock, in man or in animals.

Examples of pharmaceutically acceptable salts of kynurenic acid are sodium or potassium kynurenate.

The term "shock" in accordance with standard medical literature (e.g. Merck Manual, 17^th ed., p.1704) means any state in the patient whereby blood flow to the peripheral tissues and the perfusion thereof are inadequate for vital functions; this deficit situationVoccur following to various causes, such as insufficient cardiac -K output or disturbed distribution of peripheral blood flow, hypotension, etc. Accordingly, there are number of forms of shock, for example hypovolemic shock, vasodilatory shock, cardiogenic shock, septic shock, obstructive shock, hemorrhagic shock, etc. The present invention includes the treatment of all forms of shock: septic shock, particularly that originating from toxins, is the type preferably treated. Furthermore, by eliminating the triggering pathology, the present use extends to the treatment of all the symptoms and/or complications caused by shock, such as fever, tremors, hyperventilation, alkalosis, pulmonary oedema, etc. The dosage of kynurenic acid or its salts depends on the degree of severity and progression of the shock, as well as the age and general condition of the patient; as a non-limiting reference, active doses are within the range 0.1 - 3 g. The present invention also relates to pharmaceutical compositions useful for the prevention and treatment of shock, particularly septic shock, containing kynurenic acid or a pharmaceutically acceptable salt thereof in association with pharmaceutically inert organic or inorganic carriers or excipients. The pharmaceutical compositions are preferably divided into single administration units, containing a quantity of kynurenic acid or a salt thereof between 0.1 and 3 g. They may contain kynurenic acid or a salt thereof as the only active principle or be combined with one or more drugs commonly used in the treatment of shock, thus obtaining useful pharmacological synergies: examples of said additional drugs, depending on the type of shock to be treated, include antibiotics or antibodies, vasopressors (e.g. norepinephrine or dopamine), inotropic agents, cardiostimulants, thrombolytics, etc.

The compositions can be in solid, liquid or semisolid form, appropriate to the various administration routes.

Intravenous administration is preferred, typically in buffered isotonic solutions; this route is the most suitable since treatment of shock normally requires urgent intervention with immediate effectiveness. However, administration via other routes is possible, for example oral or transdermal, and is indicated for less urgent symptomatologies or for preventative or maintenance applications. Intravenous formulations contain the active compound dissolved, suspended or emulsified in a sterile carrier, possibly in the presence of emulsifying agents, stabilizers, buffering agents and other conventional additives; they are normally divided into vials or bottles for infusion; they can be stored as dry products to be reconstituted with water or with a suitable carrier prior to use. Solid pharmaceutical compositions can be tablets, capsules, powders, granules, pills, reconstitutable powders etc; they can contain conventional excipients such as binders, fillers, diluents, compressing agents, lubricants, detergents, disintegrants, colorants, flavourings and wetting agents. The tablets can be coated in accordance with methods well known in the art. Suitable fillers include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate. Suitable lubricants include for example magnesium stearate. Suitable wetting agents include sodium laurylsulfate.

Liquid pharmaceutical compositions for oral use can be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as dry products for reconstitution with water or a suitable carrier prior to use; they can contain conventional additives, for example suspending agents such as sorbitol, syrup, methylcellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel or hydrogenated edible fats, emulsifiers such as lecithin, sorbitan monooleate, or gum arabic; non-aqueous carriers (which can include edible oils), such as almond oil, fractionated coconut oil, oily esters such as glycerine esters, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired, conventional flavourings or colorants.

The invention is illustrated hereinafter by means of the following non-limiting examples.

Experimental part

The following example provides the results of tests undertaken in models of shock, which show that the administration of kynurenic acid drastically reduces mortality in this serious pathological condition.

Example 1

Effect of kvnurenic acid on survival in a model of septic shock a) Two groups, each consisting of 31 CD-1 mice weighing about 20 g, were treated with 10 mg/kg i.p. of LPS extracted from the cell walls of Escherichia coli.

The first group was then immediately treated with a subcutaneous saline injection, while a high dose of kynurenic acid (500 mg/kg) dissolved in buffered saline was administered to the second. After 72 hours, 10 of the 31 mice treated with LPS and saline had died while only 2 of the 31 animals treated with kynurenic acid had died. This difference is statistically significant and indicates that kynurenic acid significantly reduces mortality in models of shock. b) A second series of experiments concerned the administration of 30 mg/kg i.p. of LPS and three treatments with kynurenic acid (immediately after, after 3 hours and after 6 hours). In this case the survival was also evaluated after 72 hours. The results are given in the following table and confirm that kynurenic acid administration drastically reduces mortality in models of shock.

Dead mice / Total mice LPS 30 mg/kg i.p .

Saline 7/15

Kynurenic acid 200 mg/kg s.c. X 3 1/15*

The following further examples describe both in vivo and in vitro experiments, conducted to evaluate the specific biological effects of kynurenic acid, useful for the purposes of treating shock. Example 2

Effect of kvnurenic acid on the concentration of nitrites in serum of animals treated with LPS

As referred to above, an increased synthesis of nitric oxide is considered one of the main causes of vasodilation present in shock. To evaluate whether the administration of kynurenic acid would modify nitric oxide production in models of shock, the concentration of nitrites (metabolic products of nitric oxide) was measured using the Greiss method in control animals, in animals treated with LPS and in animals treated with LPS plus kynurenic acid (200 mg/kg s.c, three times). As shown in figure 1, while LPS administration drastically increased serum concentration of nitrites, treatment with kynurenic acid significantly reduced the concentration of said metabolites in animals that had received LPS (P<0.01). The concentration of nitrites in the controls was 1.4 ± 0.4 μM. In the mice treated with 30 mg/kg LPS, said concentration increased to 45 ± 7 μM (P<0.0001). Treatment with kynurenic acid reduced LPS-induced increase of nitrites to 18 ± 4 μM (P<0.01). Example 3

Effect of kynurenic acid on human macrophage activation

One of the causes of nitrite accumulation in the plasma of animals in the state of shock is the induction of the enzyme iNOS (nitric oxide synthetase) in macrophages. The Applicant has demonstrated that iNOS induction can be achieved in vitro in macrophages exposed to LPS. Usually, iNOS induction is associated with that of another enzyme, indoIeamine-2,3-dioxygenase (IDO), which not only can be regarded as an indicator of macrophage activation, but is also involved in kynurenic acid synthesis. Indeed, the synthesis of kynurenic acid from tryptophan takes place by the combined action of two enzymatic activities: IDO, which opens the indole ring to form kynurenine and kynurenine aminotransferase, which leads directly to the neosynthesis of kynurenate. In seeking to evaluate the effects of kynurenic acid on macrophage activation, pure cultures of human macrophages were prepared which were exposed to E. coli LPS (1 μg/ml for 24 hours) in the presence and absence of kynurenic acid (30- 100 nM). IDO activity was then evaluated by incubating 100 μl of the cell lysate with 100 μl of a buffer containing 2 mM tryptophan and measuring the quantity of kynurenine produced, after 60 minutes at 37°C. As shown in figure 2, IDO activity thus measured proved to be 20.2+2.7 pmol/min/mg.prot. in the control macrophages. When the cultures were exposed to 1 μg/ml of LPS, activity increased to 86+21 pmol/min/mg.prot (P>0.001). When, in addition to being exposed to LPS, the cultures were also exposed to kynurenic acid (30-100 nM), IDO activity was significantly reduced. These observations indicate that even extremely low concentrations of kynurenic acid are able to reduce macrophage activation.

It is clear from the aforegiven results that the administration of kynurenic acid is able to reduce macrophage activation, in particular reducing expression of the enzymes iNOS, responsible for nitric oxide synthesis, and IDO, involved in the synthesis of kynurenic acid itself, and that its utilization for the prevention and treatment of shock, in particular septic shock, is justified.

Claims

1. Kynurenic acid or pharmaceutically acceptable salts thereof, for use in therapy for the treatment of shock.

2. Kynurenic acid or salts thereof according to claim 1 , for use in the treatment of septic shock.

3. Pharmaceutical composition useful for the treatment of shock, comprising kynurenic acid or pharmaceutically acceptable salts thereof, alone or in combination with other drugs useful in therapy for shock.

4. Composition according to claim 3, wherein the other drugs are chosen from antibiotics, antibodies, vasopressors, inotropic agents, cardiostimulants, thrombolytics.

5. Composition according to claims 3 and 4, in a dosage unit form containing from 0.1 to 3 g of kynurenic acid or salt thereof.

6. Pharmaceutical composition according to claims 3-5, characterised by being formulated for intravenous administration.

7. Use of kynurenic acid or pharmaceutically acceptable salts thereof in the preparation of drugs useful for the prevention and/or treatment of shock.

8. Use according to claim 7, for the prevention and treatment of septic shock.