WO2020012373A1

WO2020012373A1 - New drugs chronic invalidating pathologies and to slow aging, based on a new metabolic tryptophan route and production process thereof

Info

Publication number: WO2020012373A1
Application number: PCT/IB2019/055869
Authority: WO
Inventors: Vincenzo Politi; Giovanni Politi; Andrea Margonelli
Original assignee: Polindols S.R.L.S.
Priority date: 2018-07-10
Filing date: 2019-07-10
Publication date: 2020-01-16

Abstract

A system is shown for the use of the enol tautomer of IPA (3-Indolyl pyruvic Acid) in pharmacologically and therapeutically effective pharmaceutical forms, overcoming the drawbacks deriving from the chemical- physical characteristics of the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid now commercially available as well as the use thereof, as a precursor and/or promoter of NAD (Nicotinamide-Adenine Dinucleotide), for the treatment of dysfunctions dependent on oxidative stress, present in the cells, and especially at the mitochondrial level, and generally from metabolic disorders which are typical of old age, and, as a possible binder of the tautomerase function present in the molecules of MIF ((Macrophage Motility Inhibiting Factor), circulating in the blood, for the treatment of serious infectious, inflammatory, autoimmune diseases and tumors, including rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus.

Description

NEW DRUGS AGAINST CHRONIC INVALIDATING PATHOLOGIES AND

TO SLOW AGING, BASED ON A NEW METABOLIC TRYPTOPHAN ROUTE AND PRODUCTION PROCESS THEREOF

DESCRIPTION

SUMMARY OF THE INVENTION

The present invention relates to the use of the Z- isomer of the enol tautomer of 3-Indolyl pyruvic acid having an enol purity ranging from 99% to 99.9% referred to the total acid, in pharmaceutical and nutraceutical formulations, for human and animal use, which exclude the presence of water or other solvents in their production process, so as to ensure their stability over time, and ensure the use, by patients and volunteers, of this compound and derivatives thereof, at the highest degree of purity and stability, as a therapeutic agent capable of:

being used, in the presence of a persistent activity of oxygen free radicals in the cells, in the treatment of dysfunctions dependent on oxidative stress, present in the cells, and especially at the mitochondrial level, and in general of metabolic disorders which are typical of old age, due to the ability to transform itself, directly or indirectly, into NAD (Nicotinamide-Adenine-Dinucleotide) ; and

being used as a possible binder of the tautomerase function present in the molecules of MIF (Macrophage Motility-Inhibiting Factor) circulating in the blood, or as a stabilizing factor of the MIF, in a long list of serious diseases of infectious, inflammatory, autoimmune and tumor type, including rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus, in accordance with the trial carried out and illustrated below.

Said Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid which will be referred to briefly as "ZENOLIPA", was obtained with a synthesis method described in patent EP 1 501 503 which allows production thereof also in industrial amounts.

Its structure formula is as follows:

Its brute formula is C₁₁H₉NO₃, and it has a molecular weight of 203.20.

The invention also relates to the preparation process in a controlled environment and the pharmaceutical and veterinary products thus obtained containing ZENOLIPA and the possible excipients, in formulations which are stable in biological fluids, transportable through biological membranes and usable in the different organs of mammals.

FIELD OF THE INVENTION

The present invention originates from the discovery made by the same inventors of a new metabolic pathway, which although existed for millions of years in nature, has never been reported in scientific publications, which relates to indole derivatives selected in the course of evolution, and present in all living beings (higher organisms, plants and animals, including humans) .

This discovery allows us to understand basic phenomena for the life of cells and organisms, when these are subjected to oxidative or metabolic stress or to mitochondrial alterations.

On the other hand, the ensuing set of knowledge has allowed the inventors themselves to identify a family of new compositions with pharmacological activity, based on the presence, as an active ingredient, of said "ZENOLIPA" compound, and salts thereof, which are useful both to develop new therapies for the treatment of serious diseases, acute and chronic, of mammals, including humans, and to investigate the pathophysiological mechanisms which affect the life of higher animals.

This family of compositions with pharmacological activity is characterized by being obtained with a production process in a controlled environment as regards humidity and protected from the presence of water or other solvents, such as to ensure the stability over time of the active ingredient consisting of the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, obtained by chemical synthesis, with an enol purity of from 99% to 99.9% with respect to acid, and ensure the use thereof by patients and volunteers at the highest degree of purity. In particular, the dry granulation system in gas flow is used, which is the object of the European patent EP 2364183, to which reference shall be made for full description.

Finally, the discovery of the new metabolic pathway allows acquiring new knowledge regarding the phenomena of the aging of cells and of entire living organisms, useful for developing new strategies to hinder, or delay in time, the loss of functionality of cells, tissues and organisms in old age.

DESCRIPTION OF THE PRIOR ART

Tryptophan

is the only one in the series of 20 natural amino acids, i.e. it forms part of the vegetable and animal protein chains, to have an indole ring in its structure, and this uniqueness has provided for many decades the opportunity to put it at the center of the scientific interests of chemists, biologists and clinicians from all over the world, interested in exploring the role of this particular molecule in the evolution of life on earth, and its possible participation in important physiological or pathological phenomena concerning humans . Since about 70 years ago, researchers interested in the study of Tryptophan have formed an international scientific society (ISTRY, International Study Group For Tryptophan Research) , which follows and publishes the progress achieved on the subject in laboratories around the world, so that each new discovery concerning the amino acid and derivatives thereof (collectively referred to as TRYCATs) can be discussed, and lead to convergences on which are the metabolic pathways, dependent on tryptophan, used for millions of years by living beings, and above all by humans, in physio- pathological situations.

With reference to mammals only, fundamental discoveries were made when the presence of a first metabolic pathway (the so-called indole pathway) was unanimously recognized, which, starting from the free tryptophan in circulation, gives rise to Serotonin (an important neurotransmitter of the nervous, central and peripheral system) and melatonin (a hormone released by the pineal gland, essential for the sleep-wake cycle, for balancing immune responses and for other hormonal functions) . The indole pathway is always hindered, in humans, by situations of physical or psychological stress due to the release of cortisol in circulation: the production of the enzyme TDO (Tryptophan

Dioxygenase) in the liver is in fact induced through this hormone, which opens the indole ring of Tryptophan, reducing the availability thereof in circulation. When TDO is chronically activated, a deficit of biological functionalities dependent on serotonin and melatonin is seen: several diseases (such as insomnia, anxiety, depression and appetite disorders) are recognized as dependent on reduced blood levels of tryptophan and indole derivatives thereof, due to an excessive activity of the TDO.

A few decades ago, a second enzyme capable of opening the indole ring of Tryptophan, and also of other indole derivatives (those containing a nitrogen atom in the side chain of the molecule) was identified and described: this enzyme was also referred to as IDO (Indolamine Dioxygenase), and is produced directly by different types of cells, when these are stimulated to react to external signals, causing inflammatory or immune reactions . The activation of IDO leads to a second important metabolic pathway of Tryptophan, the pathway of Kynurenine, which is very complex due to the several TRYCATs being produced, and is still the subject of hundreds of scientific works each year, tending to clarify the effects thereof, positive or negative, on the fundamental functions of life, and on the dynamics of a large number of serious pathological situations .

Summing up very complex and debated phenomena, it can be said that at the moment there is an almost unanimous consensus among those skilled in the field on the fact that from the beginning of evolution, Tryptophan has the fundamental function of increasing the production of NAD (Nicotinamide-Adenine- Dinucleotide) , a coenzyme necessary to produce and use the fundamental energy for cell life, in the cells: this process starts from the activation of IDO. Furthermore, for some decades, it has been ascertained that NAD has a decisive role on the stabilization of cellular structures, through the family of "Sirtuins", also involved in the control of cellular aging.

A metabolic pattern of TRYCATs is now commonly accepted and described by researchers which, starting from kynurenine (an amino acid which originates from the opening of the indole ring of tryptophan by IDO or TDO) , leads to its possible bond with a particular receptor (AHR, Aryl Hydrocarbon Receptor) , directly involved in the genesis and evolution of inflammatory and/or immune phenomena of cells and tissues. When Kynurenine enters the bloodstream, it triggers a series of enzymatic reactions which lead to other TRYCATs, more or less correlated to physiological or pathological activities: two TRYCATs with neurotoxic activity ( 3-OH-anthranilic acid and quinolinic acid), and one with neuroprotective activity (Kynurenic acid, antagonist of excitatory amino acids) .

The scientific researches carried out on the TRYCATs are mostly oriented to find synthetic molecules, or mechanisms, useful to increase the substantially neuro-protective activity of Kynurenic acid, or to hinder the neurotoxic activity of 3-OH-Kynurenine or Quinolinic acid. However, while the inhibition of 3-OH- anthranilic acid can be advantageous, and is therefore pursued by many researchers through the synthesis of complex molecules, the inhibition of Quinolinic acid synthesis is considered by researchers to be a taboo and an undesirable objective, because this molecule, in the metabolic pathways described, is considered as the most immediate precursor in the pathway leading to NAD. Blocking the formation of quinolinic acid would therefore mean reducing the levels of NAD, and therefore hindering the availability of an essential metabolite for the vital functions of the cells. The situation appears so intricate that some researchers define Quinolinic Acid as the necessary and inevitable toxin for vital functions.

BACKGROUND OF THE INVENTION

A new metabolic pathway

It has now been surprisingly found by the inventors of the present invention that in the organism of mammals, including humans, the metabolic destiny of Tryptophan is not solely dependent on the TDO and IDO enzymes, which open the indole ring and lead to the formation of Kynurenine, but that there is also a third metabolic pathway, never previously described in the literature, which is automatically activated to protect organisms when they are in situations of systemic stress, or cellular oxidative stress.

This pathway, which can be defined as the pathway of defense in the oxidative emergency, starts from tryptophan and, after transformation by common transaminases (e.g. Tryptophan aminotransferase, such as EC 2.6.1.7; EC 2.6.1.64; EC 2.6.1.65), makes 3- Indolyl pyruvic acid (IPA) available to the cell for a short time. This is an alpha-ketoacid with a powerful antioxidant effect which, subjected to the action of free radicals, spontaneously turns into Kynurenic Acid, recognized as the most important physiological inhibitor of excitatory amino acids (main responsible for neuronal death in degenerative diseases) .

In the following scheme, the lower part represents the classical pathway described in all textbooks, while in the upper part the transformation sequence is that of the pathway dependent on Enol - IPA, which is recognized as important to avoid the toxicity of Kynurenine .

Not only: it has now been seen by the inventors themselves that, in the presence of a persistent activity of oxygen free radicals in the cells, as happens in many pathological situations, IPA is transformed, directly or indirectly, also in NAD, (Nicotinamide adenine Dinucleotide) .

A possible transformation sequence is shown in scheme 2, below:

SCHEME 2

The discovery of this metabolic pathway has a fundamental value for overcoming one of the most obvious paradoxes of biochemical texts.

In fact, as it appears in scheme 1, according to the classical pathway the formation of kynurenic acid would be solely dependent on kynurenine which, in turn, is the "mother" of several other derivatives, including the neurotoxic 3-OH-Kynurenine and Quinolinic acid. Therefore, according to the classical pathway, in order to have kynurenic acid (the most important natural antagonist of excitatory amino acids), the way leading to an increase in neurotoxicity should first be taken.

It is now apparent instead that the cell, as can be seen in scheme 2, has an alternative pathway of protection, so that in the presence of free radicals, Tryptophan is transaminated to Enolipa, which is transformed directly into Kynurenic Acid, blocking the neurotoxic activities of excitatory amino acids.

Continuing in this new pathway, NAD is obtained without having to go through the neurotoxic intermediate Quinolinic acid.

Starting from the results of animal pharmacology, which show the increased turnover of Serotonin in the CNS and of Melatonin in the epiphysis, after administration of Enolipa, the first clinical work on the compound was logically directed towards insomnia, anxiety and depression, in which an improvement can be assumed through the strengthening of the central serotonergic activity.

The results of Clinical Phase I are summarized in the following table 1 : TABLE 1

SUMMARY OF PILOT CLINICAL STUDIES WITH ENOLIPA

Two Phase II clinical trials were then carried out: the first to evaluate the anxiolytic effects took place in two Italian Centers (Perugia and Bari) involving 100 patients (50 treated with drug and 50 with placebo, double-blind) . The second one, to assess the effects on mild or moderate depression, was carried out in several hospitals in Cuba, involving 200 patients (100 treated with drug and 100 with placebo) . In all the clinical studies carried out, Enolipa showed an excellent safety and the reported secondary effects were always comparable to those of placebo.

A further step, essential for understanding the role of Enolipa on cellular metabolism, was made when it was surprisingly seen that the compound, when found in a strongly oxidizing biological environment, directly produces large amounts of NAD which, as is known, is the fundamental element for cellular energy balance and, through the Sirtuins, controls the phenomena related to vitality, senescence and cell death. Many recent studies have shown that NAD levels decrease with age and that the deterioration of NAD metabolism promotes several diseases associated with late age, including metabolic and neurodegenerative diseases, and different types of cancer (Aging Research Review 47, 1 -17, 2018) . Also in this case, the discovery is important for the solution of another paradox in the official pathway, concerning the metabolism of Tryptophan. In this case, Quinolinic Acid, a powerful neurotoxin capable of destroying neurons, is always indicated as a necessary precursor for the formation of NAD. It has instead been seen that, in the presence of Enolipa and free radicals, the production of large amounts of NAD is directly obtained: Enolipa therefore appears as an essential element to allow the survival of cells in critical situations due to oxidative stress, and to antagonize the evolution of serious diseases associated with aging. This new metabolic pathway of tryptophan, which can also be referred to as an IPA pathway due to the strategic role played by ketoacid, allows suffering tissues to maintain sufficient basic metabolic activity, first blocking the destructive effects of free radicals and MIF, then supplying energy through NAD and recovering mitochondria functionality, and ultimately minimizing cellular functional needs by activating Sirtuins .

The IPA pathway can therefore be considered as a natural shortcut, which allows the body of higher animals to have a direct and fast route leading from tryptophan to NAD, avoiding the neurotoxic trap of quinolinic acid. It also allows a rapid recovery of cellular energy activity, once the excitatory stimuli due to inflammatory and/or immune reactions are neutralized. This pathway can therefore allow the recovery of functional activities of the cell, both in the case of diseases deriving from metabolic dysfunctions of any kind (especially the neurodegenerative ones), and during cellular aging.

TECHNICAL PROBLEM OF THE INVENTION

Therefore, IPA has all the features to be used as an important pharmacological tool in basic biological research .

However, since it has been shown that the molecule is active in mammalian cells only in the enol tautomeric form thereof, the task of the present invention is also to provide a system to be able to have the IPA enol tautomer in pharmacologically and therapeutically effective pharmaceutical forms, overcoming the drawbacks deriving from the chemical- physical features of the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid currently available on the market .

It is noted that this enol tautomer of IPA is industrially obtained in solid form by chemical synthesis, as described in European patent EP 1 501 503 Bl, which however, as will be widely highlighted later, make the "drugability" thereof, i.e. the transformation of the powder obtained by chemical synthesis into potential drugs for oral use, difficult without the use of special protections.

DETAILED DESCRIPTION OF THE INVENTION

Before explaining the direct transformation of IPA into NAD in detail, it is appropriate to summarize below the two metabolic pathways of ketoacid known to date .

1 ) Direct transformation of IPA into kynurenic acid

The direct transformation of IPA into Kynurenic acid has already been widely described in the past, to highlight the possibility that cells have to protect themselves from excitatory amino acids, without having to go through the formation of Kynurenine .

For example, U.S. Pat. No. 5,002,963 describes the direct transformation of indole pyruvic acid into kynurenic acid, both in tissue homogenates and after administration by injection in experimental animals.

In the work published in 1989 on Biochemical Pharmacology (vol. 38, page 2405-9), Russi et al . showed increased kynurenic acid in all organs considered (blood, heart, kidney, liver and brain) after IPA i.p. administration. The increase is about 10 times (compared to the basal values) in the kidneys, 8 in the liver and 2 in the brain after 1 hour.

Subsequent works have shown that the transformation of IPA into Kynurenic acid does not occur enzymatically through Tryptophan, because even simple saline solutions, in the presence of free radicals, are able to transform one molecule into another, through a mechanism of capture of free radicals .

Finally, it has been shown that only the enol tautomer of IPA is able to transform itself into Kynurenic Acid, while the ketonic tautomer is inactive and easily degradable in water. According to the suggested metabolic scheme, an attack of oxygen-free radicals on the indole ring of IPA leads to opening of the indole: the unstable compounds which originate lead to immediate subsequent reclosing to form the ring of Kynurenic acid, without enzyme intervention.

2. Transformation of Kynurenine into NAD

The classical metabolic pathway, which leads to the formation of Nicotinamide, and therefore of NAD, starting from Kynurenine, has been described in many scientific works, over several decades (a good recent description is that of Bryleva and Brundin, in Neuropharmacology 112, 324, 2017) . It is known that Kynurenine, subjected to the action of a specific mono oxygenase, leads to the formation of 3-Hydroxy- Kynurenine, which has no particular biological activity This molecule can then be attacked by a Kynureninase, which leads to the formation of 3-hydroxy-anthranilic acid, which compound has direct effects on the immune responses of the cell, and is also responsible for neurotoxic activity, especially on cholinergic neurons. Subjected to a specific oxidase, 3-hydroxy-anthranilic acid is transformed into the compound 2-amino-3- carboxymuconic-6-semialdehyde (ACMS) . In turn, the ACMS can be subjected to the action of the ACMSD enzyme (a decarboxylase) , with formation of Picolinic Acid, for which interaction properties with immune and/or inflammatory reactions have been found. However, if there is a lack of ACMSD, ACMS can form Quinolinic acid directly, in a non-enzymatic manner, with the suggested closing of the molecule ring. In turn, Quinolinic Acid (a compound with neuro-excitatory and neurotoxic activity, thought to be responsible for the death of neurons in many degenerative diseases), is a substrate of the enzyme QPRT (Quinolinate Phosphoribosyltransferase) from which Nicotinic Acid and NAD are then obtained.

The steps from 3-hydroxy-anthranilic acid to picolinic acid and/or quinolinic acid are not yet fully clarified in detail, but it is commonly believed that this whole metabolic pathway is fed into the cells by the pressure of a continuous flow of Kynurenine, originating from inflammatory and/or immune stimuli.

3 ) Transformation of IPA into NAD

Investigating the antioxidant properties of IPA, already described in US 5, 075, 329, it has now been found that human cells, subjected to prolonged oxidative stress, can surprisingly follow alternatively an immediate metabolic pathway, which leads directly, by IPA itself, to an increase of NAD which, as is known, is a fundamental element for cellular energy balance and through Sirtuins controls phenomena related to vitality, senescence and cell death.

4 ) Experimental tests

The experimental method used was that described in Eur . J . Biochem . 267, 684-9, 2000, which concerns the evaluation of energy metabolism and lipid peroxidation of human erythrocytes, as a function of an increased oxidative stress. In short, human blood cells taken directly from healthy volunteers were centrifuged and the erythrocytes, properly treated, were incubated with oxygenated water.

After deproteinization, centrifugation and filtration, the supernatant was subjected to HPLC chromatography to determine the levels of compounds relevant for energy metabolism, both in the presence and absence of IPA. The results obtained are described in the following table 2, in which the numbers refer to the area of the chromatographic peaks obtained:

TABLE 2

These completely unexpected results have very important practical consequences, because they confirm that IPA is not only an excellent antioxidant, protecting erythrocytes from degeneration of membrane phospholipids (MDA is virtually absent), but the concentration of NAD, when IPA is added, is increased by more than 25 times compared to the control (compare the value of the peroxydized control 24022 with the concentration of the NAD after adding the IPA which is equal to 619.208) and this cannot be due to simple protection of NAD existing in the cells, but it must necessarily come from new and consistent biosynthesis of the molecule, following the presence of IPA (which can function as a precursor, and/or stimulate the production of NAD through parallel recovery pathways) and of a highly oxidizing cellular environment. We therefore arrive at the conclusion that, in the presence of strong oxidizing agents (oxygen free radicals), IPA acts as a protector from cell degeneration, both by transforming itself, without the intervention of enzymes, into Kynurenic acid (thus antagonizing the toxic effects of excitatory amino acids), which is transformed, directly or indirectly, into NAD, even here without the intervention of enzymes, avoiding the simultaneous presence of the neurotoxic effects of Quinolinic acid.

Although the molecular mechanism through which the conversion of PAH to NAD occurs is for the moment only hypothetical (it is believed that heme-dependent proteins, abundantly present in erythrocytes, may be implicated, towards which the IPA has shown to be able to interfere at the level of coenzyme) , this is likely to be another non-enzymatic process, similar to that accepted in the synthesis of Quinolinic acid starting from ACMS, and which uses the reactivity of oxygen-free radicals to form new more chemically stable species .

5) Consequences of the discovery of the new metabolic pathway

The discovery that IPA can increase cellular levels of NAD has very important consequences for international biomedical research, because it paves the way for the development of less invasive drugs for the treatment of important chronic diseases, and also in the disorders widely present in the old age. Indeed, while it is already known from inventions and publications (such as US Pat. 4,551,471; Acta Physiol . Scand .138,97,1990; Europ . J . Pharmacol .

187,345, 1990; and others) that IPA acts on the first metabolic pathway of tryptophan (that of indoles), functioning as a precursor of Tryptophan, Serotonin and Melatonin; and that it interferes with the metabolic pathway of Kynurenine transforming itself, directly and without enzymes, into Kynurenic Acid (e.g. US Pat. 5,002,963; Neurol . Neurobiol . 46,629,1988; Pharmacol . Res 21,633,1989; Neurosci . Res . Commun . 8,137,1991), and therefore reducing the neurotoxic activity of excitatory amino acids, the discovery that NAD can be produced through IPA, when the cell is metabolically suffering or under attack by oxidizing agents, allows us to consider ketoacid as one of the most important protective resources of organisms, developed in the course of evolution to allow animal cells to survive in inhospitable oxidizing environments, of extreme danger for survival .

Focusing scientific interest on human life, in recent years it has emerged that NAD is a determining regulator of cellular physiology, and also a direct participant in the metabolism of the cell. In fact, while acting in innumerable oxide-reduction enzymatic reactions, NAD can also function as a substrate of enzymes involved in intracellular signals, such as Sirtuins, poly (ADP-ribosyl) polymerase, mono (ADP- ribosyl) transferase and CD38 (Progress in Molecular Biology and Translation Science 154, 71-104, 2018) . In turn, Sirtuins function as Deacylases of NAD-dependent proteins, and catalyze the NAD reaction with the Acyl- Lysine group, to remove the acyl group from the substrate proteins. This deacetylation operation provides a regulatory system which integrates the cellular metabolism of NAD within a wide spectrum of cellular processes, such as cell metabolism and its survival, cell cycle, apoptosis, DNA repair, homeostasis and mitochondrial biogenesis, and also the prolongation of cellular life in many lower animal species .

Regarding the therapeutic role of NAD (Nicotinamide Adenine Dinucleotide) in mitochondrial or aging-related diseases, it is widely known that the ratio of the oxidized coenzyme (NAD+) to the reduced one (NADH) is the determining factor which allows normal functioning of basic metabolic pathways, such as Glycolysis, the Krebs cycle and the oxidation of fatty acids, which ensure the energy balance of the cell.

The increased concentration of NAD+ leads to the activation of Sirtuins, which regulate the cell's responses to changes in energy balance and stress situations (Clin . Trans .Med. 5, 25, 2016). Thus, the decline in NAD+ levels is indicative of mitochondrial dysfunction, and a reduction in the NAD+/NADH ratio is considered as the basis for mitochondrial function disorders, which in turn is the cause of many pathological situations seen in cellular aging. In contrast, an increase in NAD+ is related to cell survival under stress or toxicity conditions.

6) ZENOLIPA, tool to verify the "IPA pathway" and potential drug

Like all alpha-keto acids found in nature, IPA can exist in solvents in a ketonic form (keto-IPA) and in an Enol form (Enol-IPA) . Moreover, for the Enol form of IPA we can speak of two isomers, E and Z, depending on the arrangement of the OH and COOH groups around the double bond of the side chain.

It has already been described in detail in European Patent EP 1501503 B1 that the pure Enol-IPA (between 99% and 99.9%), consisting solely of the Z- isomer of Ketoacid, can be obtained with a new synthesis method, with which one can also obtain industrially usable amounts of the product.

It is therefore legitimate to define a new chemical compound, "ZENOLIPA" (Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid) , as an ideal candidate to define the characteristics of the "IPA pathway" in physiological and pathological situations, even following the action of compounds with antioxidant activity, and possibly to introduce a drug with a broad spectrum of activity in invalidating pathologies.

However, the aforementioned European patent also clearly highlights the chemical and physical features of ZENOLIPA, which make its "drugability", i.e. the transformation of the powder obtained by chemical synthesis into potential drugs, problematic without the use of particular precautions.

It is known, in fact, that the substances obtained by chemical synthesis physically appear, for the most part, as amorphous powders, which is not possible to compress directly, nor insert into the enclosures of the capsules, in order to obtain stable products over time. Therefore, the usual pharmaceutical manufacturing techniques provide for the suspension of the powders in water, and therefore a drying step at about 80 °C at the end of which granules are obtained which are suitable both for the compression and for the filling of the capsules (wet granulation technique) .

In the case of the Enolipa, the wet granulation process is not indicated, because suspension in water would quickly lead to the disappearance of the enol tautomer, and its transformation into the ketonic tautomer and degradation products, ineffective from a pharmacological point of view.

To obtain a finished product, which can be administered orally, with the certainty that the Enolipa content is at least 99%, it is therefore necessary to use dry granulation systems, in which in addition to the absence of any solvent it is possible to continuously monitor both the environmental humidity and the amount and direction of the gas flow used for granulation .

Precisely because of the marked antioxidant properties thereof, Enolipa has shown to be a powerful des-mutagenic agent in cell cultures (Ames test) : administered to experimental animals, both by mouth and by injection, the compound largely follows the anabolic pathway of indoles, increasing the turnover of Tryptophan and Serotonin in the CNS, and of Melatonin in the pineal gland. However, if oxidative stress conditions prevail in cells and tissues, Enolipa undergoes a completely unexpected metabolic fate, described in detail in the following pages. A detail of the pharmacological effects obtained on animals with the use of Enolipa is shown in Table 3 below .

Moreover, while the ZENOLIPA obtained from chemical synthesis appears remarkably stable, if kept in perfectly dry environments and in a micro crystalline form, its solubilization in neutral or acid environments is impossible, while in slightly alkaline solutions, as physiological ones, a rapid conversion in the ketonic tautomer is obtained in short, which decomposes or polymerizes, while the enol tautomer is reduced to insignificant levels, which cannot be used for pharmacological or clinical tests.

On the other hand, it has been shown in pharmacokinetic tests that ZENOLIPA is identifiable for several hours in mouse tissues, when administered by injection, and can be seen for several hours in the blood of human patients, treated with oral formulations in capsules or in tablets. This presupposes the presence in mammals of specific receptors, localized on proteins of the circulatory system, probably with a tautomerase function, which make the substance stable in biological fluids, transportable through biological membranes, and usable in the different organs of mammals .

Therefore, in addition to having a pure starting material free of the Ketonic tautomer, the development of ZENOLIPA, both as a pharmacological tool and as a potential drug, requires the use of pharmaceutical formulations which exclude the presence of water or other solvents in the process, so as to ensure its stability over time, and ensure the use by patients and volunteers of ZENOLIPA and derivatives thereof at the highest degree of purity and stability.

A granulation system adapted to the formulation of the Enolipa is described in patent application PCT/IT/2009/000498 , extended in most of the economically advanced Countries (Aerodynamic Granulation System - AGS) in which the dry granulation continuously takes place, while both environmental humidity, amount, quality and direction of the gas flow are under strict control.

7) Industrial uses of ZENOLIPA

As a product of direct transamination of tryptophan, IPA is certainly present in all living cells, although with differentiated functions, depending on the species. Starting from the archaebacteria, and continuing with extremophile or common bacteria, all the microorganisms use IPA in vital reactions, connected with the energy production. In plants, it is known that Tryptophan is then transaminated to IPA (Keto-IPA) , which is immediately transformed into Indoleacetic Acid (IAA) , the fundamental growth factor of plant organisms.

US patent 4,551,471 claimed IPA as a substance potentially useful in humans, to increase the turnover of cerebral serotonin, and therefore to cure diseases deriving from neurotransmitter deficiencies, such as insomnia and depression.

US patent 4,808,728 describes a method of chemical synthesis of IPA, useful for having large amounts of the substance which could be used in animal and clinical pharmacology tests.

US patent 5,002,963 has suggested IPA and derivatives as agents which are capable of neutralizing the toxic effects of excitatory amino acids.

Finally, US Patent 5,447,951 has suggested IPA as an anti-stress substance, because it is able to reduce the levels of glucocorticoids in the blood of mammals.

Patent EP 1 501503 B1 has suggested the enolic form of IPA for therapeutic use at the level of the central nervous system, cardiovascular pathologies and in the treatment of neuronal cell buds and tumors.

Based on the considerations illustrated in the previous pages, ZENOLIPA, formulated in granules through dry granulation methods such as that described in the previously mentioned PCT/IT/2009/000498 patent, appears as an important tool to investigate the application potential of the derivatives of Tryptophan, and to define the intervention of various TRYCATs in phenomena related to health and diseases of human beings .

Furthermore, the discovery of a direct activity of ZENOLIPA on phenomena related to the resistance of cells and organisms to oxidative stress, allows us to consider the molecule as an important pharmacological tool in experimental animal models of cell and neuron degeneration, and finally as a potential drug for the treatment of several and important human pathological situations which are added to those known so far:

a) As a promoter of NAD, ZENOLIPA can be suggested as a drug for the treatment of dysfunctions dependent on oxidative stress, present in the cells, and especially at the mitochondrial level, and in general from metabolic disorders which are typical of old age. b) As a possible binder of the tautomerase function present in the molecules of MIF circulating in the blood, ZENOLIPA can be used as a pharmacological tool for the research of new substances able to antagonize the serious pathological effects expressed by the cytokine, and as a therapeutic agent in a long list of serious infectious, inflammatory, autoimmune and tumor diseases, including rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus .

Claims

1) A Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, having an enol purity between 99% and 99.9% with respect to the total acid, characterized in that it is formulated in granules obtained through dry granulation methods, being stable in biological fluids, transportable through cell membranes and usable in various organs of mammals for medical use.

2) A Z-isomer of the enol tautomer of the 3- indolyl pyruvic acid according to claim 1, characterized in that it is in the form of tablets, capsules and granules to be dispersed in water immediately before the administration to patients.

3) Salts and simple derivatives of the Z-isomer of the enol tautomer of pure 3-Indolyl-pyruvic acid according to claim 1, characterized in that they are obtained in the form of water-soluble granules, adapted to be injected into experimental animals and/or in patients .

4) A Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, having an enol purity between 99% and 99.9%, with respect to the total acid, according to any one of claims 1 and 2, and/or salts and simple derivatives thereof according to claim 3, for use as a promoter of NAD in a strongly oxidizing cellular environment .

5) A Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, having an enol purity between 99% and 99.9%, with respect to the total acid, according to any one of claims 1 and 2, and/or salts and simple derivatives thereof according to claim 3, for use in a therapeutic treatment of pathologies dependent on deficiency of cellular NAD, deficiency of circulating serotonin and/or melatonin, excessive presence of excitatory amino acids in nerve terminations, and excess of activity in the circulation of Cytokine MIF .

6) A Z-isomer of the enol tautomer of the 3- Indolyl pyruvic acid, having an enol purity between 99% and 99.9%, with respect to the total acid, according to any one of claims 1 and 2, and/or salts and simple derivatives thereof according to claim 3, for use in a treatment of Metabolic Diseases, such as Type 2 diabetes, obesity, appetite disorders, gastro-duodenal ulcers, pathological aging.

7) A Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, having an enol purity between 99% and 99.9%, with respect to the total acid, according to any one of claims 1 and 2, and/or salts and simple derivatives thereof according to claim 3, for use in a treatment of Neurodegenerative Diseases, such as Alzheimer's, Huntington's disease, Parkinson's disease, multiple sclerosis, Amyotrophic Lateral Sclerosis, AIDS dementia complex, Depression, Schizophrenia, Consequences of Malaria.

8) A pharmaceutical and/or nutraceutical composition for human and animal use, in the form of tablets, capsules and granulates containing the Z- isomer of the enol tautomer of the 3-Indolyl pyruvic acid, having an enol purity between 99% and 99.9%, with respect to total acid, and/or salts and simple derivatives thereof, characterized in that its production takes place by dry granulation in a controlled environment with respect to humidity, and protected from the presence of water or other solvents in the mixture containing the active substance and any excipients .

9) A pharmaceutical composition according to claim 8 for use in a therapeutic treatment of pathologies dependent on deficiency of cellular NAD, deficiency of circulating serotonin and/or melatonin, excessive presence of excitatory amino acids in nerve terminations, and excess of activity in the circulation of Cytokine MIF.

10) A pharmaceutical composition according to claim 8 for use in a treatment of Metabolic Diseases, such as Type 2 diabetes, obesity, appetite disorders, gastro-duodenal ulcers, pathological aging.

11) A pharmaceutical composition according to claim 8 for use in a treatment of Neurodegenerative Diseases, such as Alzheimer's, Huntington's, Parkinson's, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, AIDS dementia complex, Depression, Schizophrenia, Malaria Consequences.

12) A pharmaceutical composition according to claim 8 for use in a mortality protection treatment due to sepsis, excessive stimulation of immune and inflammatory systems, rheumatoid arthritis, asthma, atherosclerosis .

13) A process to produce drugs and nutraceutics for human and veterinary use, containing the Z-isomer of the enol tautomer of the 3-indolyl pyruvic acid (or ZENOLIPA) having an enol purity between 99% and 99.9%, with respect to total acid, and/or salts and simple derivatives thereof, formulated in tablets, capsules and granulates, characterized in that it comprises the following steps :

providing a powder mass of ZENOLIPA obtained by chemical synthesis in a solid, semi-crystalline, chemically stable state having an enol purity between 99% and 99.9% based on the total acid;

introducing said powder mass into a dry granulation apparatus operating under the influence of carrier gas for the production of granules to be compressed in tablets or to be used to fill capsules, in the absence of water or other liquids, wherein the gas flow is constant and the gas direction is the same as that of the mass which moves within the granulation system;

using the granules thus obtained for manufacturing, in a controlled environment with regard to humidity and protected from the presence of water or other solvents in the active ingredient-containing mixture and possible excipients, of tablets or capsules or granules, adapted to be immediately dispersed in water before administration .

14) A pharmaceutical and/or veterinary product obtained according to the method of the preceding claim, characterized in that the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, and/or salts and simple derivatives thereof, exerts the function of direct precursor of tryptophan, serotonin and melatonin in the mammalian organism in every situation of physical and psychological stress.

15) A pharmaceutical and/or veterinary product obtained according to the method of claim 13, characterized in that the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, and/or salts and simple derivatives thereof, has, as a precursor and/or promoter of NAD, therapeutic activity for the treatment of dysfunctions due to oxidative stress and in general to metabolic disorders which are typical of old age.

16) A pharmaceutical or veterinary product obtained according to the method of claim 13, characterized in that the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, and/or salts and simple derivatives thereof, has therapeutic efficacy in serious diseases of infectious type, as a possible binding of the tautomer function present in the MIF molecules circulating in the blood.

17) A pharmaceutical or veterinary product obtained according to the method of claim 15, characterized in that the Z-isomer of the enol tautomer of 3-Indolyl pyruvic acid, and/or salts and simple derivatives thereof, has therapeutic efficacy, as a direct precursor of the kynurenic acid, in all diseases caused by excess of activity of excitatory amino acids.

18) Pharmaceutical products obtained according to the method of claim 13, which can be used for the treatment of pathologies dependent on deficiency of cellular NAD, deficiency of circulating serotonin and/or melatonin, excessive presence of excitatory amino acids in nerve terminations, and excess of activity in the circulation of Cytokine MIF.

19) Pharmaceutical products containing the Z- isomer of the enol tautomer of 3-Indolyl pyruvic acid, and/or salts or simple derivatives thereof, obtained according to the method of claim 13, in combination with other therapeutic agents used in the pathologies described in claims 8-11, obtained by dry mixing both the active ingredients and the excipients.