ES2288450B1 - PLEIOTROPHIN (PTN) AND MIDKINA (MK) FOR THE TREATMENT OF TOXICITY PRODUCED BY ABUSE DRUGS. - Google Patents

Abstract

Use of cytokines Pleiotrophin (PTN) and Midkina (MK) in the preparation of drugs for the treatment of toxicity produced in humans by the use of drugs of abuse, based on the overexpression of these cytokines that takes place in different Central Nervous System areas damaged by drugs of abuse in order to repair them.

Description

Pleiotrophin (PTN) and Midkina (MK) for Treatment of toxicity produced by drugs of abuse.

The invention whose patent protection National requested is the use of cytokines Pleiotrophin (PTN) and Midkina (MK) in drug preparation  for the treatment of toxicity produced in humans by the Abuse drug use, based on overexpression that takes place of said proteins in the central nervous tissue damaged by drugs of abuse to replace it, which has been demonstrated here.

This new drug treatment against toxic effects on the central nervous system of drugs such as cocaine, opioids or amphetamines, is an alternative or effective complement to the current therapies of uninhabitation, which, regardless of their greater or lesser effectiveness, they do not act directly on the neurotoxic effects produced by these drugs

The invention therefore finds application in the field of the pharmaceutical industry, in the preparation of medicines and pharmaceutical compositions whose active ingredients be the Pleiotrophin and / or Midkina proteins, alone or in combination with other agents

State of the art

Pleiotrophin (PTN) proteins Pleiotrophin) and Midkina (MK; from English Midkine) were discovered in the late eighties by two groups of independent research. PTN is a cytokine of 136 amino acids (Milner et al., 1989; Li et al., 1990) which is 50% homologous in regards to its amino acid sequence with the MK cytokine (Kadomatsu et al., 1988; Li et al., 1990), what it does that both produce the same effects and have similar functions  in most of the contexts in which they have been studied.

The following diagram shows the alignment of the amino acid sequence of PTN and MK, which is faithful reflection of its structural homology and is the basis of its high functional similarity.

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It is important to note that, to date, no there are approved therapeutic uses of PTN or MK, which does not it's strange because of its relatively recent discovery (1988-1990). However, the study of both protein in basic research has been growing exponentially, having already established strategies therapeutic based on these proteins, some of which have been the subject of patent applications, especially in relation with tissue recovery in cancer treatment.

Mainly, the PTN and the MK are being investigated in the field of oncology, for their role in the tumor development In adults, under normal conditions, both proteins have very low expression levels in any organ, excluding the central nervous system, but when a tumor occurs, the malignant cells begin to synthesize high levels of PTN and MK, which has led to Oncology field researchers to study the role of both proteins in the development of those tumors. So, it has was able to verify that PTN and MK are powerful factors angiogenic that although they are necessary for this quality in phenomena such as repair of damaged tissue, when overexpress in tumors favor their growth by promoting angiogenesis (see reviews by Deuel et al., 2002 and from Kadomatsu and Muramatsu, 2004). You could say that 90% of the basic research that is being carried out at this time with Pleiotrophin and Midkina it is in this field due to the possible interest that a pharmacological intervention might have about these proteins in cancerous phenomena, which has been translated in the recent publication of some requests for patents on the use of these proteins as active ingredients of medicines for the treatment of cancer, such as US Patent Application US2002182242, dated publication 05/12/2005, related to compositions based on Pleiotrophin for the repair of damaged or diseased tissues of bone and cartilage.

Although in a much lower number of studies related to the functions of PTN and MK in cancer, the rest of research projects that have been taken or are being taken to out today are aimed at understanding the roles of PTN and MK in the development of the Nervous System, since it has proven that the highest levels of expression of these proteins occur during embryonic development in the System Central Nervous (Silos-Santiago et al., 1996; Kadomatsu and Muramatsu, 2004).

PTN and MK are expressed during development in neurons and glia (Silos-Santiago et al., 1996; Kadomatsu and Muramatsu, 2004), which suggests that they have important  functions in these cells. In fact, it has been proven that both cytokines stimulate neuron differentiation (see review of Deuel et al., 2002) and of the neuronal progenitors (Hienola and col., 2004).

In adults, the expression of PTN and MK is very limited to certain neuronal populations and glia; but nevertheless, both proteins overexpress themselves significantly in inflammatory processes and processes of tissue repair in the central nervous system (Yeh et al., 1998; Sakakima et al., 2004; Kikuchi-Horie et al., 2004; Herradon et al., 2005a). It has also been proven that the PTN and MK increase the survival of neurons catecholaminergic and induce cell differentiation progenitors to catecholaminergic neurons, increasing significantly your levels of Tyrosine Hydroxylase, the enzyme limitation in the synthesis of catecholamines (Kikuchi et al., 1993; Hida et al., 2003; Jung et al., 2004).

In these studies that are being carried out about the roles of PTN and MK in the Central Nervous System, it is noteworthy that there are practically no therapeutic uses established of these proteins, or at least there is no approved medication that has any of these in its composition proteins However, it is important to note that with the recent discovery of the significant increase in PTN levels in the brain area of the Nigra Substance of patients with the Parkinson's disease, its possible application has been suggested therapeutic in this disease (Marchionini et al., 2007). Also, due to the ability of PTN to differentiate cells progenitors to neurons, its possible use for regenerate the spinal cord motor nerves damaged by trauma (Mi et al., 2007).

As proof of the above, it should be said that doing an exhaustive search for patents worldwide in the Worldwide database, only one patent was found on the use of MK for a therapeutic purpose in the nervous system central; is the European patent (EP1057489) relating to a pharmaceutical composition containing the midkina protein as active ingredient to treat ischemic disorders, in prevention of cerebral infarction, cerebrovascular spasm, disease Alzheimer, senile dementia of Alzheimer's Type, and others cerebrovascular diseases such as, for example, Parkinson's

What is clear is that as for treatments used to alleviate the harmful effects of consumption  Chronic drug abuse (cocaine, opioids, amphetamines ...) a referred to in the present invention, there is no treatment specific to alleviate the toxic effects of drugs of abuse about the Central Nervous System. These toxic effects have no because always lead to neuronal death, since some drugs of abuse such as cocaine produce cellular alterations that they do not lead to his death, but they are the cause for example of compulsive drug-seeking behavior (Kalivas and Volkow, 2005).

So far, all therapeutic efforts in the field of drug addictions are aimed at the detoxification of the individual with the ultimate goal of extinction of the behavior that leads him to use drugs even after prolonged periods of withdrawal. In this strategy of cessation, multiple therapies have been used pharmacological with greater or lesser success, such as medications antidepressants, dopamine agonists, system stimulants Central nervous, opioid receptor agonists and antagonists, etc. However, as has been shown, relapses in the Drug use are very frequent. This, together with the report made public by the Ministry of Health in 2007 in which Spain appears at the head of countries with greater number of addicts to various drugs, especially in the case of cocaine, advises a serious rethinking of the therapeutic approach to addiction to drugs

Given that each year, the number of people addicted to some type of drug abuse increases and that current de-use therapies fail in a significant number of cases, it would be important to have therapeutic strategies to reduce the neurotoxic effects of drugs of abuse , of course without abandoning the existing therapies of disinhabitation, in order to prevent subsequent sequelae that affect the Nervous System
Central.

As already mentioned, it does not currently exist no drug treatment against the toxic effects of drugs of abuse in the Central Nervous System. Therefore, without damage of the existing therapies of disinhabitation, is imposed more and more the development of pharmacological treatments against the neurotoxic effects of drugs of abuse, in order to diminish the emergence of long-term sequelae due to the neurodegeneration induced by these drugs.

This has been the aim that has guided the studies and experiments that have given rise to the present invention, based on the results of recent studies that have related the addictive or neurotoxic effects of drugs of abuse with alterations of the central catecholaminergic pathways. Thus, for example, addiction and neurotoxicity associated with the consumption of methamphetamine and other psychostimulants appear to be related to an initial increase in central dopaminergic activity and damage to the dopaminergic terminals of the striatum and dopaminergic neurons of the substance nigra , respectively (Thomas et al., 2004; Riddle et al., 2005). In addition to the dopaminergic function, noradrenergic neurotransmission also seems to play an important role in both phenomena; Thus, in the case of opioids such as morphine, the sustained decrease in noradrenergic tone could have negative effects on cell survival or neurogenesis in brain areas such as the hippocampus (Kulkarni et al., 2002), and this may be related to a possible neurotoxic effect; In addition, various studies have shown a close relationship between the effects of opioids on noradrenergic pathways and their ability to produce physical dependence and generate abuse (see review by Alguacil and Morales,
2004).

Therefore, one of the keys to find the Pharmacological treatment against neurotoxicity in drug dependence is based on the identification of those factors involved in the survival and differentiation of neurons catecholaminergics whose levels of expression in the system central nervous are sensitive to drug administration of abuse.

In our laboratory we have verified that the chronic administration of yohimbine, an antagonist of α2-adrenergic receptors, increases significantly MK levels in the rat hippocampus (Ezquerra et al., 2007). This result is very relevant in our  context since, as we have shown in our laboratory During the last decade, yohimbine prevents toxicity, search behaviors and the changes that opiates produce about some parameters related precisely to the function of the noradrenergic pathways that innervate the hippocampus (see mode in summary the review of Sheriff and Morales, 2004 and the recent article by Alonso et al., 2007). Therefore, the set of these results allowed us to think once again about a possible connection between the effects of opiates, the activity of the pathways Central catecholaminergics and the expression of the cytokine MK.

PTN is overexpressed in the cerebral area of the nucleus accumbens of rats treated with amphetamine (Le Greves, 2005), while MK is overexpressed in the hippocampus of rats treated with morphine (Ezquerra et al., 2007) and in the prefrontal cortex of alcoholic patients and smokers (Flatscher-Bader and Wilce, 2006). Taking into account all the observations mentioned, it did not seem risky to suggest that the organism could be increasing the brain levels of PTN and MK to alleviate the negative effects of these different drugs of abuse, since as noted above, PTN and MK they overexpress themselves in damaged nerve tissue to repair it, increasing for example the survival of catecholaminergic neurons. In order to confirm or rule out this hypothesis, we conducted a series of experiments aimed at determining the possible protective effect of PTN on cocaine-induced toxicity in cell cultures. The results of these experiments, which are presented and discussed in the embodiment section of the invention, demonstrate, by two different techniques, on the one hand that cocaine dramatically decreases the viability of cell cultures, and on the other, the increase Significant cell viability of cell cultures treated together with cocaine and pleiotrophin. These results would be extrapolated to the MK since, as has been mentioned, both proteins, PTN and MK, are very structurally redundant (see the sequence diagram above) and also in terms of their functions and effects (see reviews of Deuel et al., 2002 and de Kadomatsu and Muramatsu, 2004). The degree of functional redundancy of both cytokines is such that one of the authors of the present invention demonstrated in a recent article that in such a way that even genetically modified mice lacking endogenous MK dramatically over-express PTN in numerous organs with in order to compensate for the absence of MK (Herradon et al.,
2005b).

The invention

Therefore, confirmed the fact that the Pleiotrophin (PTN) and Midkina (MK) are overexpress in different brain areas of experimental animals and human patients damaged by drug abuse effects (presumably to repair them), the object of the present invention is the use of these two cytokines, alone or in combination of both and / or with other agents, in the Drug preparation for the treatment of toxicity produced in humans by the use of such drugs, such as Cocaine, or opioids such as morphine or amphetamines, among others.

Said use of Pleiotrophin (PTN) and Midkina (MK) must be used to obtain pharmaceutical compositions intended for a limiting and curative therapy of toxicity produced by the various drugs of abuse, sued as alternative or complement to the cessation therapies. These compositions may be usable either in injectable form, in capsules, dragees or tablets, or by any means of the usual in clinic, and can also be used in any dose by individual and day.

The mechanism of action by which PTN and MK exert their cellular and tissue effects in different contexts (in our case, alleviate the negative effects of drugs from abuse in the central nervous system of humans that consume), has already been established. Both cytokines bind to Tyrosine protein phosphatase receptor (RPTP)? / \ zeta, inactivating its phosphatase activity and therefore, increasing phosphorylation levels of RPTP? / \ zeta substrates such as? -catenin and GIT1 / Cat-1. Recently, in other jobs in which contributed significantly one of the authors of the present invention it was shown that Fyn and the β-aducina are other effectors of the pathway of PTN / RPTP signaling? / \ zeta. The rising levels of phosphorylation of these signal translators as well as others second cellular messengers such as MAP kinases and Akt, causes signals to be sent to the cell nucleus where they are activated transcription factors that in turn increase transcription of genes involved in tissue damage repair processes.

Figure 1 shows a summary diagram of the explained mechanism of action of the PTN and the MK through which They exert their effects.

The advantage provided by the present invention in The pharmaceutical field is to have the active ingredients PTN and MK as the basis for the development of compositions specific pharmacological against neurotoxic effects drug-induced abuse, being an alternative or important complement to the current therapy therapies (both pharmacological and psychotherapy), aimed at the individual gradually reduce drug abuse until reaching extinguish it, a process that can last several years.

The use of PTN and MK drugs in proposed in this patent application report would reduce the neurotoxicity, either causing neuronal death or cellular alterations, produced by drugs of abuse. By therefore, it is expected that it also reduces the important consequences caused by chronic drug abuse on the nervous system of the individual.

Embodiment

To propose the use of cytokines PTN and MK in the treatment of toxicity produced by drug abuse was necessary to prove the hypothesis, based on the studies so far conducted of the addictive effects or neurotoxic drugs of abuse in relation to alterations of the central catecholaminergic pathways, of which said cytokines could play a protective role on toxic effects of such drugs, when over-expressing itself in the tissue nerve damaged to repair it. For this a series were made of experiments aimed at determining the possible effect PTN protector on cocaine-induced toxicity in Cell cultures.

The experiments were carried out in neuroglioblastoma cells (NG108-15) that were incubated with cocaine in high concentration (5 mM) in the presence, or not, of two concentrations of PTN (3 and 6 µM). These concentrations were chosen because they correspond to 50 and 100 ng / ml treatments, which have already shown biological activity in other in vitro assays (Marchionini et al., 2007) and which are much lower than the concentration of 30 µM (500 ng / ml), used to differentiate progenitor cells to catecholaminergic neurons (Jung et al., 2004). Two standard techniques were used to estimate the effects of incubations on cell viability, the Neutral Red cytotoxicity test (INVITTOX IP-64, adapted to NG108-15 cells) and the MTT cytotoxicity test (INVITTOX IP-17 , adapted to NG108-15 cells).

Using the neutral red test, in which measures the ability of the cell to incorporate the dye neutral red cationic, which only occurs in living cells, we could verify that the PTN at the concentration of 6 µM significantly protected cells against toxicity Cocaine-induced (5 mM), without exclusive incubation with PTN had no effect on cell viability, as It is reflected in the graphs of Figures 2 and 3.

The graph in Figure 2 shows how the Pleiotrophin (PTN) exerts a protective effect on toxicity Cocaine-induced in cell cultures, doubling cell survival at PTN concentration of 6 µM (neutral red test) * P <0.05 vs. Cocaine (statistical test of Student's t).

The graph in Figure 3 shows how Pleiotrophin (PTN, 6 µM) does not exert a significant effect " per se " on the viability of cells in culture (neutral red test).

On the other hand, using the MTT test, in which mitochondrial activity is measured as a measure of viability cellular, we could see how the toxicity induced by cocaine (5 mM) was significantly reduced even using a lower concentration of PTN (3 µM) than used in the test of neutral red (6 µM), no effect being detected significant in cells incubated exclusively with PTN (3 µM), as shown in the graphs of Figures 4 and 5.

The graph in Figure 4 shows how the Pleiotrophin (PTN) at the concentration of 3 µM, exerts an effect Protective against cocaine-induced toxicity in crops cell phones, reaching cell viability in the cultures supplemented with 80% PTN compared to cells without treat (control; 100%) (MTT test). * P <0.05 vs. Cocaine (statistical test of Student's t).

The graph in Figure 5 shows that Pleiotrophin (PTN, 3 µM) does not exert a significant effect " per se " on the viability of cells in culture (MTT test).

In short, the results of Experiments performed demonstrate, by two techniques different, on the one hand that cocaine dramatically decreases the viability of neuroglioblastoma cell cultures, and by another, the significant increase in cell viability of those cell cultures treated together with cocaine and Pleiotrophin; results that are extrapolated to Midkina, being a protein similar structurally to Pleiotrophin, with Similar effects and functions.

Industrial application

The previous results constitute the basis for the industrial preparation of pharmaceutical formulations that contain the cytokines Pleiotrophin and / or Midkina, alone or in combination with other components that complement or synergize your action, as active matter against the neurotoxicity produced in humans for drug abuse.

Claims (3)

1. Use of the cytokines Pleiotrophin (PTN; English Pleiotrophin) and Midkina (MK; English Midkine), alone or in combination of both and / or with other agents, in the preparation of drugs for the treatment of toxicity produced in human by the abuse of drugs of abuse, characterized this therapeutic functionality by the significant over-expression of these cytokines that takes place in various areas of the Central Nervous System damaged by drugs of abuse in order to repair it. 2. Pharmaceutical compositions whose principles active be Pleiotrophin (PTN) and / or Midkina (MK), according to first claim, usable either in injectable form, in capsules, dragees or tablets, or by any means of the usual ones in clinic. 3. Pharmaceutical compositions whose principles active be Pleiotrophin (PTN) and / or Midkina (MK), according to previous claims, usable in any dose by individual and day.