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Phosphocreatine complexes

The invention relates to novel derivatives of phosphocreatine suitable for use in therapeutic applications, in particular for the treatment of diseases of the central nervous system (CNS) .

Oxygen or glucose deficiency at the neuronal level prevents or limits the ability of neurons to synthesize ATP. A decrease of ATP has been shown after anoxia or ischemia both in vivo and in vitro. The creatine/phosphocreatine system can to some extent compensate for the oxygen and glucose deficiency. In normal brain functioning , creatine (Cr) is synthesized in the brain and is used to form phosphocreatine (PCr) with a reaction catalysed by creatin-kinase (CK) . When the phosphate group is detached from PCr, 45 KJ/mol of free energy are released, which represent a very high amount compared with the 31.8 KJ/mol ' released from ATP dephosphorilation. Thus, under conditions of ATP depletion (such as anoxia and ischemia) , PCr can donate its phosphate group to ADP to resynthesize ATP. This reaction too is catalysed by CK. In this way PCr allows ATP synthesis even in the absence of oxygen and glucose. Accordingly, it was shown long time ago that the content of PCr in neurons correlated with the maintenance of tissue polarization during oxygen deprivation. However, neuronal PCr content is limited, therefore in the case of complete anoxia or ischemia PCr is rapidly depleted too. Decrease in the PCr content precedes the fall of ATP, thus showing that PCr is used up by the cell to maintain as long as possible ATP concentration.

The amount of brain PCr is substantially increased by the treatment with creatine (Cr) . In fact, the CK reaction can shift from left to right and viceversa according to the gradient of concentration of its products. When excess Cr is available, the reaction shifts toward the synthesis of PCr. In the Eighties, Wittingham and Lipton [T. S. Wittingham and Lipton, Cerebral synaptic transmission during anoxia is protected by creatine, J. Neurochem. 37 (1981) 1618-1621] showed that by incubating in vitro rat hippocampal slices with a high Cr concentration (25mM) , their PCr content increased four-fold, from 40μmol/g of proteins to 150μmol/g of proteins. The same group also showed that this treatment was able to prevent the irreversible loss of synaptic transmission in rat hippocampal slices after a 10 minutes' deprivation of oxygen [Kass I.R. and P. Lipton, Mechanism involved in irreversible anoxic damage to the in vitro rat hippocampal slice. J. Physiol. (London) 332 (1982) 459-472]. Such a protection was accompanied by a partial conservation of ATP in anoxic slices: in Cr treated slices, ATP after anoxia fell to 7.9 μmol/g of proteins, while in the control slices it fell to 3.6μmol/g of proteins (control preanoxic content: 13.9 μmol/g of proteins) [Kass I.R. and P. Lipton, 1982, above] . Afterwards it was shown -that pretreatment with creatine also prevented anoxia-induced decrease of protein synthesis in rat hippocampal slices. Furthermore, on the ground of solid and repeated experimental evidences, it was shown that creatine administration protects the brain tissue from anoxia in experimental conditions.

In addition to the possible usefulness of creatine administration in human therapy, three hereditary disorders which are characterized by brain creatine deficiency have been described (Guanidino-Acetate-Methyl-Transferase or GAMT1 hereditary deficiency, Arginine-Glycine Amidino-Transferase or AGAT2 hereditary deficiency, creatine transporter3 hereditary deficiency) , two of them (GAMT or AGAT deficiency) being at last partially ameliorated by creatine treatment.

Finally, experiments on animal models also suggested that creatine administration can be useful in some degenerative diseases of the central nervous system (Huntington's Chorea, Lateral Amiothrophic Sclerosis and Parkinson Disease) , and human anamnestic data suggest a certain efficacy also in mitochondrial encephalopathies too .

Nevertheless, in spite of the high number of very promising indications, the use of creatine as a medicament in central nervous system diseases is still unsatisfactory. Small-scale clinical experimentations have not been able to confirm the efficacy of creatine in Huntington disease and in lateral amiothrophic sclerosisAt variance with the clear evidences available on creatine efficacy in in vitro models, pretreatment with creatine in an in vivo stroke model has provided unclear results up to now. Only in two of the hereditary syndromes of primary lack of creatine (deficiency of GAMT and AGAT) it was possible to demonstrate that creatine administration results in certain relief, though incomplete. However, in the third one, i.e., the lack of creatine transporter, the administration of creatine is not effective and such a condition is at present incurable.

One of the problems noticed in vivo is the very low bioavailability of creatine at the brain level . Creatine is a very polar molecule, which therefore crosses very poorly the blood brain barrier. Some of the present inventors already showed that the sistemic administration even of very high doses of creatine in any case results in very low brain levels of creatine. Data in the literature confirm that, in laboratory animals long-term oral administration of creatine causes only a very low increase in the brain, and in some of the tested species this increase is not even statistically significant. In previous experiments one of the present inventors could not show any increase in brain creatine content even after 2 weeks of oral administration of creatine in adult rats. On the contrary, a very high increase of the cerebral content of creatine (increase of 67%) was shown when this compound was administered by the intracerebroventricular route, thus circumventing the blood brain barrier. Through cerebral MRI spectroscopy, an increase (4-15%) in the human grey matter of the brain after oral administration of creatine was demonstrated. Nevertheless, in all the well documented cases of hereditary deficit of the enzymes Guanidino-Acetate-Methyl-Transferase (GAMT) and Arginine-Glycine-Amidino-Transferase (AGAT) , the restoration of the cerebral content of creatine is very slow and ultimately only partial .

In mammalians, creatine is transported through the blood brain barrier by means of a specific transporter, the creatine transporter. There are indications that most or all the creatine crosses the blood brain barrier in vivo through the creatine transporter. Accordingly, in genetic conditions which are characterized by a deficiency in such a transporter, cerebral creatine is absent and cerebral administration of creatine cannot restore the creatine supply. The deficit of the creatine transporter is still an incurable disease.

Therefore, to improve the therapeutic potential of creatine in central nervous system diseases, it would be desirable to improve the ability of this compound to cross the blood brain barrier, since the intracerebroventricular administration is not feasible in humans.

In the past, the attempt to improve the ability of creatine to cross the blood brain barrier by reducing the polarity of the molecule led to the synthesis of cyclocreatine . Nevertheless, the experiments made to study the actual protective effect of such a derivative yielded equivocal results .

Other derivatives of creatine are described in Us patent No. 6,114,379. This document discloses some chelates of creatine to be used as oral supplements. The chelates in US 6,114,379 are resistant to acid cyclization in the stomach and are therefore capable of being adsorbed in the intestine as intact molecules .

International patent application WO 96/14063 discloses the use of creatine and analogues thereof, including phosphocreatine, for the treatment of diseases of the nervous system. However, this document does not address the problem of improving blood brain barrier crossing by creatine.

One object of the present invention^ is to provide novel derivatives of phosphocreatine which are effective in the treatment of diseases of the central nervous system.

Another object of the present invention is to provide novel derivatives of phosphocreatine which are effective in the treatment of diseases of the central nervous system implying deficiency of energy, deficiency of creatine or deficiency the creatine transporter. The latter disorder is currently incurable .

A further object of the present invention is to provide novel derivatives of phosphocreatine with improved ability to cross the blood brain barrier.

Still another object of the present invention is to provide novel derivatives of phosphocreatine which are capable of crossing the blood brain barrier independently of the creatine transporter.

These and other objects are achieved by novel phosphocreatine complexes having the following general formula:

formula (I)

In the phosphocreatine complexes according to the invention of formula (I) , the molar ratio between the ligand (phosphocreatine) and the metal is 1:1.

In formula (I) M is a pharmaceutically acceptable metal, preferably, but not exclusively, selected from the group consisting of Magnesium (Mg) , Calcium (Ca) , Cobalt (Co) , Manganese (Mn) , * Zinc (Zn) , Cadmium (Cd) , Copper (Cu) , Barium (Ba) , Strontium (Sr) , Iron (Fe) , and An" is an organic or inorganic anion capable of neutralizing the positive charge of the complex. Preferred anions for this purpose are acetate, butirrate, propionate, and the anions provided with free radical scavenger neutralizing activity, sucha as for example ascorbate, benzoate, sulfonium and di-sulfonium anions .

The phosphate group of the phosphocreatine complexes of the present invention may be in an acidic form or in the form of a salt. In the latter case, the phosphate group is salified with monovalent positive ions, preferably, but not exclusively, selected from Na+, K+, NH4+, Li+, choline ion, Ag+. Sodium ions (Na+) are particularly preferred among them.

A particularly preferred embodiment of the phosphocreatine complexes of the invention, is the complex of formula (Ia) , hereinafter designated as "phosphocreatine-Mg disodium salt complex" :

formula (Ia)

As it will be shown in more detail in the section relating to the examples, the phosphocreatine complexes of formula (I) of the present invention possess a good ability to penetrate across the cell plasma membranes and consequently to cross the blood brain barrier, which renders them effective for use as active ingredients for preparing a medicament for the treatment of diseases of the central nervous system, in particular the diseases which imply an energy deficiency or a creatine deficiency.

Moreover, the administration of the phosphocreatine derivatives of the invention instead of creatine is preferable, in that the compound does not need to be phosphorylated by creatin-kinase and can immediately release the energy available in the phosphate link of PCr.

The inventors also showed that the ability of the complexes of formula (I) to cross the cell plasma membranes is independent of the presence of a creatine transporter, thus making it possible to envisage their application also for the treatment of diseases involving the deficiency of the creatine transporter which were up to know incurable.

Thus, a second aspect of the invention is to use the phosphocreatine complexes of formula (I) for preparing a medicament for the treatment of diseases of the central nervous system, preferably selected from the group consisting of diseases of the central nervous system which imply energy deficiency, diseases of the central nervous system which imply deficiency of creatine and diseases of the central nervous system which imply deficiency of the creatine transporter.

Illustrative examples of such diseases are:
- hereditary deficiency of the creatine transporter: this rare disease causes severe brain disfunction causing mental retardation and movement disorders;
hereditary deficiency of guanidinoacetate-methyltransferase, or hereditary deficiency of arginine-glycine-amidinotransferase: these diseases too cause severe brain disfunction in children leading to mental retardation, autism, movement disorders and epilepsy;
- conditions known to be associated to a high risk of brain ischemic damage: among them, by way of example, open heart surgery (e.g. aortic-coronaric by-pass) which are often followed by cognitive disorders caused by cerebral oligaemia during surgery; transient ischemic attacks; and other conditions;
- anoxic encephalopathy of the new borns;
- ischemic stroke;
- neurodegenerative, diseases (Alzheimer disease, Parkinson disease, Huntington's Chorea).

In a particularly preferred embodiment, said disease is the disease caused by creatine transporter deficiency.

A subject-matter of the present invention is also a pharmaceutical composition comprising a pharmaceutically effective amount of at least one phosphocreatine complex of formula (I) and a pharmaceutically acceptable carrier or diluent .

The pharmaceutical composition of the invention may be administered by the oral, intravenous, intra-arterial or intrathecal route .

Dosage forms suitable for oral administration are for example tablets, capsules, vials, solutions. Dosage forms suitable for intravenous, intra-arterial or intrathecal administration are for example injectable solutions or suspensions. Such dosage forms may be prepared using conventional preparation methods and excipients which are well-known to the person skilled in the art, for example using conventional binders, carriers, diluents, fillers, extending agents, emulsifying agents, solvents solvents, suitable for the selected dosage form.

The phosphocreatine complexes of the present invention may be administrated at a dosage generally comprised between 50 and lδOOmg/Kg, which however can be varied according to the specific conditions, both in relation to the disease to be treated and iii relation to the to patient's conditions and type.

The following examples are provided by way of illustration only and are not intended to limit the scope of the invention as defined by the appended claims.

Example 1: Preparation of phosphocreatine-Mg disodium salt

(formula Ia)

The complex phosphocreatine-Mg disodium salt of formula (Ia) was prepared using the protocol for creatine magnesium complex described in US patent 6,114,379 by combining the following components: 50 ml of water, 3,5 g (13,72 mmol) of phosphocreatine disodium salt monohydrate and 2,94 g (13,72 mmol) of magnesium acetate, tetrahydrate . The reaction mixture was stirred at room temperature for 1 hour and freeze-dried. The resulting white powder was used without further purification.

Example 2 : Protective effect of phosphocreatine Mg disodium salt complex towards brain ischemic damage

Rat hippocampal in vitro slices, prepared and studied as described in M. Balestrino et al., Exogenous creatine delays anoxic depolarization and protects from hypoxic damage: dose-effect relationship, Brain Res. 816 (1999) 124-130, were used. The effects of phosphocreatine Mg disodium salt complex during the period of time comprised between the onset of anoxia and the anoxic depolarization were studied.

Latency period between the onset of anoxia and the anoxic depolarization was the following: (in seconds, mean ± standard deviation) : controls, 255 ± 91 (N 15) ; phosphocreatine Mg disodium salt complex ImM, 390 ± 175 (N = 6, p = 0.03 vs. controls, t-test) .

These results demonstrate that phosphocreatine Mg disodium salt is able to replicate the protective effect of creatine in in vivo models of cerebral anoxia. Delayed anoxic depolarization during anoxia is known to be associated to a better functional recovering after ischemia. It is known that creatine is able to delay anoxic depolarization, and our data cited herein show that phosphocreatine Mg disodium salt complex also replicates this effect. Thus, the biological effectiveness of phosphocreatine Mg disodium salt is comparable to that of creatine, despite the modifications of the molecule.

Example 3 : Cell plasma membrane penetration by phosphocreatine Mg disodium salt complex

The creatine transporter is known to be blocked by guanidinopropionic acid (GPA) .

Mouse hippocampal slices were incubated In vitro in the presence of creatine (2mM) and ImM, 4mM, or 1OmM GPA. Uptake of* creatine within brain cells decreases to 52 ± 15% of the controls with ImM GPA, to 55 + 7% of the controls with 4mM GPA and to 44 + 14% of the controls with 1OmM GPA incubated slices (mean ± standard deviation, N = 4 for each group, p< 0.0001 AMOVA for paired data). These results show that in vitro hippocampal slices uptake of creatine is carrier-dependent .

Then, the increase of creatine in mouse hippocampal slices after incubation with creatine and with phosphocreatine Mg disodium salt complex was studied.

The creatine content was (in μg/mg of proteins, mean ± standard deviation) : controls, 23 ± 12 (N = 23) , creatine 2mM, 58 ± 29 (N = 20) , phosphocreatine Mg disodium salt complex 2mM, 40 ± 12 (N 21) . Both creatine- and phosphocreatine-induced increase < are statistically significant .

Then, the effects of GPA on the uptake were studied. Fig. 1 shows the effects of increasing GPA concentrations on creatine uptake. In this figure, tissue creatine concentration (ordinates) is expressed as a percentage of the concentration measured in paired controls incubated without GPA. Asterisks show statistically significant differences from paired controls (incubated without GPA) . One asterisk : p<0.05 (t-test) . Two asterisks: p<0.01 (t-test) . N = 6. As shown, it is confirmed that the creatine transporter blocker (GPA) reduces the of creatine uptake at the brain level.

Fig 2 shows the effects of increasing concentrations of GPA on the uptake of the phosphocreatine Mg disodium salt complex. As it can be seen, the same concentration of GPA did not prevent creatine to increase in mouse hippocampal slices incubated with phosphocreatine Mg disodium salt complex 2mM (N = 6, p = 0.96 ANOVA for paired data). This finding is interpreted to indicate that t'he uptake of the phosphocreatine Mg disodiuπi salt complex of the invention is carrier-independent. Without being bound by any particular theory, we think that this effect is due to the greater lipophilicity of the phosphocreatine complexes of the invention.