MX2013002884A - Compositions useful for treating neuropathic pain, which comprise endogenous antagonists of the n-methyl-d-aspartate receptor and inhibitors of the organic acids carrier. - Google Patents

Abstract

The present invention describes compositions that comprise an endogenous antagonist of the N-methyl-D-aspartate receptor and an inhibitor of the organic acids carrier, such as kynurenine with probenecid, which result very useful and effective for an effective treatment of neuropathic pain without side effects in the motor activity. The compositions of the invention reduce the excretion of kynurenic acid and promote the accumulation in the central nervous system, which results useful for treating the neuropathic pain.

Description

Compositions useful for the treatment of neuropathic pain comprising endogenous N-methyl-D-aspartate receptor antagonists and inhibitors of organic acid transporter Field of the invention.

The present invention relates to the treatment of neuropathic pain, particularly to pharmaceutical compositions for the treatment of neuropathic pain comprising combinations of endogenous N-methyl-D-aspartate receptor antagonists and organic acid transporter inhibitors, particularly kynurenine and probenecid, which are useful to reduce the excretion of kinuric acid and promote its accumulation in the central nervous system, which allows the treatment of neuropathic pain.

BACKGROUND OF THE INVENTION The nervous system plays a vital role in the protection of organisms. It is responsible for detecting, responding and remembering any harmful stimulus by translating it into an unpleasant experience known as pain. Pain plays an extremely important role in the intact organism, as it forces the body to withdraw from physical, chemical or mechanical stimuli that endanger its integrity. However, when there is an abnormal functioning in the structures that regulate this mechanism, the pain exceeds its function and becomes pathological.

The sensation of pain involves physiopathological and psychological components that are often difficult to interpret, so defining it becomes very complicated. Currently, the International Organization for the Study of Pain (IASP) has the most accepted definition for this term and defines it as "an unpleasant sensory and emotional experience associated with real or potential tissue damage that can be described. in terms of the magnitude of the damage. " Neuropathic pain is the consequence of an imbalance between excitatory factors and inhibitors of nociception, which occurs as a failed mechanism of adaptation to a nerve injury that generates long-term plastic changes and perpetuates the painful experience without any protective role. Neuropathic pain was considered for a long time as intractable because it was resistant to the usual analgesic drugs (NSAIDs and opiates). Drugs that attenuate central sensitization by action on N-methyl-D-aspartate receptors are an option for the treatment of neuropathic pain. However, the wide distribution of the N-methyl-D-aspartate receptor in the brain limits the clinical use of antagonists on these receptors due to the significant number of side effects that accompany the analgesic effect.

Among the existing patent documents relating to the use of kinuric acid is US6265442 which relates to the administration of metabolic precursors of kinuric acid which includes kynurenine and tryptophan to treat neurological diseases such as Huntington's disease.

WO2010 / 128345 discloses kinuric acid derivatives and their pharmaceutically acceptable salts, amides, stereoisomers including diastereoisomers and / or enantiomers, as well as a preparation process and pharmaceutical compositions containing them and their use in the treatment of headaches particularly migraine non-neurodegenerative.

Patent EP0603301 discloses a pharmaceutical composition and method for the coadministration of an uricosuric agent such as probenecid or sulfinpyrazone and an excitatory amino acid for treating neurological or neurodegenerative diseases. The probenecid is administered in an amount of 500 mg.

Patent application WO2009 / 040849 relates to the use of the amino acid L-kinurenine and derivatives thereof in combination with a pharmaceutically acceptable excipient such as β-interferon to treat inflammatory diseases.

Despite the above and the treatment options that exist for neuropathic pain, it is considered that such pharmacological treatment is ineffective, so it is important to provide efficient pharmacological solutions for the treatment of neuropathic pain.

BRIEF DESCRIPTION OF THE INVENTION The current pharmacological treatment for neuropathic pain is ineffective. Drugs that attenuate central sensitization by action on N-methyl-D-aspartate receptors are an option for the treatment of neuropathic pain. Kininic acid is an endogenous antagonist of N-methyl-D-aspartate receptors by binding to the allosteric glycine site. Systemic administration of its precursor L-kynurenine (endogenous receptor antagonist N-methyl-D-aspartate) in combination with probenecid (inhibitor of the organic acid transporter) reduces the excretion of kinuric acid and promotes its accumulation in the central nervous system. The objective of the present invention was to analyze the participation of kinuric acid in the processing of neuropathic pain induced by spinal cord ligation L5 and L6 in the rat. The i.p. (intraperitoneal) of kynurenine (50-200 mg / Kg) and probenecid (100 mg / Kg) individually did not produce antialodynic effect, while the combination of kynurenine with probenecid at 100 mg / Kg but not at 10 mg / Kg presented an effect antialodynic maximum at three hours and was preserved even until 24 hours after its administration. The area under the curve of the time course showed that the L-kynurenine / probenecid combination of the present invention significantly reduces tactile allodynia. Intrathecal administration of kinuric acid (1-30 pg) produced a dose-dependent effect that declined after one hour of administration, while its co-administration with probenecid i.p. potentiated this effect and maintained it until 5 h after administration. The evaluation of the adverse effects in the rota-rod showed that the kinurenine / probenecid combination of the present invention did not affect the motor coordination at the same dose and time that the maximum antialodynic effect occurred, while the MK-801, an antagonist Exogenous N-methyl-D-aspartate receptors significantly decreased the time in the rota-rod at the same dose that had antialodynic effect. Consistent with the presence of antialodynic effect, administration of L-kynurenine (200 mg / Kg) and probenecid (100 mg / Kg) individually did not increase the concentration of kinuric acid in the cerebrospinal fluid. In contrast, the L-kynurenine / probenecid combination of the present invention increased the concentration of kinuric acid in the cerebrospinal fluid more than 2 times compared to the control group. The results indicate that the increase in concentration of kinuric acid at the central level, either by intraperitoneal administration of the L-kynurenine / probenecid combination of the present invention, or by exogenous administration of spinal kinuric acid, reduces neuropathic pain in mammals, for example in rats without the presence of adverse effects in their motor activity at the doses evaluated.

Brief description of the figures.

Figure 1. The experimental design for evaluating the antiallodynic effect of the combination of L-kynurenine and probenecid of the present invention at different doses is shown.

Figure 2. The experimental design is shown to evaluate the antiallodynic effect of exogenous spinal kinuric acid and its co-administration with probenecid.

Figure 3. The experimental design for the quantification of kinuric acid and for the evaluation of the adverse effects present at the motor level by the L-kinurenine / probenecid combination of the invention is shown.

Figure 4. The evaluation of allodynia in naive and sham rats without treatment and administered neuropathic rats i.p. with L-kynurenine (K), probenecid (P) or vehicle (0.1 N NaOH, pH 8.0). The withdrawal response of the left hind paw of the rat was evaluated by stimulation with the von Frey filaments. The data were expressed as 50% withdrawal threshold (g) and the average + standard error was plotted (n = 6). Those withdrawal responses with a value less than 4 g were considered allodynic.

Figure 5. The evaluation of allodynia in administered neuropathic rats i.p. with the combination of L-kynurenine (K) at different doses (25, 50, 100 and 200 mg / kg) plus probenecid (P) at 10 mg / Kg. The withdrawal response of the left hind paw of the rat was evaluated by stimulation with the von Frey filaments. Data were expressed as 50% withdrawal threshold (g) and the mean ± standard error was plotted (n = 6). Those withdrawal responses with a value less than 4 g were considered allodynic.

Figure 6. The evaluation of allodynia in administered neuropathic rats i.p. with the combination of L-kynurenine (K) at different doses (50, 150, 175 and 200 mg / kg) plus probenecid (P) at 100 mg / Kg. The withdrawal response of the left hind paw of the rat was evaluated by stimulation with the von Frey filaments. Data were expressed as 50% withdrawal threshold (g) and the mean ± standard error was plotted (n = 6). Those withdrawal responses with a value less than 4 g were considered allodynic.

Figure 7. The antiallodynic effect of the i.p. administration is shown. of the L-kynurenine / probenecid (K + P) combination of the invention in neuropathic rats compared to the control group (C), the neuropathic group without treatment (N) and the vehicle group (Veh). The figure expresses the area under the curve (ABC) calculated from the time course of the different groups. The bars are the mean ± the standard error (n = 6). * Significant difference (p <0.05) compared to untreated groups, determined by one-way ANOVA followed by the Student-Newman-Keuls test.

Figure 8. The evaluation of allodynia in neuropathic rats administered by spinal route with vehicle (0.1 N NaOH, pH 8.0), kinuric acid (AK) independently or in combination with probenecid i.p. (P, 100 mg / kg). The withdrawal response of the left hind paw of the rat was evaluated by stimulation with the von Frey filaments. The data were expressed as 50% withdrawal threshold (g) and the average + standard error was plotted (n = 6). Those withdrawal responses with a value less than 4 g were considered allodynic.

Figure 9. The antiallodynic effect of spinal administration of kinuric acid (AK) alone and in combination with probenecid (P) in neuropathic rats is shown. The figure expresses the area under the curve (ABC) calculated from the time course of the different groups. The bars are the average ± the standard error (n = 6). * Significant difference (p <0.05) compared to the vehicle group. # Statistically significant difference (p <0.05) compared to the rest of the groups, determined by one-way ANOVA followed by the Student-Newman-Keuls test.

Figure 10. The evaluation of motor activity in naive rats and neuropathic rats without treatment (N) or administered i.p. with L-kynurenine (K), probenecid (P), vehicle (N + V) and MK-801. The residence time in the rota-rod was evaluated at a speed of 4-40 rpm for 10 min. The time of -1.5 represents the basal values of motor activity. The data were expressed as the dwell time in seconds and the average of 3 evaluations per rat was plotted for each time ± the standard error (n = 6).

Figure 11. The evaluation of motor activity in naive rats (C) and neuropathic rats without treatment (N) or administered i.p. with vehicle (V), L-kynurenine (K), probenecid (P) and MK-801 (MK). The figure expresses the area under the curve (ABC) calculated from the time course of the different groups. The bars are the mean ± the standard error (n = 6). *Significant difference (p <0.05) compared to the rest of the groups, determined by one-way ANOVA.

Figure 12. The comparison of the antiallodynic effect in neuropathic rats administered i.p with the combination L-kynurenine (K) / probenecid (P) of the invention or with MK-801 is shown. The withdrawal response of the left hind paw of the rat was evaluated by stimulation with the von Frey filaments. Data were expressed as 50% withdrawal threshold (g) and the mean ± standard error was plotted (n = 6). Those withdrawal responses with a value less than 4 g were considered allodynic.

Figure 13. The concentration of kinuric acid in cerebrospinal fluid of naive rats (Na) and neuropathic rats without treatment (N) or administered i.p. with vehicle (V, 0.1 N NaOH), L-kynurenine (K, 200 mg / kg) and probenecid (P, 100 mg / kg). The figure expresses the concentration of kinuric acid (AK) in cerebrospinal fluid. The bars are the mean ± the standard error (n = 6).

Detailed description of the invention.

The present invention provides pharmaceutical compositions comprising the combination of kynurenine with probenecid (inhibitor of the organic acid transporter), which when administered systemically to a subject with neuropathic pain, reduces the excretion of kinuric acid and promotes its accumulation in the nervous system central, which allows the effective treatment of neuropathic pain.

In contrast to the solutions proposed up to the present invention for the treatment of neuropathic pain, the compositions described herein raise the concentrations of kinuric acid in the central nervous system by the systemic administration of kynurenine, particularly L-kynurenine, in combination with probenecid. , since kinurenic acid is an endogenous antagonist of the N-methyl-D-aspartate receptor, it has a low capacity to produce motor alterations. For the Thus, the compositions of the present invention constitute an alternative for the effective treatment of neuropathic pain.

According to Woolf's classification, neuropathic pain is a type of spontaneous pain accompanied by hypersensitivity that is generated by lesions in the nervous system, both peripherally (diabetic polyneuropathy or post-herpetic neuralgia) and at the central level (lesion in the spinal cord or multiple sclerosis); Likewise, it is considered a type of pathological pain (also called chronic pain) that is usually poorly adaptive, persistent and has no defensive function or other useful effects.

Neuropathic pain is defined as an unpleasant sensory or emotional experience associated with a dysfunction or injury of the nervous system. It is considered the most debilitating type of pain and affects a large and growing number of people around the world. The patients describe it as a burning sensation, pickets and electric shocks, which translates into the presence of allodynia, hyperalgesia, paresthesia and dysesthesia. These sensory alterations are the expression of neuropathic pain and its intensity indicates the degree of neuronal damage present.

Prior to the present invention, the pharmacological treatment for neuropathic pain was nonspecific and ineffective, the main therapeutic tool being the use of antidepressants, anticonvulsants and opioids.

The mechanisms that contribute to the generation and maintenance of neuropathic pain are multiple, have different duration and occur at different levels such as the peripheral nervous system, the spinal cord, the medulla, and even the brain. The peripheral mechanisms that have been best described so far include the generation of ectopic discharges as a result of an increase and redistribution of sodium channels, alterations in the expression of ion channels in the neuroma (site of injury or nerve damage), alteration in the expression of ion channels (Na + and K +), adrenergic receptors, SP, vasoactive intestinal peptide, galanin and BDNF in the primary afferent, collateral propagation of primary afferent neurons, propagation of sympathetic neurons within the dorsal root ganglion and sensitization of the nociceptor .

The central sensitization that occurs in the dorsal horn of the spinal cord is an event that is considered essential for the generation of neuropathic pain. Phosphorylation of the N-methyl-D-aspartate receptor occurs during central sensitization. When phosphorylated, it increases its distribution and moves from intracellular deposits to the synaptic space. Once in the membrane, the increase in action potentials increases the sensitivity to glutamate and the flow of Ca2 + in said receptor. This causes an increase in the excitability of the cells, a reduction in the trigger threshold and the system modifies its response, since it now responds to normally innocuous stimuli while responding exaggeratedly to noxious stimuli. In neuropathic pain these sensory alterations (hyperalgesia and allodynia) are maintained even when the lesion that originated it has healed, suggesting the intervention of mechanisms involved in synaptic plasticity.

Taken together, these phenomena suggest that the molecules involved in synaptic plasticity may be therapeutic targets for the treatment of neuropathic pain. The glutamate receptor of the N-methyl-D-aspartate type plays a vital role during the synaptic plasticity and turns out to be one of the candidates for the treatment of this type of pain. In addition, these evidences suggest the development of therapeutic strategies aimed at the modulation of N-methyl-D-aspartate receptors for the treatment of neuropathic pain.

The N-methyl-D-aspartate receptor is an ionotropic glutamatergic receptor that controls a cationic channel highly permeable to monovalent ions and calcium. These receptors play a major role in excitatory synaptic transmission and plasticity as well as in various processes ranging from the formation of memory to the establishment of chronic pain. The key mechanism that involves N-methyl-D-aspartate receptors in synaptic plasticity is its voltage-dependent characteristic. These receptors are inactive at resting membrane potentials, because they possess a blockage in the pore of the Mg2 + channel even in the presence of glutamate. N-methyl-D-aspartate receptors require that two events occur simultaneously, one is the release of glutamate and the other is that the postsynaptic membrane must be depolarized so that Mg2 + blockage is removed and glutamate exerts its action. Once activated, the receptor allows the entry of Ca2 +, which activates a series of signaling molecules including protein kinases, protein phosphatases and transcription proteins. This event leads to the induction and maintenance of synaptic plasticity.

The N-methyl-D-aspartate receptors are distinguished from the a-amino-3-hydroxy-5-methyl-4-isoxazole propionic and kainate by their high permeability to Ca2 +, blocking Mg2 + during resting potentials and the requirement of a glycine molecule as a co-agonist for its activation. In addition to the glycine binding site, the receptor possesses binding sites to certain polyamines such as spermine, Ifenprodil and protons that can enhance or inhibit the opening currents of the gate in the channel.

Several investigations show the participation of N-methyl-D-aspartate receptors in pain processes. The N-methyl-D-aspartate receptors play an important role in hyperalgesia processes. Peripheral administration of CP-101, an antagonist of N-methyl-D-aspartate receptors, decreases the shaking behavior in the formalin test. Also the peripheral administration of the MK-801 antagonist decreases the activity of dorsal horn neurons of the spinal cord in rats. In addition, the number of N-methyl-D-aspartate receptors increases in peripheral nerve fibers during inflammation.

In this sense, the use of N-methyl-D-aspartate receptor antagonists represents a possible therapeutic tool for the treatment of neuropathic pain if we consider the participation of central sensitization and the localization of these receptors in structures such as the brain, the ganglia of the dorsal root and the spinal cord. For example, kinuric acid is an endogenous antagonist of the glycine site of N-methyl-D-aspartate receptors., which plays an important role in processes of functional pain and has recently been shown to have an analgesic effect on inflammatory pain. The kinurenine pathway is considered the main route of tryptophan degradation and is carried out in macrophages, microglia cells and astrocytes. This pathway has been described in both the liver and brain of humans, non-human primates, rodents and smaller mammals. The kinurenine pathway is considered as a route for the transformation of peripheral tryptophan to nicotinadenindinucleotide (NAD) and nicotinadenindinucleotide phosphate (NADP) in mammalian metabolism.

All metabolites of the kinurenine pathway are derived directly or indirectly from L-kynurenine. It is transported through the blood-brain barrier by the neutral amino acid transporter. L-kynurenine can be converted into two main metabolites with neuroactive properties. One of them is the quinolinic acid with the function of agonist of the N-methyl-D-aspartate receptor with neurotoxic effects and neuronal damage, while also the L-kinurenine gives rise to the formation of the kinuric acid with the function of antagonist of the N-receptor. methyl-D-aspartate and neuroprotective effects.

Quinolinic acid is characterized as a weak but specific receptor agonist for N-methyl-D-aspartate. For this quality, it is considered as a powerful excitotoxin. The quinolinic acid induces the depletion of α-aminobutyric acid, increases the concentrations of intracellular Ca2 +, also induces peroxidation of lipids, the production of reactive oxygen species and consequently cell death.

For its part, kinurénico acid is an antagonist of receptors to excitatory amino acids. At micromolar concentrations (Cl50 = 7.9 μ?) It acts as a receptor antagonist for excitatory amino acids capable of inhibiting the hyperexcitability of N-methyl-D-aspartate receptors by binding them to the allosteric glycine site. At higher concentrations, it has an antagonism on a-amino-3-hydroxy-5-methyl-4-isoxazole propionic and kainate receptors and is able to inhibit the nicotinic alpha-7 acetylcholinergic receptors present at the presynaptic level. The peculiarity of its antagonistic effect lies in the presence of kinuric acid endogenously. However, under normal conditions its concentration is very low and it is unable to act on said receptors.

Due to the characteristics of kinuric acid to inhibit overexcitation derived from glutamatergic transmission, researchers in several areas describe its influence on physiological and pathological processes with possible therapeutic effects in neurological disorders. There are some studies that establish the participation of kinuric acid in pain and give evidence that antagonism of kinuric acid on the glycine site of the N-methyl-D-aspartate receptor is related to analgesic properties in the rat. Some reports describe an indirect participation, where they observe that non-steroidal anti-inflammatory drugs (NSAIDs) produce anti-hyperalgesic effects by altering the metabolism of tryptophan and the accumulation of kinurenate. Such is the case of diclofenac that facilitates the accumulation of kinuric acid in plasma and kidney, apparently inhibiting its renal elimination. There is also evidence that describes a more direct participation of kinuric acid in pain processes, such as the attenuation of nociceptive responses induced by formalin and capsaicin. It has been demonstrated that kinurénico acid is able to abolish the glutamatergic stimulation in the nucleus of rafé, suggesting a modulating effect in the nociceptive processing at brain stem level. Accordingly, kinuric acid decreases nociceptive behavior in the tail flick and hot plate tests.

Unfortunately, kinuric acid does not cross the blood-brain barrier, so the best strategy to increase its concentrations at the central level is, for example, the systemic (intraperitoneal) administration of its precursor L-kinurenine, which is capable of crossing said barrier. The kynurenine by an irreversible transaminase reaction mediated by kinurenine amino transferase II (KAT-II), results in the formation of kinuric acid at the central level.

Kinurenine amino transferase II is an enzyme found mainly in the astrocytic mitochondrial fraction of the brain of mammals, suggesting that the L-kynurenine pathway is preferably carried out in the glia and not in the neurons. This has made it possible to postulate that the secretion of kinuric acid occurs from the glial cells that are in immediate vicinity towards certain specific synaptic contacts in the extracellular space. It should be noted that L-kinurenine amino transferase II is a highly selective enzyme for its substrate (L-kynurenine), compared to its low affinity for L-tryptophan, L-a-glutamate and L-aminoadipate. It is logical to think that L-kynurenine, being an intermediate metabolite in the kinurenine pathway, would not only lead to the formation of neuroprotective agents such as kinuric acid, but could also lead to the synthesis of neurotoxic metabolites such as 3-hydroxyquinurenine and later the quinolinic acid. The production of 3-hydroxykinurenine from L-kinurenine is catalyzed by kinurenine 3 hydroxylase, monoxigenase with high affinity for its substrate (Km 1 μ), suggesting that under physiological conditions it metabolizes most of kynurenine. However, it has been demonstrated that infusion via dialysis of L-kynurenine in the brain does not produce changes in the concentration of quinolinic acid in the extracellular fluid. The systemic administration of extremely high doses of L-kynurenine (600 mg / Kg) only increases the levels of quinolinic acid in the extracellular fluid by 6%, suggesting that L-kynurenine is first metabolized to kinuric acid in the central nervous system.

Probenecid (p-di-n-propylsulfamyl) benzoic acid) is a drug that was developed in the 1950s to prolong the duration of action of antibiotics, by inhibiting their excretion, by a mechanism that described originally the inhibition of renal tubule excretion and the biliary excretion of various carboxylic and sulphonic acids. Currently, probenecid is mainly used as an uricosuric agent for the treatment of gout and hyperuricemia. The probenecid is filtered at the level of the renal glomerulus and then it is secreted in the proximal convoluted tubule and finally reabsorbed at the level of the distal convoluted tubule. The anion transporter reabsorbs the uric acid from the kidney urine and returns it to the plasma. The probenecid interferes with that carrier. Being an organic acid, probenecid binds to the renal anion transporter so that uric acid has no binding site, preventing it from returning to the blood plasma and ensuring its renal excretion. This tends to improve the levels of uric acid in the blood.

The administration of probenecid increases concentrations of kinuric acid at the central level, which helps to show that kinuric acid is purified from the central nervous system by an organic acid transporter.

According to the present invention, when administering the compositions described herein comprising a combination of an endogenous antagonist of the N-ethyl-D-aspartate receptor and an inhibitor of the organic acid transporter, such as for example L-kynurenine in combination with probenecid, the excretion of kinuric acid is reduced and its accumulation in the central nervous system is promoted, which allows efficient treatment of neuropathic pain. Different studies have reported the combination of L-kinurenine and probenecid as a therapeutic strategy in neurodegenerative processes and more recently in inflammatory pain, however, until before the present invention, there were no studies evaluating the effect of kinuric acid or, for example, L-kynurenine / probenecid combination in neuropathic pain.

After a process of inflammation or nerve damage, usually dramatic alterations occur in the somatosensory system that amplify the responses and increase the sensitivity to peripheral stimuli. This causes that the painful process is exacerbated and can be activated by innocuous stimuli (allodynia) or by low intensity nociceptive stimuli (hyperalgesia).

As can be seen below, the present invention analyzes the participation of kinuric acid in the modulation of neuropathic pain.

The present invention also allows to increase the concentrations of kinuric acid in the central nervous system by means of the systemic administration of L-kinurenine and probenecid, causing an antiallodynic effect mediated by the antagonism of the N-methyl-D-aspartate receptors in a pain model. neuropathic, which allows to provide a highly effective treatment for neuropathic pain.

One embodiment of the present invention is the determination of the antiallodynic effect of the combination L-kynurenine and probenecid.

Another additional embodiment of the present invention is the use of kinuric acid as a favorable alternative in the elimination or reduction of neuropathic pain, due to being an endogenous antagonist of the N-methyl-D-aspartate receptor, it has a low capacity to produce alterations motor Therefore, raising the concentrations of kinuric acid in the central nervous system by systemic administration of an endogenous N-methyl-D-aspartate receptor antagonist such as for example L-kynurenine in combination with an inhibitor of the organic acid transporter, such as for example probenecid, allows to provide an effective treatment for neuropathic pain.

For purposes of the present invention, the pharmaceutical compositions of the invention comprise an endogenous antagonist of the N-methyl-D-aspartate receptor and an inhibitor of the organic acid transporter, wherein the amount of the endogenous antagonist is 0.5 to 2 times the amount of the inhibitor of the transporter, with the proviso that the amount of said inhibitor that is administered is at least 14 mg / Kg of body weight.

In accordance with that described herein, the endogenous N-methyl-D-aspartate receptor antagonist can be selected from the group comprising kynurenine, L-kynurenine, N-formylkinurenine, L-tryptophan, their intermediates, metabolic precursors and mixtures thereof, although L-kynurenine is preferred, while the organic acid transporter inhibitor is selected from probenecid, Likewise, the compositions of the present invention can be designed according to the routes of administration that are suitable for the treatment of neuropathic pain, for example orally or parenterally, in which the combination of active principles (endogenous N receptor antagonist) -methyl-D-aspartate + organic acid transporter inhibitor, particularly L-kynurenine + probenecid) can be administered through compositions defined according to common and known practices in pharmacology for the design of pharmaceutical compositions, i.e., for example, in conjunction with vehicles and pharmacological agents that are convenient for its administration, as well as through the pharmaceutical form that is administered and considered convenient.

As can be seen in the examples, the amounts used of kynurenine and probenecid are higher due to the animal model used (rats), while for observe the same dose response effect in humans, the amounts tend to be 7 times lower, according to the experience of a technician with average knowledge in the field.

According to the present invention, the compositions described herein can be administered systemically to a subject suffering from neuropathic pain by means of a scheme of administration such as to treat said condition. In this sense, suitable administration schemes of the compositions of the invention comprise its administration, for example parenterally, with daily administrations of 2 to 3 times a day per Kg of weight of the subject to be treated, or for a sufficient time until the administration is effective in the treatment of neuropathic pain of the subject who suffers.

In contrast to the treatments known to date for treating neuropathic pain, the administration of the compositions of the present invention does not cause adverse effects on the motor activity of the treated subject, an undesirable effect that is always generated with the administration of, for example, other antagonists. Classical N-methyl-D-aspartate receptor, such as MK-801 (see example 5, figure 10), which allows them to have greater security in the antiallodynic effect they generate in the subject suffering from neuropathic pain.

For the purpose of illustrating the present invention, the following examples are included, without implying limitations within its scope.

Example 1. Materials and methods.

Female rats of the Wistar strain with a body weight of 140 to 160 g were used. All the animals were provided and kept in the Cinvestav's habitat (South Headquarters) under controlled air, temperature and relative humidity conditions and 12:12 h light-dark cycles with free access to water and food. All the experiments were carried out according to the guidelines on ethical aspects for the investigation of experimental pain in animals (IASP, 1983). Additionally, the Institutional Committee for the care and use of animals of Cinvestav approved the study (protocol 146-03). Each rat was used only once and was sacrificed in a chamber with CO2 at the end of each experiment.

In the present invention, the following drugs were used: L-kinurenine sulfate, probenecid and kinuric acid. All drugs were obtained from Sigma Aldrich (St.

Louis MO, USA). All drugs were prepared on the day of the experiment and in all cases they were dissolved in 0.1 N NaOH and the pH was adjusted to 8.0.

Example 2. Induction of neuropathic pain.

Induction of neuropathic pain was carried out by the surgical procedure described by Kim and Chung. Under conditions of general anesthesia (Ketamine 50 mg / Kg-Xylazine 10 mg / Kg i.p.) the animals were placed in a stereomicroscope and the spine was exposed in their lower back, in order to section the vertebra corresponding to L6 and leave exposed the left spinal nerves L5 and L6 that were linked with a 6-0 silk suture in the distal part of the dorsal root ganglion. For the falsely operated rats (Sham) the nerves were not ligated, only the surgical procedure was performed including the manipulation of said nerves without damaging them. Finally, the incision was sutured and the animals remained in recovery for 10 days, this being the time necessary for the generation of the neuropathic state. For animals that underwent intrathecal cannulation, the recovery time was only 7 days.

Example 3. Intrathecal cannulation The intrathecal cannulation was carried out according to the technique described by Yaksh and Rudy. Rats that previously underwent spinal nerve ligation were anesthetized with a mixture of Ketamine 50 mg / Kg-Xylazine 10 mg / Kg i.p. and they were placed in a stereotaxic equipment to fix the head. The occipital muscles separated from their point of attachment and retracted caudally until exposing the atlanto-occipital membrane at the base of the skull. Subsequently, a small incision was made and a polyethylene catheter of number 10 (PE-10) with a length of 7.5 cm was inserted to the lumbo-sacral level of the spinal cord. After the surgery, the rats were allowed to recover for 5 more days to subsequently administer the drugs or the vehicle. The animals that presented motor deficiencies such as paraplegia or paralysis were discarded from the study.

Example 4. Determination of tactile allodynia.

The rats were individually placed in plastic boxes with a metal mesh bottom for 30 min. The tactile threshold test induces the removal of the left leg to the be stimulated with the von Frey filaments. The stimulated area was the middle part of the plantar surface, avoiding the paw pad. The 50% withdrawal threshold was determined using the up-down method. In the up-down paradigm, the test was started with the 4.31 g filament, half of the von Frey 20 filament series, with logarithmic increments of stiffness or hardness and was ascended or descended (1.65 -6.65 g) in function of the animal's response. The withdrawal threshold was measured taking as a positive response the withdrawal of the paw stimulated in a lapse of 10 seconds. The negative response is one in which, when stimulating the leg, it was not withdrawn. After the first positive response, the stimulation was carried out another 4 times, taking a series of 6 patterns of positive and negative responses and the last filament used. The 50% response threshold was calculated using the formula: 50% Threshold (g) = (10 [xf + k6]) / 10,000 (1) where: X is the value of the last von Frey filament used (in log units); k is the correction factor based on the response patterns of the calibration table and the tabulated value based on the pattern of positive and negative responses and d that refers to the average differences between stimuli (log units).

Example 5. Determination of adverse effects.

The adverse effects derived from the antagonism of kinuric acid on the N-methyl-D-aspartate receptor by kinuric acid were determined by the evaluation of motor coordination and compared against the effects produced by a classical antagonist of the same receptor (MK-801 , 0.5 mg / Kg). To determine motor coordination, the dwell time in the Rota-rod was quantified for a maximum of 10 min at a speed of 4-40 rpm with increments of 1 rpm every 15 seconds. The animals were previously acclimated by placing them daily for 5 min for two days. On the third day, the basal motor activity of all the groups was evaluated. Subsequently, the corresponding treatment was administered to each group and the temporal course of motor activity was evaluated until 3 h (time of maximum antiallodynic effect). Each rat was evaluated three times in each time and the average of each rat was obtained in order to obtain the mean and the standard error of each group.

Example 6. Extraction of cerebrospinal fluid.

Cerebrospinal fluid was removed by puncture in the cisterna magna. The rats were anesthetized with a mixture of Ketamine 50 mg / Kg-Xylazine 10 mg / Kg i.p. and they were placed in a stereotaxic equipment to locate the cisterna magna by touch. With the help of a P50 cannula attached to a 22G thick needle, the cisterna magna was inserted and the cerebrospinal fluid was extracted in a volume of between 60 and 100 μ with a 1 ml syringe. approximately. Samples containing traces of blood were immediately centrifuged at 4 ° C and 12,000 rpm for 2 min, discarding those that did not show a transparent appearance. Subsequently, the samples were kept frozen at -20 ° C until the day of quantification.

Example 7. Determination of kinuric acid.

It was carried out in the Department of Neurochemistry of the National Institute of Neurology and Neurosurgery "Manuel Velasco Suárez", where the standardized method for the detection of kinuric acid by high resolution liquid chromatography (HPLC) is available. This method meets the requirements of linearity, precision, accuracy, limit of quantification, limit of detection and stability according to the official Mexican standard (NOM-177).

Cerebrospinal fluid samples were thawed at room temperature. To precipitate any protein residue, 20 μ? of cold methanol and centrifuged at 4 ° C and 14,000 rpm for 4 min. Finally, they were filtered through a 0.22 μ? T microfilter? and were placed in individual vials for analysis. The chromatographic analysis was carried out in a high resolution liquid chromatograph equipped with quaternary pump, auto-sampler and fluorescence and UV detectors with diode array using Chemstation version 5.0 software. 50 μ? of cerebrospinal fluid in the equipment using a mobile buffer phase of 10 mM zinc acetate (89%), 7% acetonitrile and 4% methanol at a flow rate of 1.1 ml / min and a stationary phase consisting of a column of reverse phase of 4.6 x 150 mm and particle size 3.0 μ? t ?. Detection was performed by fluorescence at a wavelength of 246 nm excitation and 400 nm emission. To achieve a better detection of the metabolite, each sample was injected together with 50 μ? from a standard kinuric acid to a known concentration in simulated cerebrospinal fluid. The total of the samples It was analyzed in 3 different runs with independent calibration curve for each one. The calculation of the concentration was done by interpolating the values of each sample in the linear regression formula of its respective calibration curve. Finally, at the concentration obtained, the value of the concentration of the added standard was subtracted to obtain the real concentration of each sample.

Example 8. Evaluation of the antiallodynic effect of the administration of the kynurenine / probenecid combination of the invention.

Dose-response curves were performed with 4 doses of L-kinurenine and 2 doses of probenecid administered via i.p. according to the scheme of figure 1.

Example 9. Evaluation of the antiallodynic effect of the spinal administration of kinuric acid.

Dose-response curves were performed with 4 doses of intrathecal kinurénico acid (i.t.) and 1 dose with the co-administration of probenecid i.p. according to the scheme of figure 2.

Example 10. Evaluation of adverse effects and determination of kinuric acid concentration by the administration of the L-kynurenine / probenecid combination of the invention.

The dose that presented the best antiallodynic effects was used and the respective determinations were made according to the scheme of figure 3.

The results were expressed as the 50% average of the withdrawal threshold (in g) ± the standard error (e.e.) for each experimental group (n = 6). Temporary courses were developed for the effects observed at the different doses and the area under the curve (ABC) was calculated by the trapezoid method. ABC was considered as a global expression of the intensity and duration of the effect.

The differences between the averages of the treated groups and their controls were determined by one-way analysis of variance (ANOVA), followed by a Student Newman Keuls (SNK) test. A p < 0.05 was considered significant.

According to the present invention, the i.p. of kynurenine at different doses (25, 50, 100 and 200 mg / Kg) or probenecid (100 mg / Kg) did not produce antialodynic effect in the model of the ligation of spinal nerves L5 and L6 (figure 4). In addition, the i.p. of the kinurenine combination (25, 50, 100 and 200 mg / Kg) with probenecid (10 mg / Kg) also did not produce antiallodynic effect (figure 5). In contrast, the i.p. of the combination of kynurenine (50, 150, 175 and 200 mg / Kg) with probenecid (100 mg / Kg) of the present invention, produced an antiallodynic effect in a dose-dependent manner. The maximum effect was reached at three hours and was retained until 24 hours after the administration of the combination (Figure 6). The area under the curve of the time course of the different treatments shows that the combination L-kynurenine (50, 150, 175 and 200 mg / Kg) / probenecid (100 mg / Kg) of the invention presents a significant difference (p <0.05) from the lowest dose, being the highest dose of the combination L-kynurenine (200 mg / Kg) / probenecid (100 mg / Kg) which has values closer to that of a normal rat (figure 7) .

To corroborate the hypothesis that kinuric acid is responsible for the observed antiallodynic effect, exogenous kinuric acid (1, 3, 10, and 30 pg) was administered spinally. Spinal administration of kinuric acid produced a dose-dependent antiallodynic effect. The maximum effect was reached after 1 h of the administration, but fell rapidly after that point and disappeared at approximately 3 h. In contrast, spinal administration of kinuric acid (10 pg) in combination with i.p. of probenecid (100 mg / Kg) produced a maximum antiallodynic effect from 0.5 h and remained until 5 h after administration (figure 8). The area under the curve of the time course of the different doses showed significant difference (p <0.05) from the lowest dose (1 pg). It was observed that the group administered with 10 pg of kinuric acid increased its area value under the curve more than twice when co-administered with probenecid (figure 9).

The i.p. of kynurenine (200 mg / kg) and probenecid (100 mg / kg) produced no adverse effects at the motor level in the spinal nerve ligation model L5 and L6 when both drugs were administered independently or in combination. In contrast, the systemic administration of MK-801 decreased motor activity. It was observed that the time course in the rota-rod was maintained in a very similar time range for all the groups evaluated, except for the group treated with MK-801, which presented shorter duration (Figure 10).

The area under the curve of the time course of the different treatments showed that the L-kinurenine / probenecid combination does not present a significant difference when compared to the rest of the groups. In contrast, the group treated with MK-801 presented a significant difference (p <0.05) in comparison with all the groups (figure 11). Additionally, the anti-lodynic effect was evaluated in the group of rats treated with MK-801 and it was observed that the dose that produces adverse effects at the motor level produces an anti-lodynic effect although this was very low and of short duration. The appearance of adverse effects correlated with the appearance of the antiallodynic effect, since at the same time that it had the shortest time in the rota-rod it has the maximum antiallodynic effect. In contrast, the dose of the kynurenine / probenecid combination had values of allodynia close to those of a normal rat at the same time without altering the activity (Figure 12).

The i.p. of the combination L-kynurenine (200 mg / Kg) / probenecid (100 mg / Kg) of the present invention was the only treatment that increased the concentration of kinuric acid in the cerebrospinal fluid. It was observed that this increase is greater than 200% compared to the group of neuropathic rats without treatment. The administration of L-kynurenine or probenecid independently did not modify the concentration of kinuric acid in cerebrospinal fluid with respect to any of the control groups (Figure 13).

Finally, it is of interest to mention that the present invention also suggests the treatment with the systemic administration of L-kinurenine / probenecid as a possible therapeutic tool for the treatment of neuropathic pain without apparent risk of adverse effects on motor activity.

Claims (15)

Claims

1. A pharmaceutical composition for the treatment of neuropathic pain, characterized in that it comprises an endogenous antagonist of the N-methyl-D-aspartate receptor and an inhibitor of the organic acid transporter, in a pharmaceutically acceptable carrier.

2. The pharmaceutical composition of claim 1, characterized in that the endogenous N-methyl-D-aspartate receptor antagonist is selected from the group comprising kynurenine, L-kynurenine, N-formylkinurenine, L-tryptophan, its intermediates, metabolic precursors and mixtures of the same.

3. The pharmaceutical composition of claim 2, characterized in that the endogenous N-methyl-D-aspartate receptor antagonist is L-kynurenine.

4. The pharmaceutical composition of claim 1, characterized in that the inhibitor of the organic acid transporter is probenecid.

5. The pharmaceutical composition of claim 1, characterized in that the amount of the endogenous N-methyl-D-aspartate receptor antagonist is 0.5 to 2 times the amount of the organic acid transporter inhibitor.

6. The pharmaceutical composition of claim 1, characterized in that it does not generate adverse effects on motor activity.

7. A method for the treatment of neuropathic pain which comprises administering to a subject suffering said pain, the composition of claim 1.

8. The method of claim 7, wherein the amount of the organic acid transporter inhibitor that is administered to the subject by the composition is at least 14 mg / Kg of body weight.

9. The method of claim 7, wherein the administration of the composition of claim X, does not generate adverse effects on motor activity.

10. The use of a combination of an endogenous antagonist of the N-methyl-D-aspartate receptor and an inhibitor of the organic acid transporter for the treatment of neuropathic pain.

11. The use of claim 10, wherein the endogenous N-methyl-D-aspartate receptor antagonist is selected from the group comprising kynurenine, L-kynurenine, N-formylkinurenine, L-tryptophan, its intermediates, metabolic precursors and mixtures thereof .

12. The use of claim 11, wherein the endogenous N-methyl-D-aspartate receptor antagonist is L-kynurenine.

13. The use of claim 10, wherein the inhibitor of the organic acid transporter is probenecid.

14. The use of claim 10, wherein the amount of the endogenous N-methyl-D-aspartate receptor antagonist is 0.5 to 2 times the amount of the organic acid transporter inhibitor.

15. The use of claim 10, wherein the amount of the organic acid transporter inhibitor that is administered to a subject suffering said pain by the composition is at least 14 mg / Kg of body weight.