Ibudilast as a neuroprotector in patients with early-stage parkinson's disease

Abstract

The present invention describes ibudilast for use as a neuroprotective drug for preventing and / or treating Parkinson's disease in a subject, preferably human, in an early stage of Parkinson's disease. The invention also relates to a composition comprising same and to a method for selecting therapeutic agents useful in treating and / or preventing Parkinson's disease in a subject in an early stage of Parkinson's disease.

Description

[0001] IBUDILAST AS A NEUROPROTECTOR IN PATIENTS WITH EARLY STAGE PARKINSON'S DISEASE

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention falls within the field of Medicine. Specifically, it relates to the prevention and treatment of neurodegenerative diseases, and more specifically, Parkinson's disease.

[0005] PRIOR ART

[0006] Synnucleopathies comprise a group of diseases in which the main pathological feature is the presence of insoluble fibrillar aggregates of α-synuclein, called Lewy bodies, in specific brain cell populations. The main synnucleopathies are Parkinson's disease (PD), Lewy body dementia, and multiple system atrophy. In PD, Lewy bodies are present in the substantia nigra and the locus coeruleus, an anatomical region in the brainstem. α-Synuclein is a small protein of 140 amino acids, expressed from five exons of the SNCA gene, located on the long arm of chromosome 21.

[0007] Parkinson's disease (PD) is the second most prevalent neurodegenerative disease worldwide. Clinically, it is characterized by parkinsonism, defined as bradykinesia (slowness of movement) along with rigidity (increased muscle tone) and / or tremor. Pathologically, it is characterized by the degeneration of dopaminergic neurons in the substantia nigra, and the consequent dopamine deficiency in the nigrossional pathway, responsible for the motor symptoms, and some non-motor symptoms, of the disease.

[0008] The nigrostriatal pathway is the part of the extrapyramidal nervous system that controls movement and various cognitive functions. It connects two main brain regions: the substantia nigra and the striatum. The substantia nigra is divided into two subregions: the pars compacta and the pars reticulata, both with specific functions in motor information processing. In Parkinson's disease (PD), the substantia nigra pars compacta is affected, and the most severe neuronal loss occurs in the ventrolateral region. The striatum is composed of two interconnected subregions that are part of the basal ganglia system: the caudate nucleus and the putamen. These structures are essential for planning and executing precise movements, as well as for integrating sensory and motor information in the motor control process.A distinctive feature of the nigrostriatal pathway is the presence of dopaminergic neurons in the substantia nigra. These neurons are responsible for the synthesis and release of dopamine, a crucial neurotransmitter in the regulation of motor and cognitive functions. Dopamine acts as a chemical messenger in the nigrostriatal pathway, modulating the activity of neurons in the striatum and facilitating the transmission of movement-related signals. As previously mentioned, in Parkinson's disease (PD), there is a progressive degeneration of these dopaminergic neurons. The etiology of PD is likely multifactorial, resulting from a variable combination of genetic and environmental factors, with aging being the main risk factor for its development.This combination of factors triggers various pathophysiological processes at the cellular and molecular level (aberrant aggregation of α-synuclein, inflammation, mitochondrial dysfunction, among others) that would ultimately culminate in neuronal death.

[0009] Currently, treatment for Parkinson's disease (PD) is symptomatic, aiming to provide exogenous dopamine to alleviate the disease's symptoms. However, there is no effective treatment capable of halting the progressive loss of dopaminergic neurons in patients with PD.

[0010] Glial cell line-derived neurotrophic factor (GDNF) has been extensively studied for both its potential therapeutic effects and its neuroprotective properties. GDNF is a small protein encoded by the GDNF gene that potently promotes the survival of many types of neurons, with a notable capacity to support the survival of dopaminergic neurons, the neuronal populations that die during Parkinson's disease (PD). The neuroprotective role of GDNF in the nigrosstal pathway is well established; it is able to reduce overall neuronal loss during development, rescue cells from axotomy-induced cell death, and prevent chronic degeneration. GDNF was originally isolated from a rat glioma cell line, and the original study demonstrated its ability to promote the survival and growth of cultured dopaminergic neurons.GDNF acts primarily on dopaminergic (DA) terminals in the putamen and caudate nucleus, also known as the striatum, which originate from the substantia nigra pars compacta. GDNF activates the RET-GFRa1 receptor complex and triggers intracellular second messengers, mainly through the MAPK / ERK and PI3K / AKT pathways, activating trophic pathways in dopaminergic neurons and promoting dopamine synthesis [1]. Administration of GDNF to the brain has been shown to protect dopaminergic neurons from selective degeneration induced by toxins in several animal models, including rodents and monkeys [2].

[0011] To date, clinical trials of exogenous GDNF treatment for PD have yielded mixed results. The main limitation is that GDNF does not cross the blood-brain barrier, so these trials have relied on administering GDNF directly into the brains of PD patients using intracerebral pumps [3] or gene therapy [4]. Some studies reported positive effects on motor symptoms and neuronal survival [3, 5], while others showed no significant improvements or raised safety concerns [6, 7].

[0012] Other researchers have studied methods to increase endogenous GDNF production using gene therapy techniques (introducing genes into cells to produce GDNF), but these approaches are still in the experimental phase and require further research.

[0013] It is worth noting that GDNF is primarily produced by parvalbumin (PV) GABAergic interneurons, a subpopulation of inhibitory interneurons, in the mouse striatum [8]. Its production is necessary for the survival of the nigrostriatal pathway in the adult brain [9]. The use of pharmacological compounds has been proposed to specifically increase the synthesis and release of GDNF from PV interneurons. To this end, a comparison of the transcriptomic signature was carried out between PV interneurons (from the striatum) that express GDNF and other PV interneurons (from the cerebral cortex) with the same embryonic origin but lacking GDNF. This study in mice revealed specific receptors and enriched pathways that could potentially be modulated to activate GDNF synthesis

[0010] . It was established that the intracellular cyclic adenosine monophosphate (cAMP) pathway could be a relevant pharmacological target for modulating GDNF synthesis.

[0014] Phosphodiesterase (PDE) inhibitors are a class of drugs that prevent the degradation of cAMP and cyclic guanosine monophosphate (cGMP), thereby increasing their intracellular levels. These PDE inhibitors have been proposed as potential treatments for various neurodegenerative diseases

[0011] , including Parkinson's disease

[0012] ,

[0013] . However, only one study

[0014] has been found to directly link PDE inhibitors to the regulation or modulation of GDNF.

[0015] Ibudilast is an inhibitor of several human PDE isoforms: PDE3, PDE4, PDE10 and PDE11

[0015] , which can cross the blood-brain barrier, and has therefore been proposed as a therapeutic option to improve the treatment of neurodegenerative diseases

[0016] .

[0016] In preclinical models, ibudilast has been described as having neuroprotective effects in various in vitro neurodegeneration models

[0017] , as in vivo

[0018] ,

[0017] In the clinical setting, ibudilast is approved for use in respiratory diseases in Japan. Its use has also been evaluated in other systemic pathologies associated with inflammation and in neurodegenerative diseases such as multiple sclerosis and amyotrophic lateral sclerosis, and for the relief of neuropathic pain. However, to date, although preclinical models suggest a potential benefit of ibudilast in PD, the biological effect and efficacy of ibudilast have not been evaluated in clinical trials in patients with PD.

[0018] On the other hand, another aspect to consider is at what stage of the disease the administration of ibudilast could be most effective in patients with PE.

[0019] In this context, some preclinical studies suggest that administering ibudilast in the early stages of Parkinson's disease (PD) may not be effective. A study

[0014] conducted in mice with acute administration of 1-methyl-4-phenyl,6-tetrahydropyridine (MPTP) as a model of dopaminergic neuron degeneration showed that ibudilast had no protective effect during the acute phase of injury. The study revealed an increase in mRNA levels encoding GDNF, but no significant neuroprotective effects were observed in the nigrostriatal pathway according to the parameters analyzed: dopamine and metabolite content, and tyrosine hydroxylase levels in the striatum. However, a decrease in the production of neuroinflammatory factors was detected, accompanied by a reduction in astrocyte markers.It is important to emphasize that MPTP-induced injury, particularly in the acute phase following administration of the compound, is a suitable model of dopaminergic neuronal degeneration for assessing the early stages of neurodegeneration in Parkinson's disease. The anti-inflammatory and neurotrophic supportive effects of ibudilast may contribute to an improved quality of life for patients in advanced stages, but they do not appear to play a role in preventing or protecting against the early neuronal damage characteristic of disease onset. The experimental protocol used in this study was based on a model of acute nigrostriatal pathway degeneration, with subcutaneous administration of ibudilast at doses of 10 to 50 mg / kg, and a different approach to measuring nigrostriatal impairment compared to the experiments conducted by the applicants for this patent, which are discussed below.

[0020] Thus, the effects of ibudilast only appeared relevant in the stages following MPTP administration that correspond to the intermediate and advanced stages of PD according to the Neuronal Disease Staging System by α-synuclein (NSD-ISS) classification

[0015] , specifically between stages 3 and 5. According to these studies, the use of ibudilast would not be appropriate for the early stages of the disease, which correspond to stages 1 and 2 of PD development according to the NSD-ISS system. The NSD-ISS system is based on biological anchors and the degree of functional impairment caused by clinical signs or symptoms.Stages 0-1 occur without signs or symptoms and are defined by the presence of pathogenic variants in the SNCA gene (stage 0), S alone (stage 1A), or both S and D (stage 1B), where S corresponds to the presence of pathological n-asyn species detected in vivo and D to dopaminergic neuronal dysfunction. The presence of clinical manifestations marks the transition to stage 2 and beyond. Stage 2 is characterized by subtle signs or symptoms, but without functional impairment. Stages 2B to 6 require both S and D, as well as an increase in stage-specific functional impairment.

[0021] In the intermediate and advanced stages of PD development, the focus is on reducing the progression of dopaminergic dysfunction and symptoms, whereas in the early stages there is still no clinically significant neuronal dysfunction or functional impairment, suggesting a window of opportunity for the use of ibudilast due to its anti-inflammatory and neuroprotective action

[0016] . These early stages are characterized by focusing on the early identification of pathological biomarkers, such as α-synuclein aggregation and incipient dopaminergic dysfunction, without extensive inflammation or neuronal damage, where ibudilast could have an active role.

[0022] DESCRIPTION OF THE INVENTION

[0023] Once it was established that ibudilast crosses the blood-brain barrier after systemic administration and that it exerts a positive action on striatal GDNF synthesis, the effect of ibudilast was tested in a preclinical model of dopaminergic neuron degeneration, which corresponds to the early stages of patients with PD.

[0024] The results obtained demonstrate a neuroprotective effect of ibudilast in mice treated with the neurotoxin MPTP, an animal model of the early stages of nigrostriatal degeneration, and that this neuroprotective effect on dopaminergic neurons occurs during the acute phase after treatment with MPTP.

[0025] Therefore, the administration of ibudilast is proposed as a neuroprotective agent in PD, mainly based on the increase in endogenous GDNF and also on the reduction of neuroinflammation, oxidative stress and mitochondrial dysfunction, so that it can slow the degeneration of dopaminergic neurons and modify the course of the disease and even prevent it.

[0026] In addition to ibudilast, several selective PDE inhibitors were tested to try to promote GDNF expression in the MPTP-treated mouse striatum, including cilostamide, roflumilast, and PF0518099. Although roflumilast and PF0518099 successfully stimulated GDNF synthesis in brain slices maintained under in vitro survival conditions, they did not demonstrate efficacy in live animal models. Ibudilast was the only PDE inhibitor with a stimulatory effect on GDNF production in the preclinical (mouse) in vivo model.

[0027] The effect of ibudilast was particularly relevant in the initial moments following MPTP administration, during the acute phase of MPTP-induced injury. This acute phase of MPTP-induced injury corresponds to a model for the early stages of neurodegeneration in Parkinson's disease (PD), and therefore ibudilast is proposed as a treatment in the early stages of PD, especially for stages 1 and 2 according to the previously described NSD-ISS staging system.

[0028] Ibudilaste is proposed as a treatment for patients with nigrostriatal degeneration in the early stages of Parkinson's disease (PD), specifically stages 1 and 2 according to the NSD-ISS staging system, particularly in patients with early signs of synucleopathy (detected by cerebrospinal fluid [CSF] analysis) and striatal involvement (detected by single-photon emission computed tomography [SPECT] of the brain). It is also proposed as a preventative treatment to avoid the disease manifesting or progressing to more severe forms.

[0029] Ibudilast, formerly known as MN-166, is an oral drug with activity in several biological pathways. It is a PDE inhibitor that has been studied for various therapeutic applications, primarily for its anti-inflammatory, neuroprotective, and immunomodulatory properties. It has been used in Japan and Korea for several years to treat bronchial asthma and vertigo symptoms, but more recently it has attracted interest for its potential in other areas of medicine, especially in neurology, due to its neuroprotective effect in multiple sclerosis and its use in the treatment of drug addiction. Currently, ibudilast is approved for the treatment of non-neurological diseases and is in clinical trials for the treatment of other diseases (amyotrophic lateral sclerosis, among others).

[0030] Its activity is based on the suppression of three cytokines that promote inflammation: IL-1B, TNF-α and IL-6.

[0031] Ibudilast is the compound with formula (I), CI4H 18 N2O (CAS Number: 50847-11-5) shown below:

[0032]

[0033] Formula (I) Therefore, in a first aspect of the present invention, ibudilast is described for use as a neuroprotective medicament in subjects in the early stages of Parkinson's disease. Specifically, ibudilast is described for use as a medicament for the prevention and / or treatment of Parkinson's disease in human subjects in the early stages of Parkinson's disease, understood as subjects who are in Phase I or II of the Neuronal α-synuclein Integrated Staging System (NSD-ISS) or with less than 5 years since the onset of the first motor symptoms of Parkinson's disease.

[0034] A second aspect of the invention relates to a composition comprising ibudilast for use in the prevention and / or treatment of Parkinson's disease in patients in the early stage of Parkinson's disease.

[0035] Preferably, said composition is a pharmaceutical composition and more preferably, further comprises a pharmaceutically acceptable vehicle and / or pharmaceutically acceptable excipients. Preferably, the subject is a human.

[0036] As used herein, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, relief, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions.

[0037] The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or assists in its manufacture by providing consistency, shape, flavor, or any other specific functional characteristic. Thus, excipients may have the function of binding the ingredients together, such as starches, sugars, or cellulose; a sweetening function; a coloring function; a protective function, such as isolating it from air and / or moisture; a filler function for a tablet, capsule, or any other form of formulation; a disintegrating function to facilitate the dissolution of the components and their absorption; and include other types of excipients not mentioned in this paragraph.A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable") refers to any substance, or combination of substances, known in the pharmaceutical industry, used in the manufacture of dosage forms and includes, among others, solids, liquids, solvents, or surfactants. The carrier may be an inert substance or have an action similar to any of the compounds of the present invention, serving to facilitate the incorporation of the drug, as well as other compounds, allowing for improved dosage and administration, or providing consistency and form to the pharmaceutical composition. When the dosage form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound in question is permitted and evaluated to be harmless to the organisms to which it is administered.

[0038] The pharmaceutical composition of the invention can be administered via any route of administration, and as such, said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intravenously, or intraperitoneally.

[0039] The pharmaceutical composition may also include another active ingredient or compound useful in the treatment of PD. The pharmaceutical composition may consist of a single composition or separate compositions.

[0040] The pharmaceutical composition may include an effective amount of ibudilast.

[0041] The term "effective amount" used herein refers to an amount sufficient to promote the synthesis of the GDNF protein and thus prevent or treat PD in an individual at an early stage of disease development.

[0042] The effective dose can be appropriately selected for each individual by a qualified healthcare professional, taking into account the severity of the disease, the patient's age, body weight, health conditions, sex, drug sensitivity, duration of administration, route of administration, excretion rate, treatment duration, and other factors, including whether the drug is used in combination with or concurrently with other pharmaceutical formulations, and other factors known in the field of medicine. Therapy is considered "personalized" when the compound administered to an individual to treat a disease is specifically tailored to the genotypic and phenotypic characteristics of the individual being treated, thus avoiding the waste of time with ineffective therapies.

[0043] In another aspect of the invention, the administration regimen of ibudilast is daily. Preferably, the daily dose of ibudilast is at least 1 mg / day, preferably 5 mg / day, 10 mg / day, 15 mg / day, 20 mg / day, 25 mg / day, 30 mg / day, 35 mg / day, 40 mg / day, 45 mg / day, 50 mg / day, 55 mg / day, 56 mg / day, 57 mg / day, 58 mg / day, 59 mg / day, 60 mg / day, 61 mg / day, 62 mg / day, 63 mg / day, 64 mg / day, 65 mg / day, 70 mg / day, 75 mg / day, 80 mg / day, 85 mg / day, 90 mg / day, 95 mg / day, 96 mg / day, 97 mg / day, 98 mg / day, 99 mg / day, 10 ... mg / day, 101 mg / day, 102 mg / day, 103 mg / day, 104 mg / day, 105 mg / day, 110 mg / day, 115 mg / day, 120 mg / day, 125 mg / day, 130 mg / day, 135 mg / day, 140 mg / day, 145 mg / day, 150 mg / day, 155 mg / day, 160 mg / day, 165 mg / day, 170 mg / day, 175 mg / day, 180 mg / day, 185 mg / day, 190 mg / day, 195 mg / day, 200 mg / day, or doses higher than 200 mg / day.

[0044] In a preferred embodiment, the ibudilast dosage regimen is 60 mg / day, which is considered a moderate dose. Alternatively, in another embodiment of this aspect of the invention, the ibudilast dosage regimen is 100 mg / day, which is considered a high dose.

[0045] Preferably, the administration of daily ibudilast doses is divided into two doses per day.

[0046] Therefore, in a preferred embodiment of the pharmaceutical composition of the invention, the effective amount of ibudilast in the composition is at least 1 mg, preferably 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 40 mg, 45 mg, 46 mg, 47 mg, 48 mg, 49 mg, 50 mg, 51 mg, 52 mg, 53 mg, 54 mg, 55 mg, 56 mg, 57 mg, 58 mg, 59 mg, 60 mg, 61 mg, 62 mg, 63 mg, 64 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 96 mg, 97 mg, 98 mg, 99 mg, 100 mg, 101 mg, 102 mg, 103 mg, 104 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, or higher.

[0047] Even more preferably, the effective amount of ibudilast in the composition is 30 mg, 50 mg, 60 mg, or 100 mg, according to the preferred ibudilast administration regimens described above. The total duration of treatment with ibudilast is preferably 12 months. More preferably, follow-up is performed for at least 6 months.

[0048] The term "prevention", as used herein, refers to the ability of the compound of the invention or the pharmaceutical composition of the invention to prevent, minimize, or hinder the progression of PD.

[0049] The term "treatment", as used herein, refers to the ability of the compound of the invention or the pharmaceutical composition of the invention to minimize, reduce or totally or partially reverse the effects of PD.

[0050] The term "patient," "individual," or "subject," as used herein, refers to any animal, preferably a mammal, preferably primates and human beings. In a preferred embodiment, the subject is a human being.

[0051] A method for selecting therapeutic agents useful in the treatment and / or prevention of PD in a human subject at an early stage of PD is also described, comprising: a) determining the level of GDNF production at a set concentration of the compound to be analyzed or in the absence of said compound, and b) determining the level of GDNF production at a concentration of the compound to be analyzed different from that of a), such that compounds capable of increasing the level of GDNF production are identified as therapeutic agents against PD in human subjects at early stages of PD.

[0052] Finally, a method for selecting PD subjects who are eligible for treatment with Ibudilast is described, comprising: a) performing cerebrospinal fluid analysis on a previously isolated sample from a subject to detect early signs of synucleopathy, and b) performing a single-photon emission computed tomography (SPECT) scan of the brain to assess striatal involvement, where the subject is selected as eligible for treatment with Ibudilast when they exhibit early signs of synucleopathy and striatal involvement.In a preferred embodiment of this method, the detection of synucleopathy in cerebrospinal fluid isolated from the subject is carried out by an α-synuclein seed amplification assay, immunohistochemistry, biomarkers based on modifications of α-synuclein present in cerebrospinal fluid, measurement of α-synuclein-associated exosomes in cerebrospinal fluid, or any combination thereof.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by a person skilled in the art to which this invention pertains. Similar or equivalent methods and materials to those described herein may be used in the practice of the present invention. Throughout the description and claims, the word "comprises" and its variations are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will be apparent to those skilled in the art upon examination of the description or may be discovered through the practice of the invention. The following examples and drawings are provided for illustrative purposes and are not intended to limit the scope of the present invention.

[0054] DESCRIPTION OF THE FIGURES

[0055] Figure 1: Stimulation of GDNF synthesis in the mouse striatum by pharmacological inhibition of different phosphodiesterases. (A) Schematic of the intracellular cyclic AMP (cAMP) / protein kinase A (PKA) signaling pathway involved in the regulation of Gdnf gene expression in parvalbumin (PV) skeletal interneurons. AC: adenylyl cyclase; CRE: cAMP response element; CREB: CRE-binding protein; GPCR: G protein-coupled receptor. (B) Effect of ex vivo PKA activation using dibutyryl cAMP (dbcAMP) on Gdnf mRNA levels in skeletal slices. (C) Comparative expression levels of several PDE-encoding genes and other reference genes in PV skeletal interneurons, with arrows indicating the PDEs targeted by the selective inhibitors used in this study. (D) Effect of increasing concentrations of cilostamide (CILO) on Gdnfen mRNA levels in ex vivo skeletal tissue.(E) Dose-dependent effect of roflumilast (ROFLU) on Gdnf mRNA levels after 5 hours of ex vivo incubation. (F) Measurement of natal Gdnf mRNA levels at 4 and 8 hours after injection of ROFLU at a dose of 1 mg / kg body weight. (G) Gdnf mRNA levels in natal sections after 5 hours of incubation with varying concentrations of PF05180999 (PF05). (H) Direct effect of PF05 on PV interneurons in the presence of tetrodotoxin (TTX; 1 pM) to inhibit presynaptic input. (I) Natal Gdnf mRNA levels at 4 and 8 hours after administration of PF05 at 10 mg / kg body weight. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using a two-tailed Student's t-test (B), a two-tailed repeated Student's t-test (G), or one-way ANOVA with Tukey's multiple comparison test (D, E, F, H, I).P values ​​are reported when statistical significance is less than 0.05.

[0056] Figure 2: Ibudilast-induced stimulation of GDNF synthesis in mouse striatum. (A) Bar graph illustrating Gdnf mRNA levels in striatal tissue ex vivo after a 5-hour incubation with increasing concentrations of ibudilast (IBU). (B) Striatal sections incubated with 50 pM ibudilast in the presence of 1 pM tetrodotoxin (TTX) to block indirect afferent input.

[0057] (C) The ibudilast-induced increase in Gdnf mRNA levels is blocked by 1 pM 666-15, a CREB inhibitor. (D) Measurement of Gdnf mRNA levels in the striatum in vivo 4 hours after intraperitoneal administration of 10 mg / kg of ibudilast. (E) Striatal GDNF protein content measured by ELISA. Error bars represent the standard error of the mean (SEM). Statistical analyses include one-way ANOVA with Tukey's multiple comparison test (A, B, C) and two-tailed Student's t-test (D, E), with p-values ​​shown for each graph.

[0058] Figure 3: Effect of systemic ibudilast treatment on the nigrostriatal dopaminergic pathway in MPTP-induced neurodegeneration in mice. (A) Representative fluorescent micrographs with tyrosine hydroxylase (TH) immunostaining in 25 µm coronal sections of the midbrain, showing the substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) in mice treated for 4 weeks with saline, ibudilast, MPTP, or ibudilast + MPTP.

[0059] (B) Quantitative analysis of TH+ (dopaminergic) neurons in the SNpc, arranged along the anteroposterior axis. (C) Bar chart showing the mean total number of TH+ cells. (D) Representative sections of the coronal hemencephalon with TH staining in the striatum for each treatment group. (E) Bar chart with individual data points indicating the relative intensity of TH staining in the dorsal striatum under all treatment conditions. (F) Vertical graphs of striatal dopamine levels measured by HPLC, normalized to tissue weight. Error bars represent the standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA with Tukey's multiple comparison test, with p-values ​​for results of statistical significance less than 0.05.

[0060] DETAILED DESCRIPTION OF THE INVENTION

[0061] Mice

[0062] The animals used were bred at the Oscar Pintado Animal Research Center of the University of Seville and transferred to the Animal Facility of the Institute of Biomedicine of Seville one week prior to the experiments. Both facilities maintained controlled conditions with a temperature of 22 ± 1°C, a 12-hour light / 12-hour dark cycle, and ad libitum access to food and water. Male C57BI / 6J mice, 30–35 days old, were used for the ex vivo experiments, and 2–3 months old for the in vivo studies. All mice were cared for in accordance with European Council Directives 86 / 609 / EEC and 2010 / 63 / EU on the Care and Use of Laboratory Animals. The procedures were approved by the Ethics Committee for the Care and Use of Animals of the Virgen del Rocío University Hospital / Institute of Biomedicine of Seville and authorized by the Regional Government of Andalusia (project no. 30 / 03 / 2021 / 039).

[0063] Compounds

[0064] The toxins used in the assays were tetrodotoxin (TTX) (1069, Tocris) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (M0896, Sigma).

[0065] The PDE inhibitors tested were: cilostamide (CILO) (0915, Tocris), ibudilast (IBU) (1694, Tocris), roflumilast (roflu) (6641, Tocris), PF05180999 (PF05) (6405, Tocris).

[0066] Other compounds used in this study were: 666-15 (5661, Tocris) and dibutyryl cyclic AMP (dibutyryl-cAMP or dbcAMP) (D0627, Sigma).

[0067] The hydrophobic compounds were reconstituted in dimethyl sulfoxide (DMSO) and the water-soluble reagents were prepared in sterile 0.9% saline solution.

[0068] Preparation of ex vivo brain sections

[0069] Male mice aged between P30 and P35 were deeply anesthetized by intraperitoneal injection of thiobarbital (B. Braun, Spain) and transcardially perfused with iced cutting solution (ICS) composed of 222 mM sucrose, 11 mM glucose, 3 mM KCl, 1 mM NaH2PO4, 26 mM NaHCO3, 7 mM MgCl2, and 0.5 mM CaCl2, saturated with a gas mixture of 95% O2 and 15% CO2. After perfusion, the brain was rapidly extracted, placed in an ICS, and cut into 250 µm thick coronal sections using a vibratome (VT1200S, Leica Microsystems) equipped with an anti-vibration device. Four or five sections containing the striatum were split in half (into hemispheres) and incubated in artificial cerebrospinal fluid (aCSF) containing 4 mM sucrose, 10 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 1 mM MgCl2, 2.5 mM CaCl2, and 124 mM NaCl.Up to three brains (six hemispheres) were processed per experiment.

[0070] Hemicerebral sections were incubated in a six-well BSK6-6 Brain Slice Keeper (AutoMate Scientific, Berkeley, CA), with each well filled with 4 mL of oxygenated aCSF (continuously bubbled with 95% O2 / 5% CO2). The device was placed in a water bath to maintain a constant temperature of 36°C. After a 30-minute acclimation period, the aCSF was replaced with pre-warmed oxygenated aCSF containing the drug compounds being tested or the control solution (DMSO, if the drug had been prepared in DMSO). The sections were incubated for 5 hours, after which the stalactites were dissected from the surrounding tissue and stored at -20°C in RNAiAter solution (AM7020, Invitrogen).

[0071] In vivo drug treatment procedure

[0072] Mice received intraperitoneal injections of the test drugs, diluted in sterile 0.9% saline solution according to their body weight. For drugs prepared in DMSO, the injectable solution was freshly prepared from the stock solution just before administration. Control groups received the same volume of DMSO diluted in saline or saline alone. Animals were euthanized at different time points after injection with a lethal dose of sodium thiopental (Thiobarbital, B. Braun, Spain). The brain was rapidly removed, rinsed in iced sterile PBS made with RNase-free water, and the two striata were dissected on an iced sterile plastic plate. Tissue samples were also collected from the cortex for GDNF protein analysis. Each tissue fragment was placed in a microcentrifuge tube, frozen in liquid nitrogen, and stored at -80°C.

[0073] RNA preparation and quantitative real-time RT-PCR (qPCR)

[0074] Total RNA was extracted from streak samples using the TRIzol method (Life Technologies) according to the manufacturer's instructions. An equal amount of RNA (600 ng) was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR (qPCR) was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems) with TaqMan Gene Expression Assays (Thermo Fisher Scientific). The TaqMan probes used in this study were Gdnf (Mm00599849_m1) and Hmbs (Mm00660262_g1), with Hmbs serving as an internal control to normalize the input RNA and perform relative quantification using the AACt method.

[0075] Protein extraction and GDNF assay

[0076] Striatum and cortex samples were homogenized by sonication in 400 ml of lysis buffer (pH 7.4) containing 25 mM Tris, 50 mM p-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 100 pg / ml PMSF, 1% (v / v) protease inhibitor cocktail (P8350, Sigma), 1% (v / v) phosphatase inhibitor cocktail (P0044, Sigma), and 1% Triton X-100. The homogenates were incubated in the lysis buffer. The homogenates were incubated on ice for 15 minutes, after which they were centrifuged to collect the supernatant. The protein content in the lysates was determined using the Lowry method (DC Protein Assay, Bio-Rad), with absorbance measured at 750 nm using a multiplate spectrophotometer (Multiskan Spectrum, Thermo Fisher Scientific).Protein concentrations were adjusted to 2.7 mg / ml using lysis buffer, and samples were stored at -80°C.

[0077] Striatal GDNF protein levels were measured using a commercial ELISA kit (GDNF Emax Immunoassay System, Promega). For the assay, 50 µl of protein lysate (containing 67 pg of total protein) were used, following the manufacturer's protocol with minor modifications. To eliminate nonspecific signals, cortical protein lysate, where GDNF mRNA is undetectable, was used to account for nonspecific binding in the GDNF ELISA, as previously described

[0019] . The final reaction was measured at 450 nm using spectrophotometry, and the GDNF protein concentration was expressed as pg / mg of total protein for comparative analysis.

[0078] MPTP and ibudilast treatment

[0079] In this experiment, eight-week-old male C57BI / 6J mice were used. Each mouse underwent two rounds of injections, administered three times per week for one month (a total of 12 rounds of injections). Ibudilast or vehicle was first injected intraperitoneally (ip), followed by a subcutaneous (sc) injection of MPTP or saline solution four hours later.

[0080] Ibudilast was freshly prepared prior to administration by diluting a 20 mg / ml stock solution in DMSO at a 1:10 ratio, resulting in a working concentration of 2 mg / ml (with DMSO and saline mixed at a 1:9 ratio). Animals were administered 100 ml per 20 grams of body weight, corresponding to a dose of 10 mg / kg. For the vehicle group, animals received 10% DMSO in saline at the same ratios. MPTP solution was prepared at a concentration of 8 mg / ml in sterile saline and administered subcutaneously at a dose of 20 mg / kg, injected into the back of the neck. Control animals received only saline. Each mouse was euthanized with sodium thiopental, and the brain was rapidly removed and divided in half.The left hemisphere was fixed in cold 4% paraformaldehyde (PFA) in PBS at 4°C for 24 hours, then cryoprotected in a 30% sucrose / PBS solution overnight at 4°C, embedded in OCT compound, frozen in dry ice, and stored at -80°C. The striatum of the right hemisphere was dissected, weighed, frozen in liquid nitrogen, and stored at -80°C until later use.

[0081] Immunohistofluorescence and microscopic analysis

[0082] Coronal sections 25 µm thick were cut through the striatum and midbrain of each hemisphere using a cryostat (Leica) and collected into three sets of free-floating sections in PBS, each set containing one section from every three collected. The sections were then stored in an antifreeze solution (0.9% w / v NaCl, 30% w / v sucrose, 1% w / v polyvinylpyrrolidone [9003-39-8, Sigma], 30% v / v ethylene glycol in PBS) at -20°C until use. Before staining, the antifreeze-stored sections were thoroughly washed with PBS. Primary and secondary antibodies were prepared in PBST (PBS with 0.1% Triton X-100) with 5% normal donkey serum (Sigma). To detect dopaminergic neurons, free sections were incubated with a rabbit anti-tyrosine hydroxylase antibody (Novus, NB300-109) at a dilution of 1:2500, for 24 hours at 4°C, followed by washes with PBST.The sections were then incubated for 2 hours at room temperature with a fluorescently labeled secondary antibody, anti-rabbit donkey Alexa Fluor 568 (Invitrogen, A10042), diluted to 1:1000. Finally, the sections were stained with DAPI (Sigma, D9542), washed with PBST, mounted on glass slides, and sealed with coverslips.

[0083] Tyrosine hydroxylase (TH) staining images along the anteroposterior axis of the substantia nigra were captured using a BX-61 microscope with a 20X objective, equipped with a DP70 camera (Olympus) and dichroic fluorescent filters (excitation 530–550 nm, emission 590 nm), using CellSens CELL-A software. For quantification, TH-positive cells were manually counted in 16 images covering the region of interest along the anteroposterior axis using the Adobe Photoshop counting tool.

[0084] For the innervation of TH-positive fibers in the dorsal striatum, images were acquired using a Leica Thunder microscope with Leica LAS X software, a 575 nm Texas Red filter, and a 20X objective. Six fields were captured with the same exposure and intensity settings and saved at the highest resolution in JPEG format. The original images were converted to 32-bit grayscale and analyzed using FUI software. The mean grayscale intensity of the dorsal striatum was measured to quantify the density of TH-positive fibers.

[0085] HPLC

[0086] Striatal dopamine content was quantified by high-performance liquid chromatography (HPLC) as follows: striatal tissue was dissected fresh on ice, weighed, immediately frozen in liquid nitrogen, and stored at -80 °C until further processing. Tissue samples were asonicated in 200 lp of ice-cold solution per 10 mg of tissue containing 0.1 M HClO4, 0.02% EDTA, and 1% ethanol, followed by centrifugation at 16,000 x g for 10 minutes at 4 °C. The resulting supernatants were filtered through a 10,000 Da molecular weight cutoff membrane (VS0101, Sartorius) by centrifugation at 16,000 x g for 30 minutes at 4 °C and subsequently injected into the HPLC system (ALEXYS 100; Antee, Leyden).Dopamine was separated from other catecholamines in the sample using a 3 pm C-18 column (ALB-215; Antee Leyden) and detected electrochemically with a glassy carbon electrode paired with an in situ ISAAC reference electrode (Antee Leyden). Endogenous striatal dopamine levels were quantified against a standard curve obtained with standard dopamine (H8502, Sigma), with concentrations expressed as pmol of dopamine per mg of wet striatum.

[0087] Data analysis

[0088] Data analysis and graphical representation were performed using Prism 9.0 (GraphPad Software), and figures were created using Illustrator 2021 (Adobe). The normality of the data distribution was assessed using the Shapiro-Wilk test. For comparisons between two datasets, an unpaired Student's t-test was applied. When the control and treatment samples were obtained from the same brain, paired t-tests were used for ex vivo datasets. For multi-group analyses, one-way ANOVA followed by Tukey's post-hoc test was used. For comparisons of multiple datasets with a single control group, Dunnett's post-hoc test was applied. Data are presented as mean ± SEM, with statistical significance set at p < 0.05.

[0089] Pharmacological inhibition of striatal phosphodiesterases.

[0090] To evaluate the effect of striatal phosphodiesterase (PDE) inhibition, brainstem slices were used under ex vivo conditions in oxygenated artificial cerebrospinal fluid maintained at 36°C, as previously described

[0010] . Given the established role of the cAMP / PKA signaling pathway in regulating Gdnf gene expression (Fig. 1A), dibutyl-cAMP (dbcAMP), a cell-permeable PKA-activating analog of cAMP, was applied to the brainstem slices. Incubation with 1 mM dbcAMP for 5 hours resulted in a marked increase in striatal Gdnf mRNA levels (Fig. 1B), compared to control slices. In addition, previously obtained gene expression data for striatal PV neurons

[0010] were analyzed to highlight the relative expression levels of PDE subtypes (Fig. 1C).Several genes encoding PDE subtypes, such as Pdelb, Pde2a, Pde4b, Pde9a, Pde10a, and Pde6d, were observed to be expressed at levels comparable to Pvalb, which encodes parvalbumin (Fig. 1C). Additional reference genes, such as Actb, Hmbs, Gpr83, and Gdnf, were included to provide a relative scale for gene expression. Based on the established PDE gene expression profile, three specific PDE inhibitors were selected for ex vivo slice testing: cilostamide, roflumilast, and PF05180999.

[0091] Since Pde3a is the gene encoding the phosphodiesterase most specifically expressed in striatal PV interneurons

[0010] , cilostamide (CILO), a selective PDE3A inhibitor, was selected for initial testing. Hemibrain sections incubated with CILO at concentrations of 0.5 to 50 pM showed no effects on Gdnf gene expression after 5 hours of exposure (Fig. 1D).

[0092] Next, the effects of Roflumilast (Roflu), a selective PDE4 inhibitor

[0017] , were examined in ex vivo sections across a range of concentrations. A 5-hour incubation with 5 pM Roflu resulted in a significant increase in Gdnf mRNA levels compared to controls (Fig. 1E). Following these findings, the effect of Roflumilast (Roflu) on Gdnf expression was investigated in vivo. Adult male mice received a single injection of Roflu at a dose of 1 mg / kg or vehicle, as previously described

[0021] , and were sacrificed 4 and 8 hours post-injection (Fig. 1F). The results of Roflumilast administration in live mice were inconclusive.

[0093] Finally, the selective PDE2A inhibitor PF05180999 (or PF05)

[0022] was tested in ex vivo hemibrain slices at increasing concentrations of 1, 5, and 25 pM. In these experiments, control and treatment hemibrains were obtained from the same animal, allowing for direct comparison and paired t-test statistical analysis. Treatment with 1 pM PF05 had no effect on Gdnf mRNA levels, while 5 pM and 25 pM PF05 significantly increased Gdnf mRNA in a dose-dependent manner (Fig. 1G). To assess the direct effect of PF05 on PV interneurons, the experiment was repeated with tetrodotoxin (TTX). TTX, applied at a concentration of 1 pM, blocks afferent synoptic inputs, thereby eliminating any indirect effects of PDE2A inhibition on Gdnf gene expression.Stimulation of striatal Gdnf expression induced by PF05 persisted despite TTX, indicating a direct effect of PF05 on GDNF synthesis in PV interneurons (Fig. 1H).

[0094] Following these results, the effect of PF05 on Gdnf expression was investigated in vivo. Adult male mice received a single injection of 10 mg / kg of PF05 or vehicle

[0023] , and striatal tissue was collected at 4 and 8 hours post-injection. No changes in Gdnf mRNA levels were detected (Fig. 11).

[0095] Effect of ibudilast on striatal GDNF synthesis.

[0096] To stimulate Gdnf expression in the striatum of live mice, ibudilast, a broad-spectrum inhibitor targeting PDE3, PDE4, PDE10, and PDE11, was used

[0024] (Fig. 1C). Increasing concentrations of ibudilast were tested in ex vivo sections incubated for 5 hours. Concentrations of 2 and 5 pM showed no effect on Gdnf mRNA levels compared to untreated sections, while 10 pM and above significantly increased Gdnf expression (10 pM, p = 0.0038; 25 pM and 50 pM, p < 0.0001) (Fig. 2A).

[0097] TTX did not inhibit the ex vivo stimulatory effect of ibudilast on Gdnf expression (Fig. 2B), indicating a direct action on PV interneurons.

[0098] Since cAMP / PKA / CREB signaling regulates Gdnf transcription, the effect of ibudilast was evaluated in the presence of 666-15, a potent and selective inhibitor of CREB

[0025] . In this ex vivo experiment, the stimulatory effect of ibudilast on Gdnf expression was completely blocked by 10 pM of 666-15, indicating that the action of ibudilast is mediated through CREB activation (Fig. 2C).

[0099] The effect of intraperitoneal ibudilast (10 mg / kg) on ​​Gdnf mRNA levels was investigated in vivo. Mice treated with ibudilast and sacrificed after 4 hours showed a significant increase in Gdnf mRNA (p = 0.001) compared to vehicle controls (Fig. 2D), consistent with ex vivo results. However, GDNF protein levels showed only a slight and non-significant increase (p = 0.1895) after ibudilast treatment (Fig. 2E), suggesting that post-transcriptional factors may limit GDNF protein translation or stability, or prevent it from reaching peak GDNF levels. Ibudilast treatment provides partial neuroprotection in the MPTP model of PD.

[0100] To evaluate the therapeutic potential of ibudilast in Parkinson's disease (PD), its neuroprotective effects were examined in an MPTP-induced model of nigrostriatal dopaminergic neuron degeneration. Mice were pretreated with ibudilast for one week (3 administrations of 10 mg / kg i.p. ibudilast), followed by a 4-week course of ibudilast co-administered with MPTP. Ibudilast was administered 4–5 hours before each MPTP exposure to induce the first synthesis of GDNF. This experiment included four treatment conditions: saline, ibudilast alone, MPTP alone, and ibudilast + MPTP.

[0101] Immunostaining for tyrosine hydroxylase (TH) in the substantia nigra pars compacta (SNpc) showed a significant reduction of TH+ cells in the MPTP group compared with saline and ibudilast alone, indicating a substantial loss of dopaminergic neurons (Fig. 3A).

[0102] The images (Fig. 3A) reveal a higher number of TH+ cells in the IBU + MPTP group than in MPTP alone, consistent across anteroposterior SNpc sections (Fig. 3B). Unbiased TH+ cell counts confirmed a significant decrease in the MPTP group (1799 ± 177, p < 0.0001) compared with saline (4807 ± 83) and ibudilast (4772 ± 240), while ibudilast pretreatment significantly increased the number of TH+ cells compared with MPTP alone (3431 ± 219, p < 0.0001), indicating a neuroprotective effect of ibudilast (Fig. 3C).

[0103] In the dorsal striatum (dST), the primary target of SNpc dopaminergic projections, MPTP treatment led to a significant reduction in TH+ fiber density compared with the saline and ibudilast groups, with partial restoration observed in the IBU + MPTP group (Fig. 3D). Quantification of TH+ fiber optical density (in arbitrary units) yielded values ​​of 1.000 ± 0.048 for saline and 1.029 ± 0.077 for ibudilast, both significantly higher than those for MPTP alone (0.446 ± 0.041, p < 0.0001). The IBU + MPTP group showed a significant increase in fiber density (0.722 ± 0.041, p = 0.0184) compared with MPTP alone, although TH+ fiber arborization was not fully restored, indicating partial protection against MPTP-induced dopaminergic fiber loss by ibudilast (Fig. 3E).Striatal dopamine levels measured by HPLC were consistent with TH staining results (Fig. 3F). MPTP treatment significantly reduced dopamine levels compared with saline and ibudilast (p < 0.0001). Although dopamine levels in the IBU + MPTP group were significantly higher than in the MPTP-only group (p = 0.0008), they did not fully return to the levels observed in the saline or ibudilast-only groups.

[0104] In summary, these results suggest that ibudilast provides partial short-term neuroprotection in the MPTP-induced acute neurotoxicity model.

[0105] Clinical trial with ibudilast in patients with PD

[0106] A phase I / II, randomized, open-label clinical trial has been designed to evaluate the safety and tolerability of ibudilast treatment in patients with PD, and to evaluate the in vivo biological effect of ibudilast on GDNF in blood and cerebrospinal fluid, as well as on biomarkers of neurodegeneration and inflammation, and the efficacy of the treatment on the symptoms and progression of the disease as measured by clinical and neuroimaging variables.

[0107] The study design is open-label and consists of two treatment arms to which patients are randomized in a 1:1 ratio. Treatment consists of daily administration of ibudilast in two treatment groups: moderate doses of ibudilast (60 mg) or high doses of ibudilast (100 mg), both divided into two doses per day. The total duration of treatment is 12 months, with a subsequent follow-up of at least 6 months.

[0108] The study includes 30 patients with PD diagnosed according to the currently accepted criteria of the Movement Disorders Society

[0026] . Patients in early stages of the disease are included, defined as less than 5 years of evolution from the onset of the first motor symptoms.

[0109] Monitoring of the safety and tolerability variables of oral treatment is performed through: 1) Periodic clinical examination and medical history taking conducted by the neurologist during both in-person and remote visits; 2) Collection of adverse effects during in-person visit interviews to assess tolerability; 3) Monthly analytical monitoring with hematological and biochemical control. Variables for assessing the biological effect include biomarkers in blood and cerebrospinal fluid such as GDNF, alpha-synuclein, neurofilament light chain, and inflammatory markers. Treatment efficacy is evaluated using validated scales for motor symptoms, such as the Unified Parkinson's Disease Rating Scale, among others, and using neuroimaging biomarkers (cranial magnetic resonance imaging and F18-FDG PET).

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Claims

CLAIMS 1. Ibudilast for use as a neuroprotective drug for the prevention and / or treatment of Parkinson's disease in a subject at an early stage of Parkinson's disease.

2. Ibudilast for use according to the preceding claim where the subject is human.

3. Ibudilast for use according to the preceding claim, wherein the human subject is in Phase I or II of the α-synuclein neuronal disease integrated staging system (NSD-ISS).

4. Ibudilast for use according to claim 2 wherein the human subject has less than 5 years of evolution since the onset of the first motor symptoms of Parkinson's disease.

5. Composition comprising ibudilast for use according to any of claims 1 to 4.

6. Composition comprising Ibudilast for use according to the preceding claim, further comprising pharmaceutically acceptable excipients or carriers.

7. Method for selecting therapeutic agents useful in the treatment and / or prevention of Parkinson's disease in a human subject at an early stage of Parkinson's disease comprising: a) determine the level of GDNF production at a set concentration of the compound to be analyzed or in the absence of said compound, and b) determine the level of GDNF production at a concentration of the compound to be analyzed different from that of a), so that compounds capable of increasing the level of GDNF production are identified as therapeutic agents against Parkinson's disease in human subjects in early stages of Parkinson's disease.

8. A method for selecting subjects with Parkinson's disease who are candidates for treatment with Ibudilast, comprising: a) performing cerebrospinal fluid analysis on a previously isolated sample from a subject to detect early signs of synucleopathy, and b) perform a single-photon emission computed tomography scan of the brain to assess striatal involvement where the subject is selected as likely to be treated with Ibudilast when he presents early signs of synucleopathy and striatal involvement.

9. The method according to the preceding claim, wherein the detection of synucleopathy in cerebrospinal fluid isolated from the subject is carried out by means of an α-synuclein seed amplification assay, immunohistochemistry, biomarkers based on modifications of α-synuclein present in the cerebrospinal fluid, measurement of α-synuclein-associated exosomes in the cerebrospinal fluid, or any combination thereof.

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