Mitochondrial modulation with leriglitazone as a potential treatment for Rett syndrome.

BACKGROUND: Rett syndrome is a neuropediatric disease occurring due to mutations in MECP2 and characterized by a regression in the neuronal development following a normal postnatal growth, which results in the loss of acquired capabilities such as speech or purposeful usage of hands. While altered neurotransmission and brain development are the center of its pathophysiology, alterations in mitochondrial performance have been previously outlined, shaping it as an attractive target for the disease treatment. METHODS: We have thoroughly described mitochondrial performance in two Rett models, patients' primary fibroblasts and female Mecp2tm1.1Bird-/+ mice brain, discriminating between different brain areas. The characterization was made according to their bioenergetics function, oxidative stress, network dynamics or ultrastructure. Building on that, we have studied the effect of leriglitazone, a PPARγ agonist, in the modulation of mitochondrial performance. For that, we treated Rett female mice with 75 mg/kg/day leriglitazone from weaning until sacrifice at 7 months, studying both the mitochondrial performance changes and their consequences on the mice phenotype. Finally, we studied its effect on neuroinflammation based on the presence of reactive glia by immunohistochemistry and through a cytokine panel. RESULTS: We have described mitochondrial alterations in Rett fibroblasts regarding both shape and bioenergetic functions, as they displayed less interconnected and shorter mitochondria and reduced ATP production along with increased oxidative stress. The bioenergetic alterations were recalled in Rett mice models, being especially significant in cerebellum, already detectable in pre-symptomatic stages. Treatment with leriglitazone recovered the bioenergetic alterations both in Rett fibroblasts and female mice and exerted an anti-inflammatory effect in the latest, resulting in the amelioration of the mice phenotype both in general condition and exploratory activity. CONCLUSIONS: Our studies confirm the mitochondrial dysfunction in Rett syndrome, setting the differences through brain areas and disease stages. Its modulation through leriglitazone is a potential treatment for this disorder, along with other diseases with mitochondrial involvement. This work constitutes the preclinical necessary evidence to lead to a clinical trial.

Development of PPARγ Agonists for the Treatment of Neuroinflammatory and Neurodegenerative Diseases: Leriglitazone as a Promising Candidate.

Increasing evidence suggests that the peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, plays an important role in physiological processes in the central nervous system (CNS) and is involved in cellular metabolism and repair. Cellular damage caused by acute brain injury and long-term neurodegenerative disorders is associated with alterations of these metabolic processes leading to mitochondrial dysfunction, oxidative stress, and neuroinflammation. PPARγ agonists have demonstrated the potential to be effective treatments for CNS diseases in preclinical models, but to date, most drugs have failed to show efficacy in clinical trials of neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. The most likely explanation for this lack of efficacy is the insufficient brain exposure of these PPARγ agonists. Leriglitazone is a novel, blood-brain barrier (BBB)-penetrant PPARγ agonist that is being developed to treat CNS diseases. Here, we review the main roles of PPARγ in physiology and pathophysiology in the CNS, describe the mechanism of action of PPARγ agonists, and discuss the evidence supporting the use of leriglitazone to treat CNS diseases.

PPAR gamma agonist leriglitazone improves frataxin-loss impairments in cellular and animal models of Friedreich Ataxia.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is characterized by degeneration of the large sensory neurons and spinocerebellar tracts, cardiomyopathy, and increased incidence in diabetes. The underlying pathophysiological mechanism of FRDA, driven by a significantly decreased expression of frataxin (FXN), involves increased oxidative stress, reduced activity of enzymes containing iron­sulfur clusters (ISC), defective energy production, calcium dyshomeostasis, and impaired mitochondrial biogenesis, leading to mitochondrial dysfunction. The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcriptional factor playing a key role in mitochondrial function and biogenesis, fatty acid storage, energy metabolism, and antioxidant defence. It has been previously shown that the PPARγ/PPARγ coactivator 1 alpha (PGC-1α) pathway is dysregulated when there is frataxin deficiency, thus contributing to FRDA pathogenesis and supporting the PPARγ pathway as a potential therapeutic target. Here we assess whether MIN-102 (INN: leriglitazone), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid ß-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.

Clinical pharmacokinetics of leriglitazone and a translational approach using PBPK modeling to guide the selection of the starting dose in children.

Leriglitazone is a unique peroxisome proliferator-activated receptor-gamma (PPARγ) agonist that crosses the blood-brain barrier in humans and clinical trials have shown evidence of efficacy in neurodegenerative diseases. At clinical doses which are well-tolerated, leriglitazone reaches the target central nervous system (CNS) concentrations that are needed for PPARγ engagement and efficacy; PPARγ engagement is also supported by clinical and anti-inflammatory biomarker changes in the Cerebrospinal fluid in the CNS. Plasma pharmacokinetics (PK) of leriglitazone were determined in a phase 1 study in male healthy volunteers comprising a single ascending dose (SAD) and a multiple ascending dose (MAD) at oral doses of 30, 90, and 270 mg and 135 and 270 mg, respectively. Leriglitazone was rapidly absorbed with no food effect on overall exposure and showed a linear PK profile with dose-exposure correlation. A physiologically based pharmacokinetic (PBPK) model was developed for leriglitazone based on phase 1 data (SAD part) and incorporated CYP3A4 (fmCYP3A4 = 24%) and CYP2C8-mediated (fmCYP2C8 = 45%) metabolism, as well as biliary clearance (feBIL = 19.5%) derived from in vitro data, and was verified by comparing the observed versus predicted concentration-time profiles from the MAD part. The PBPK model was prospectively applied to predict the starting pediatric doses and was preliminarily verified with data from five pediatric patients.

Cannabidiol ameliorates mitochondrial disease via PPARγ activation in preclinical models.

Mutations in mitochondrial energy-producing genes lead to a heterogeneous group of untreatable disorders known as primary mitochondrial diseases (MD). Leigh syndrome (LS) is the most common pediatric MD and is characterized by progressive neuromuscular affectation and premature death. Here, we show that daily cannabidiol (CBD) administration significantly extends lifespan and ameliorates pathology in two LS mouse models, and improves cellular function in fibroblasts from LS patients. CBD delays motor decline and neurodegenerative signs, improves social deficits and breathing abnormalities, decreases thermally induced seizures, and improves neuropathology in affected brain regions. Mechanistically, we identify peroxisome proliferator-activated receptor gamma (PPARγ) as a key nuclear receptor mediating CBD's beneficial effects, while also providing proof of dysregulated PPARγ expression and activity as a common feature in both mouse neurons and fibroblasts from LS patients. Taken together, our results provide the first evidence for CBD as a potential treatment for LS.

PPAR Gamma Agonist Leriglitazone Recovers Alterations Due to Pank2-Deficiency in hiPS-Derived Astrocytes.

The novel brain-penetrant peroxisome proliferator-activated receptor gamma agonist leriglitazone, previously validated for other rare neurodegenerative diseases, is a small molecule that acts as a regulator of mitochondrial function and exerts neuroprotective, anti-oxidative and anti-inflammatory effects. Herein, we tested whether leriglitazone can be effective in ameliorating the mitochondrial defects that characterize an hiPS-derived model of Pantothenate kinase-2 associated Neurodegeneration (PKAN). PKAN is caused by a genetic alteration in the mitochondrial enzyme pantothenate kinase-2, whose function is to catalyze the first reaction of the CoA biosynthetic pathway, and for which no effective cure is available. The PKAN hiPS-derived astrocytes are characterized by mitochondrial dysfunction, cytosolic iron deposition, oxidative stress and neurotoxicity. We monitored the effect of leriglitazone in comparison with CoA on hiPS-derived astrocytes from three healthy subjects and three PKAN patients. The treatment with leriglitazone did not affect the differentiation of the neuronal precursor cells into astrocytes, and it improved the viability of PKAN cells and their respiratory activity, while diminishing the iron accumulation similarly or even better than CoA. The data suggest that leriglitazone is well tolerated in this cellular model and could be considered a beneficial therapeutic approach in the treatment of PKAN.

Safety and efficacy of leriglitazone for preventing disease progression in men with adrenomyeloneuropathy (ADVANCE): a randomised, double-blind, multi-centre, placebo-controlled phase 2-3 trial.

BACKGROUND: Adult patients with adrenoleukodystrophy have a poor prognosis owing to development of adrenomyeloneuropathy. Additionally, a large proportion of patients with adrenomyeloneuropathy develop life-threatening progressive cerebral adrenoleukodystrophy. Leriglitazone is a novel selective peroxisome proliferator-activated receptor gamma agonist that regulates expression of key genes that contribute to neuroinflammatory and neurodegenerative processes implicated in adrenoleukodystrophy disease progression. We aimed to assess the effect of leriglitazone on clinical, imaging, and biochemical markers of disease progression in adults with adrenomyeloneuropathy. METHODS: ADVANCE was a 96-week, randomised, double-blind, placebo-controlled, phase 2-3 trial done at ten hospitals in France, Germany, Hungary, Italy, the Netherlands, Spain, the UK, and the USA. Ambulatory men aged 18-65 years with adrenomyeloneuropathy without gadolinium enhancing lesions suggestive of progressive cerebral adrenoleukodystrophy were randomly assigned (2:1 without stratification) to receive daily oral suspensions of leriglitazone (150 mg starting dose; between baseline and week 12, doses were increased or decreased to achieve plasma concentrations of 200 µg·h/mL [SD 20%]) or placebo by means of an interactive response system and a computer-generated sequence. Investigators and patients were masked to group assignment. The primary efficacy endpoint was change from baseline in the Six-Minute Walk Test distance at week 96, analysed in the full-analysis set by means of a mixed model for repeated measures with restricted maximum likelihood and baseline value as a covariate. Adverse events were also assessed in the full-analysis set. This study was registered with ClinicalTrials.gov, NCT03231878; the primary study is complete; patients had the option to continue treatment in an open-label extension, which is ongoing. FINDINGS: Between Dec 8, 2017, and Oct 16, 2018, of 136 patients screened, 116 were randomly assigned; 62 [81%] of 77 patients receiving leriglitazone and 34 [87%] of 39 receiving placebo completed treatment. There was no between-group difference in the primary endpoint (mean [SD] change from baseline leriglitazone: -27·7 [41·4] m; placebo: -30·3 [60·5] m; least-squares mean difference -1·2 m; 95% CI -22·6 to 20·2; p=0·91). The most common treatment emergent adverse events in both the leriglitazone and placebo groups were weight gain (54 [70%] of 77 vs nine [23%] of 39 patients, respectively) and peripheral oedema (49 [64%] of 77 vs seven [18%] of 39). There were no deaths. Serious treatment-emergent adverse events occurred in 14 (18%) of 77 patients receiving leriglitazone and ten (26%) of 39 patients receiving placebo. The most common serious treatment emergent adverse event, clinically progressive cerebral adrenoleukodystrophy, occurred in six [5%] of 116 patients, all of whom were in the placebo group. INTERPRETATION: The primary endpoint was not met, but leriglitazone was generally well tolerated and rates of adverse events were in line with the expected safety profile for this drug class. The finding that cerebral adrenoleukodystrophy, a life-threatening event for patients with adrenomyeloneuropathy, occurred only in patients in the placebo group supports further investigation of whether leriglitazone might slow the progression of cerebral adrenoleukodystrophy. FUNDING: Minoryx Therapeutics.

Characterisation of two deletions involving NPC1 and flanking genes in Niemann-Pick type C disease patients.

Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal disorder characterised by the accumulation of a complex pattern of lipids in the lysosomal-late endosomal system. More than 300 disease-causing mutations have been identified so far in the NPC1 and NPC2 genes, including indel, missense, nonsense and splicing mutations. Only one genomic deletion, of more than 23 kb, has been previously reported. We describe two larger structural variants, encompassing NPC1 and flanking genes, as a cause of the disease. QMPSF, SNP inheritance and CytoScan® HD Array were used to confirm and further characterise the presence of hemizygous deletions in two patients. One of the patients (NPC-57) bore a previously described missense mutation (p.T1066N) and an inherited deletion that included NPC1, C18orf8 and part of ANKRD29 gene. The second patient (NPC-G1) had a 1-bp deletion (c.852delT; p.F284Lfs*26) and a deletion encompassing the promoter region and exons 1-10 of NPC1 and the adjacent ANKRD29 and LAMA3. This study characterised two novel chromosomal microdeletions at 18q11-q12 that cause NPC disease and provide insight into missing NPC1 mutant alleles.

The brain penetrant PPARγ agonist leriglitazone restores multiple altered pathways in models of X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD), a potentially fatal neurometabolic disorder with no effective pharmacological treatment, is characterized by clinical manifestations ranging from progressive spinal cord axonopathy [adrenomyeloneuropathy (AMN)] to severe demyelination and neuroinflammation (cerebral ALD-cALD), for which molecular mechanisms are not well known. Leriglitazone is a recently developed brain penetrant full PPARγ agonist that could modulate multiple biological pathways relevant for neuroinflammatory and neurodegenerative diseases, and particularly for X-ALD. We found that leriglitazone decreased oxidative stress, increased adenosine 5'-triphosphate concentration, and exerted neuroprotective effects in primary rodent neurons and astrocytes after very long chain fatty acid-induced toxicity simulating X-ALD. In addition, leriglitazone improved motor function; restored markers of oxidative stress, mitochondrial function, and inflammation in spinal cord tissues from AMN mouse models; and decreased the neurological disability in the EAE neuroinflammatory mouse model. X-ALD monocyte-derived patient macrophages treated with leriglitazone were less skewed toward an inflammatory phenotype, and the adhesion of human X-ALD monocytes to brain endothelial cells decreased after treatment, suggesting the potential of leriglitazone to prevent the progression to pathologically disrupted blood-brain barrier. Leriglitazone increased myelin debris clearance in vitro and increased myelination and oligodendrocyte survival in demyelination-remyelination in vivo models, thus promoting remyelination. Last, leriglitazone was clinically tested in a phase 1 study showing central nervous system target engagement (adiponectin increase) and changes on inflammatory biomarkers in plasma and cerebrospinal fluid. The results of our study support the use of leriglitazone in X-ALD and, more generally, in other neuroinflammatory and neurodegenerative conditions.

Present and future of antisense therapy for splicing modulation in inherited metabolic disease.

The number of mutations identified deep in introns which activate or create novel splice sites resulting in pathogenic pseudoexon inclusion in mRNA continues to grow for inherited metabolic disease (IMD) and other human genetic diseases. A common characteristic is that the native splice sites remain intact thus retaining the potential for normal splicing. Antisense oligonucleotides (AO) have been shown to modulate the splicing pattern by steric hindrance of the recognition and binding of the splicing apparatus to the selected sequences. In the case of pseudoexons, AO force the use of the natural splice sites, recovering normally spliced transcripts encoding functional protein. This review summarizes the present knowledge of antisense splicing modulation as a molecular therapy approach for pseudoexon-activating mutations, with a focus in IMD. Although the feasibility of treatment for patients with IMD has yet to be proven, it appears to be clinically promising, as positive results have been reported in cellular and animal models of disease, and antisense therapy for splicing modulation is currently in the clinical trials phase for Duchenne muscular dystrophy patients. Here, we review the most recent advances in AO stability, targeting and delivery, and other issues to be considered for an effective treatment in the clinical setting. Although the number of patients who can be potentially treated is low for each IMD, it represents an excellent therapeutical option as a type of personalized molecular medicine which is especially relevant for diseases for which there is, to date, no efficient treatment.

Antisense oligonucleotide treatment for a pseudoexon-generating mutation in the NPC1 gene causing Niemann-Pick type C disease.

Niemann-Pick type C disease is an autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. While most of the mutations are missense, a few splicing mutations have also been described. We identified and characterized a novel point mutation c.1554-1009G>A located in intron 9 of the NPC1 gene in a Spanish patient. Sequencing of the cDNA from the patient showed that this intronic mutation creates a cryptic donor splice site resulting in the incorporation of 194 bp of intron 9 as a new exon (pseudoexon) in the mRNA. This new transcript bears a premature termination codon and is degraded by the nonsense-mediated mRNA decay mechanism. Experimental confirmation that the point mutation generates the inclusion of a pseudoexon in the mRNA was obtained using a minigene. A specific antisense morpholino oligonucleotide targeted to the cryptic splice site was designed and transfected into fibroblasts from the patient. Using this approach, normal splicing was restored. These results demonstrate the importance of screening deep intronic regions and support the efficacy of antisense therapeutics for the treatment of diseases caused by pseudoexon-generating mutations.

Identification and characterization of SMPD1 mutations causing Niemann-Pick types A and B in Spanish patients.

Niemann-Pick disease (NPD) types A/B are both caused by a deficiency of lysosomal acid sphingomyelinase and display autosomal recessive inheritance. These two types of the disease were described according to the presence (type A) or absence (type B) of neurological symptoms. We present a molecular analysis of 19 Spanish NPD A/B patients and two from Maghreb. Eight of the patients had type A and 13 had type B NPD. All mutant SMPD1 alleles were identified, including 17 different mutations, 10 of which were novel. The only frequent mutations in the 21 NPD patients were c.1823_1825delGCC (p.R608del) (38%) and c.1445C>A (p.A482E) (9%). Genotype-phenotype correlations were established for most of the mutations and, in particular, the p.R608del-type B association was confirmed. This mutation accounts for 61.5% of the mutant alleles in the type B subgroup of patients. Expression studies performed on six of the identified mutations confirmed them to be disease-causing due to their low enzyme activity. An allele with a mutation affecting a noncanonical donor splice site produced only aberrant mRNAs, corresponding to previously reported nonfunctional SMPD1 minor transcripts. This study is the first exhaustive mutational analysis of Spanish Niemann-Pick A/B disease patients.

Pharmacological chaperones for enzyme enhancement therapy in genetic diseases.

Pharmacological chaperone therapy (PCT) is a rather new approach consisting in targeting incorrectly folded proteins by small molecules, thus, facilitating the correct folding of the protein and inducing a recovery of its functionality. Many diseases result from mutations on specific genes; this patent review focuses on those pathologies where PCT has a potential application for enzymatic enhancement. Rare diseases are the main area where PCT has been applied and the most advanced compounds are aiming to cure lysosomal storage disorders such as Fabry, Pompe or Gaucher. Until now, most compounds used as pharmacological chaperones were based on substrate-like chemical structures but recently new nonsubstrate-like and non-inhibitory compounds have been disclosed for Gaucher and Pompe diseases. This initiates a new era for pharmacological chaperones with more diverse chemical structures and binding modes. This review covers the patents relating to enzyme enhancement on pharmacological chaperone therapy. Only an update is presented for Gaucher disease, where PCT is highly applied and recently reviewed.

Generation of a human neuronal stable cell model for niemann-pick C disease by RNA interference.

Niemann-Pick type C (NPC) disease is a fatal autosomal recessive neurodegenerative disorder caused, most commonly, by mutations in the NPC1 gene. At the cellular level, the disease is characterized by the storage of multiple lipids in the endosomal-lysosomal system, including free cholesterol, glycosphingolipids, sphingomyelin and the catabolic product of sphingolipids, sphingosine. Therapeutic options for NPC disease are relatively limited. One drawback for the development of novel therapies is the lack of suitable human neuronal cell models. In this work, a stable SH-SY5Y cell model for NPC disease was generated using short hairpin RNAs. An inhibition of the NPC1 expression of around 90% was obtained at the RNA level. The NPC1 knockdown was confirmed at the protein level. To characterize the stable cell line generated, cholesterol levels were analyzed in the NPC1-knockdown SH-SY5Y cells by filipin staining and gas chromatography-mass spectrometry. A characteristic NPC pattern and a twofold increase of the free cholesterol levels, related to intact SH-SY5Y cells, were found. Moreover, sphingolipids were analyzed by liquid chromatography-mass spectrometry and an increase in ganglioside GM2 levels was observed. The stable NPC1-knockdown SH-SY5Y cell line generated in the present study provides a human neuronal cell model for this lethal disease that could be a valuable tool for the study of future therapeutic approaches.