Efficacy and Safety of the Natural Killer T Cell-Stimulatory Glycolipid OCH-NCNP1 for Patients With Relapsing Multiple Sclerosis: Protocol for a Randomized Placebo-Controlled Clinical Trial.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that causes myelin sheath damage and axonal degeneration. The glycolipid (2S, 3S, 4R)-1-O-(α-d-galactosyl)-2-tetracosanoylamino-1,3,4-nonaetriol (OCH-NCNP1 or OCH) exerts an immunoregulatory action that suppresses T helper (Th)1 cell-mediated immune responses through natural killer T cell activation, selective interleukin-4 production, and Th2 bias induction in human CD4-positive natural killer T cells. OBJECTIVE: This trial aims to investigate the efficacy and safety of the immunomodulator OCH in patients with relapsing MS through 24-week repeated administration. METHODS: This protocol describes a double-blind, multicenter, placebo-controlled, randomized phase II clinical trial that was initiated in September 2019. The participants were randomly assigned to either a placebo control group or an OCH-NCNP1 group and the investigational drug (3.0 mg) was orally administered once weekly for the 24-week duration. Major inclusion criteria are as follows: patients had been diagnosed with relapsing MS (relapsing-remitting and/or secondary progressive MS) based on the revised McDonald criteria or were diagnosed with MS by an attending physician as noted in their medical records; patients with at least two medically confirmed clinical exacerbations within 24 months prior to consent or one exacerbation within 12 months prior to consent; patients with at least one lesion suspected to be MS on screening magnetic resonance imaging (MRI); and patients with 7 points or less in the Expanded Disability Status Scale during screening. Major exclusion criteria are as follows: diagnosis of neuromyelitis optica and one of optic neuritis, acute myelitis, and satisfying at least two of the following three items: (1) spinal cord MRI lesion extending across at least three vertebral bodies, (2) no brain MRI lesions during onset (at least four cerebral white matter lesions or three lesions, one of which is around the lateral ventricle), and (3) neuromyelitis optica-immunoglobulin G or antiaquaporin-4 antibody-positive. Outcome measures include the primary outcome of MRI changes (the percentage of subjects with new or newly expanded lesions at 24 weeks on T2-weighted MRI) and the secondary outcomes annual relapse rate (number of recurrences per year), relapse-free period (time to recurrence), sustained reduction in disability (SRD) occurrence rate, period until SRD (time to SRD occurrence), no evidence of disease activity, and exploratory biomarkers from phase I trials (such as gene expression, cell frequency, and intestinal and oral microbiome). RESULTS: We plan to enroll 30 patients in the full analysis set. Enrollment was closed in June 2021 and the study analysis was completed in March 2023. CONCLUSIONS: This randomized controlled trial will determine whether OCH-NCNP1 is effective and safe in patients with MS as well as provide evidence for the potential of OCH-NCNP1 as a therapeutic agent for MS. TRIAL REGISTRATION: ClinicalTrials.gov NCT04211740; https://clinicaltrials.gov/study/NCT04211740. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/46709.

Systemic administration of the antisense oligonucleotide NS-089/NCNP-02 for skipping of exon 44 in patients with Duchenne muscular dystrophy: Study protocol for a phase I/II clinical trial.

AIM: The purpose of this study is to evaluate the safety and pharmacokinetics of the novel morpholino oligomer NS-089/NCNP-02 which can induce exon 44 skipping, in patients with DMD. Additionally, we aimed to identify markers predictive of therapeutic efficacy and determine the optimal dosing for future studies. METHODS: This is an open-label, dose-escalation, two-center phase I/II trial in ambulant patients with DMD, presence of an out-of-frame deletion, and a mutation amenable to exon 44 skipping. Part 1 is a stepwise dose-finding stage (4 weeks) during which NS-089/NCNP-02 will be administered intravenously at four dose levels once weekly (1.62, 10, 40, and 80 mg/kg); Part 2 is a 24-week evaluation period based on the dosages determined during Part 1. The primary (safety) endpoints are the results of physical examinations, vital signs, 12-lead electrocardiogram and echocardiography tests, and adverse event reporting. Secondary endpoints include expression of dystrophin protein, motor function assessment, exon 44 skipping efficiency, plasma and urinary NS-089/NCNP-02 concentrations, and changes in blood creatine kinase levels. DISCUSSION: Exon-skipping therapy using ASOs shows promise in selected patients, and this first-in-human study is expected to provide critical information for subsequent clinical development of NS-089/NCNP-02.

First-in-human clinical trial of the NKT cell-stimulatory glycolipid OCH in multiple sclerosis.

Background: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system that causes the damage to the myelin sheath as well as axonal degeneration. Individuals with MS appear to have changes in the numbers and functions of T-cell subsets, leading to an immunological imbalance accompanied by enhanced autoreactivity. In previous preclinical studies, (2 S,3 S,4R)-1-O-(α-D-Galactopyranosyl)-N-tetracosanoyl-2-amino-1,3,4-nonanetriol (OCH), a synthetic analog of α-galactosylceramide stimulatory for invariant NKT (iNKT) cells, has shown therapeutic or disease-preventive immunoregulatory effects in autoimmune disease models such as experimental autoimmune encephalomyelitis (EAE). Objectives: This study is the first-in-human study of oral OCH to evaluate the pharmacokinetics and to examine the effects on immune cells as well as related gene expression profiles. Methods: Fifteen healthy volunteers and 13 MS patients who met the study criteria were enrolled. They were divided into five cohorts and received oral administration of various doses of granulated powder of OCH (0.3-30 mg), once per week for 4 or 13 weeks. Plasma OCH concentrations were measured by high-performance liquid chromatography. Frequencies of lymphocyte subsets in peripheral blood were evaluated by flow cytometry, and microarray analysis was performed to determine OCH-induced changes in gene expression. Results: Oral OCH was well tolerated, and its bioavailability was found to be sufficient. Six hours after a single dose of OCH, increased frequencies of Foxp3+ regulatory T-cells were observed in some cohorts of healthy subjects and MS patients. Furthermore, gene expression analysis demonstrated an upregulation of several immunoregulatory genes and downregulation of pro-inflammatory genes following OCH administration. Conclusion: This study has demonstrated immunomodulatory effects of the iNKT cell-stimulatory drug OCH in human. Safety profiles together with the presumed anti-inflammatory effects of oral OCH encouraged us to conduct a phase II trial.

Regulatory Frameworks for Gene and Cell Therapies in Japan.

The regulations for the human use of advanced therapy medical products such as gene and cell therapy products have evolved in accordance with advance of clinical experience, scientific knowledge, and social acceptance to these technologies. In Japan, two laws, the Pharmaceuticals and Medical Devices (PMD) Act and the Act on the Safety of Regenerative Medicine (ASRM), were enacted in November 2014. The PMD Act defines regenerative medical products for the first time and introduces a system for the conditional and time-limited marketing authorization of regenerative medical products. Under ASRM, the responsibilities of medical institutions to ensure the safety and provide transparency of such medical technologies are described. Amendments to accompanying guidelines for these two Acts are currently in preparation. It is expected that the new legislative frameworks will promote the timely development of new products and technologies, to bring safe and effective regenerative medicines to Japanese patients.

Structures of cyanobacteriochromes from phototaxis regulators AnPixJ and TePixJ reveal general and specific photoconversion mechanism.

Cyanobacteriochromes are cyanobacterial tetrapyrrole-binding photoreceptors that share a bilin-binding GAF domain with photoreceptors of the phytochrome family. Cyanobacteriochromes are divided into many subclasses with distinct spectral properties. Among them, putative phototaxis regulators PixJs of Anabaena sp. PCC 7120 and Thermosynechococcus elongatus BP-1 (denoted as AnPixJ and TePixJ, respectively) are representative of subclasses showing red-green-type and blue/green-type reversible photoconversion, respectively. Here, we determined crystal structures for the AnPixJ GAF domain in its red-absorbing 15Z state (Pr) and the TePixJ GAF domain in its green-absorbing 15E state (Pg). The overall structure of these proteins is similar to each other and also similar to known phytochromes. Critical differences found are as follows: (i) the chromophore of AnPixJ Pr is phycocyanobilin in a C5-Z,syn/C10-Z,syn/C15-Z,anti configuration and that of TePixJ Pg is phycoviolobilin in a C10-Z,syn/C15-E,anti configuration, (ii) a side chain of the key aspartic acid is hydrogen bonded to the tetrapyrrole rings A, B and C in AnPixJ Pr and to the pyrrole ring D in TePixJ Pg, (iii) additional protein-chromophore interactions are provided by subclass-specific residues including tryptophan in AnPixJ and cysteine in TePixJ. Possible structural changes following the photoisomerization of the chromophore between C15-Z and C15-E are discussed based on the X-ray structures at 1.8 and 2.0-Å resolution, respectively, in two distinct configurations.

The cyanobacteriochrome, TePixJ, isomerizes its own chromophore by converting phycocyanobilin to phycoviolobilin.

The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a λ(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.

Cyanobacteriochromes: a new superfamily of tetrapyrrole-binding photoreceptors in cyanobacteria.

A new group of photoreceptors has been experimentally revealed in cyanobacteria. They are phototaxis regulator SyPixJ1, TePixJ and AnPixJ, chromatic acclimation regulator SyCcaS, circadian input kinase homolog SyCikA and many other candidates, which have been found only in cyanobacteria to date. These new photoreceptors are now proposed to be "cyanobacteriochromes". They are characterized by the presence of a chromophore-binding GAF domain that is homologous to the tetrapyrrole-binding GAF domain of the phytochrome. Here, we summarized unique features of those representatives: (1) only the GAF domain is sufficient for full photoconversion, (2) the GAF domain is homologous to but distinct from the phytochrome GAF, (3) the GAF domain binds a linear tetrapyrrole pigment such as phycoviolobilin or phycocyanobilin, (4) spectral properties are very diverse from near ultra-violet to red region. We also discussed the functionality of the other candidate GAFs, structure and evolution.

A novel photoactive GAF domain of cyanobacteriochrome AnPixJ that shows reversible green/red photoconversion.

We report the discovery of a novel cyanobacteriochrome, the green/red photoreceptor AnPixJ (All1069), isolated from the heterocyst-forming cyanobacterium Anabaena (Nostoc) sp. PCC 7120. Cyanobacteriochromes are a recently emerging tetrapyrrole-based photoreceptor superfamily that are distantly related to the conventional red/far-red photoreceptor phytochromes (Phys). The chromophore-binding domains of AnPixJ produced in cyanobacterial and Escherichia coli cells both showed a reversible and full photoconversion between a green-absorbing form (lambda(max)=543 nm) and a red-absorbing form (lambda(max)=648 nm). Denaturation analysis revealed that the green-absorbing form and the red-absorbing form covalently ligated phycocyanobilin with E-configuration and Z-configuration at the C15C16 double bond, respectively. Time-resolved spectral analysis showed the formation of the first intermediate state peaking at 680 nm from the dark-stable red-absorbing form. This step resembles the first photoconversion step from the red-absorbing form to the red-shifted lumi-R intermediate state of the Phys. These results suggest that the Pr of AnPixJ is almost equivalent to that of the Phys and starts a primary photoreaction with Z-to-E isomerization in a mechanism similar to that in the Phys, but is finally photoconverted to the unique green-absorbing form.

Cyanobacteriochrome TePixJ of Thermosynechococcus elongatus harbors phycoviolobilin as a chromophore.

Cyanobacteria have several putative photoreceptors (designated cyanobacteriochromes) that are related to but distinct from the established phytochromes. The GAF domain of the phototaxis regulator, PixJ, from a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 (TePixJ_GAF) is a cyanobacteriochrome which exhibits reversible photoconversion between a blue light-absorbing form (max = 433 nm) and a green light-absorbing form (max = 531 nm). To study the chromophore, we prepared TePixJ_GAF chromoprotein from heterologously expressed Synechocystis and performed spectral analysis after denaturation by comparing it with the cyanobacterial phytochrome Cph1 which harbors phycocyanobilin (PCB) as a chromophore. The results indicated that the chromophore of TePixJ is not PCB, but its isomer, phycoviolobilin (PVB). It is suggested that the GAF domain of TePixJ has auto-lyase and auto-isomerase activities.

Characterization of cyanobacteriochrome TePixJ from a thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1.

A putative photoreceptor gene, TepixJ, of a thermophilic cyanobacterium is homologous to SypixJ1 that mediates positive phototaxis in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. The putative chromophore-binding GAF domain of TePixJ protein was overexpressed as a fusion with a polyhistidine tag (His-TePixJ_GAF) in Synechocystis cells and isolated to homogeneity. The photoreversible conversion of His-TePixJ_GAF showed peaks at 531, 341 and 266 nm for the green light-absorbing form (Pg form), and peaks at 433 and 287 nm for the blue light-absorbing form (Pb form). At 77K, the Pg form fluoresced at 580 nm, while the Pb form did not emit any fluorescence. Mass spectrometry of the tryptic chromopeptide demonstrated that a phycocyanobilin isomer binds to the conserved cysteine at ring A via a thioether bond. It is established that TePixJ and SyPixJ1 are novel photoreceptors in cyanobacteria ('cyanobacteriochromes') that are similar, but distinct from the phytochromes and bacteriophytochromes.