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Enzyme replacement therapy with weekly infused intravenous (IV) idursulfase is effective in treating somatic symptoms of mucopolysaccharidosis II (MPS II; Hunter syndrome). A formulation of idursulfase for intrathecal administration (idursulfase-IT) is under investigation for the treatment of neuronopathic MPS II. Here, we report 36-month data from the open-label extension (NCT02412787) of a phase 2/3, randomized, controlled study (HGT-HIT-094; NCT02055118) that assessed the safety and efficacy of monthly idursulfase-IT 10 mg in addition to weekly IV idursulfase on cognitive function in children older than 3 years with MPS II and mild-to-moderate cognitive impairment. Participants were also enrolled in this extension from a linked non-randomized sub-study of children younger than 3 years at the start of idursulfase-IT therapy. The extension safety population comprised 56 patients who received idursulfase-IT 10 mg once a month (or age-adjusted dose for sub-study patients) plus IV idursulfase (0.5 mg/kg) once a week. Idursulfase-IT was generally well tolerated over the cumulative treatment period of up to 36 months. Overall, 25.0% of patients had at least one adverse event (AE) related to idursulfase-IT; most treatment-emergent AEs were mild in severity. Of serious AEs (reported by 76.8% patients), none were considered related to idursulfase-IT treatment. There were no deaths or discontinuations owing to AEs. Secondary efficacy analyses (in patients younger than 6 years at phase 2/3 study baseline; n = 40) indicated a trend for improved Differential Ability Scale-II (DAS-II) General Conceptual Ability (GCA) scores in the early idursulfase-IT versus delayed idursulfase-IT group (treatment difference over 36 months from phase 2/3 study baseline: least-squares mean, 6.8 [90% confidence interval: -2.1, 15.8; p = 0.2064]). Post hoc analyses of DAS-II GCA scores by genotype revealed a clinically meaningful treatment effect in patients younger than 6 years with missense variants of the iduronate-2-sulfatase gene (IDS) (least-squares mean [standard error] treatment difference over 36 months, 12.3 [7.24]). These long-term data further suggest the benefits of idursulfase-IT in the treatment of neurocognitive dysfunction in some patients with MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

Two-thirds of patients with mucopolysaccharidosis II (MPS II; Hunter syndrome) have cognitive impairment. This phase 2/3, randomized, controlled, open-label, multicenter study (NCT02055118) investigated the effects of intrathecally administered idursulfase-IT on cognitive function in patients with MPS II. Children older than 3 years with MPS II and mild-to-moderate cognitive impairment (assessed by Differential Ability Scales-II [DAS-II], General Conceptual Ability [GCA] score) who had tolerated intravenous idursulfase for at least 4 months were randomly assigned (2:1) to monthly idursulfase-IT 10 mg (n = 34) via an intrathecal drug delivery device (IDDD; or by lumbar puncture) or no idursulfase-IT treatment (n = 15) for 52 weeks. All patients continued to receive weekly intravenous idursulfase 0.5 mg/kg as standard of care. Of 49 randomized patients, 47 completed the study (two patients receiving idursulfase-IT discontinued). The primary endpoint (change from baseline in DAS-II GCA score at week 52 in a linear mixed-effects model for repeated measures analysis) was not met: although there was a smaller decrease in DAS-II GCA scores with idursulfase-IT than with no idursulfase-IT at week 52, this was not significant (least-squares mean treatment difference [95% confidence interval], 3.0 [-7.3, 13.3]; p = 0.5669). Changes from baseline in Vineland Adaptive Behavioral Scales-II Adaptive Behavior Composite scores at week 52 (key secondary endpoint) were similar in the idursulfase-IT (n = 31) and no idursulfase-IT (n = 14) groups. There were trends towards a potential positive effect of idursulfase-IT across DAS-II composite, cluster, and subtest scores, notably in patients younger than 6 years at baseline. In a post hoc analysis, there was a significant (p = 0.0174), clinically meaningful difference in change from baseline in DAS-II GCA scores at week 52 with idursulfase-IT (n = 13) versus no idursulfase-IT (n = 6) among those younger than 6 years with missense iduronate-2-sulfatase gene variants. Overall, idursulfase-IT reduced cerebrospinal glycosaminoglycan levels from baseline by 72.0% at week 52. Idursulfase-IT was generally well tolerated. These data suggest potential benefits of idursulfase-IT in the treatment of cognitive impairment in some patients with neuronopathic MPS II. After many years of extensive review and regulatory discussions, the data were found to be insufficient to meet the evidentiary standard to support regulatory filings.

[Display omitted] • The neural network described extracts coupling constants and chemical shifts from a 2D J-resolved spectrum. • Training of the network on spectra simulated using quantum dynamics is demonstrated. • Results on simulated test sets are satisfactory, but experimental spectra present obstacles. • Future work employing pure-phase J-resolved spectra is suggested. Extracting parameters such as chemical shifts and coupling constants from proton NMR spectra is often a first step in using spectra for compound identification and structure determination. This can become challenging when scalar couplings between protons are comparable in size to chemical shift differences (strongly coupled), as is often the case with low-field (bench top) spectrometers. Here we explore the potential utility of AI methods, in particular neural networks, for extracting parameters from low-field spectra. Rather than seeking large experimental sets of spectra for training a network, we chose quantum mechanical simulation of sets, something that is possible with modern software packages and computer resources. We show that application of a network trained on 2-D J-resolved spectra and applied to a spectrum of iduronic acid, shows some promise, but also meets with some obstacles. We suggest that these may be overcome with improved pulse sequences and more extensive simulations. [ABSTRACT FROM AUTHOR]

Heparan sulfate belongs to the group of glycosaminoglycans (GAGs), highly sulfated linear polysaccharides. Heparan sulfate 2- O -sulfotransferase 1 (HS2ST1) is one of several specialized enzymes required for heparan sulfate synthesis and catalyzes the transfer of the sulfate groups to the sugar moiety of heparan sulfate. We report bi-allelic pathogenic variants in HS2ST1 in four individuals from three unrelated families. Affected individuals showed facial dysmorphism with coarse face, upslanted palpebral fissures, broad nasal tip, and wide mouth, developmental delay and/or intellectual disability, corpus callosum agenesis or hypoplasia, flexion contractures, brachydactyly of hands and feet with broad fingertips and toes, and uni- or bilateral renal agenesis in three individuals. HS2ST1 variants cause a reduction in HS2ST1 mRNA and decreased or absent heparan sulfate 2- O -sulfotransferase 1 in two of three fibroblast cell lines derived from affected individuals. The heparan sulfate synthesized by the individual 1 cell line lacks 2- O -sulfated domains but had an increase in N - and 6- O -sulfated domains demonstrating functional impairment of the HS2ST1. As heparan sulfate modulates FGF-mediated signaling, we found a significantly decreased activation of the MAP kinases ERK1/2 in FGF-2-stimulated cell lines of affected individuals that could be restored by addition of heparin, a GAG similar to heparan sulfate. Focal adhesions in FGF-2-stimulated fibroblasts of affected individuals concentrated at the cell periphery. Our data demonstrate that a heparan sulfate synthesis deficit causes a recognizable syndrome and emphasize a role for 2- O -sulfated heparan sulfate in human neuronal, skeletal, and renal development. [ABSTRACT FROM AUTHOR]

Due to the all-axial orientation of the OH-groups in the 1C 4 chair conformation considered standard for L-hexapyranosides, including l -iduronopyranoside – a component of many biologically and medically significant sulfated glycans, these monosaccharides can be anticipated to display unusual conformations upon the introduction of bulky and charged substituents. Herein we describe the synthesis of a series of iduronopyranoside derivatives with varying sulfation patterns, which were studied computationally using the DLPNO-MP2 approach and by means of analyzing their chemical shifts to ascertain the effects sulfation has on the conformation of the iduronopyranoside ring. [Display omitted] • A series of iduronosides covering all possible sulfation patterns was completed. • The effects of sulfate location on the iduronoside ring conformation were studied. • High-level DLPNO-MP2 method was used, as opposed to older works employing just DFT. • It emerges that apart from 2S O , 1S 3 may be present in the equilibrium. [ABSTRACT FROM AUTHOR]

We have demonstrated that cisplatin (CP), an anticancer drug, showed a preference for binding the sulfated-L-iduronic acid (S-L-IdoA) unit over the sulfated-D-glucuronic acid unit of heparan sulfate. The multivalency of S-L-IdoA, such as in the proteoglycan mimic, resulted in distinct modes of cell-surface engineering in normal and cancer cells, with these disparities having a significant impact on CP-mediated toxicity.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously delivered IDS is unable to cross the blood-brain barrier (BBB). Hematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (rabies virus glycoprotein [RVG] and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via hematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared with LV.IDS.ApoEII and LV.IDS in MPS II mice at 6 months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG- and LV.IDS.gh625-treated mice than in LV.IDS.ApoEII- and LV.IDS-treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis, and lysosomal swelling were partially normalized in MPS II mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalized by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared with control tissue from LV.IDS- and LV.IDS.ApoEII-transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPS II and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPS II disease than IDS alone.

L‐Iduronic acid (IdoA) plays a pivotal role in glycosaminoglycan (GAG) protein interactions. However, the structural microheterogeneity of GAG appears to impede the systematic investigation of IdoA functions. Under such conditions, oligo‐Iduronic acid (Oligo‐IdoA) are ideal and straightforward heparin mimetics to unravel the relationship between IdoA structure and functions. Herein, we report for the first‐time linear synthesis of rare oligo‐IdoA precursor utilizing anhydrous β‐L‐idopyranosyl and IdoA thiophenol building block. After screening various synthetic strategies, we have Installed successive IdoA by 5 step reactions with 25–26 % overall yield. These oligo‐IdoA are expected to be excellent probes to understand conformational plasticity of IdoA and fine tune carbohydrate–protein interactions. [ABSTRACT FROM AUTHOR]

Mucopolysaccharidoses (MPSs) make up a group of lysosomal storage diseases characterized by the aberrant accumulation of glycosaminoglycans throughout the body. Patients with MPSs display various signs and symptoms, such as retinopathy, which is also observed in patients with MPS II. Unfortunately, retinal disorders in MPS II are resistant to conventional intravenous enzyme-replacement therapy because the blood-retinal barrier (BRB) impedes drug penetration. In this study, we show that a fusion protein, designated pabinafusp alfa, consisting of an antihuman transferrin receptor antibody and iduronate-2-sulfatase (IDS), crosses the BRB and reaches the retina in a murine model of MPS II. We found that retinal function, as assessed by electroretinography (ERG) in MPS II mice, deteriorated with age. Early intervention with repeated intravenous treatment of pabinafusp alfa decreased heparan sulfate deposition in the retina, optic nerve, and visual cortex, thus preserving or even improving the ERG response in MPS II mice. Histological analysis further revealed that pabinafusp alfa mitigated the loss of the photoreceptor layer observed in diseased mice. In contrast, recombinant nonfused IDS failed to reach the retina and hardly affected the retinal disease. These results support the hypothesis that transferrin receptor-targeted IDS can penetrate the BRB, thereby ameliorating retinal dysfunction in MPS II.

Monosaccharides are essential for maintaining the normal physiological functions of living organisms. Under disease states, metabolic disorders in vivo will inevitably affect the levels of monosaccharides, which brings the possibility of monosaccharides as a biomarker of some diseases. In this study, a method was developed and validated for simultaneously determining 10 monosaccharides (glucose, galactose, mannose, rhamnose, fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine) in SD rat plasma using liquid chromatography-tandem mass spectrometry. The method employed 1-phenyl-3-methyl-5-pyrazolone (PMP) as a derivatization reagent, considerably improved the chromatographic retention and ionization efficiency of monosaccharides. After protein precipitation of plasma samples, monosaccharides and isotope internal standards were derivatized and liquid-liquid extraction was performed to remove excess PMP. To achieve the baseline separation of several isomers, the resulting derivatives were chromatographed on a Bridged ethyl hybrid (BEH) Phenyl column using gradient elution with a total run time of 8 min. The method was linear within the range of 0.0100-5.00 μg/mL for rhamnose, 0.0500-25.0 μg/mL for fucose, xylose, iduronic acid, glucuronic acid, N-acetylgalactosamine and N-acetylglucosamine, 1.00-500 μg/mL for galactose, 10.0-5000 μg/mL for mannose, and 50.0-25,000 μg/mL for glucose. And the accuracy and precision verification of surrogate matrix samples and plasma samples met the required criteria. The method has been used successfully to study the effect of hepatic insufficiency on monosaccharide levels in rats. It was found that the concentration of glucuronic acid in SD rat plasma was abnormally increased in rats with liver injury., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xiaoyan Chen reports financial support was provided by The High-level new R&D institute (2019B090904008) and the High-level Innovative Research Institute (2021B0909050003) of the Department of Science and Technology of Guangdong Province., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Multiple complex intracellular cascades contributing to Hunter syndrome (mucopolysaccharidosis type II) pathogenesis have been recognized and documented in the past years. However, the hierarchy of early cellular abnormalities leading to irreversible neuronal damage is far from being completely understood. To tackle this issue, we have generated two novel iduronate-2-sulfatase (IDS) loss of function human neuronal cell lines by means of genome editing. We show that both neuronal cell lines exhibit no enzymatic activity and increased GAG storage despite a completely different genotype. At a cellular level, they display reduced differentiation, significantly decreased LAMP1 and RAB7 protein levels, impaired lysosomal acidification and increased lipid storage. Moreover, one of the two clones is characterized by a marked decrease of the autophagic marker p62, while none of the two mutants exhibit marked oxidative stress and mitochondrial morphological changes. Based on our preliminary findings, we hypothesize that neuronal differentiation might be significantly affected by IDS functional impairment., (© 2023. The Author(s).)

Mucopolysaccharidosis type II, commonly called Hunter syndrome, is a rare X-linked recessive disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). A deficiency of I2S causes an abnormal glycosaminoglycans accumulation in the body's cells. Although enzyme replacement therapy is the standard therapy, adeno-associated viruses (AAV)-based gene therapy could provide a single-dose solution to achieve a prolonged and constant enzyme level to improve patient's quality of life. Currently, there is no integrated regulatory guidance to describe the bioanalytical assay strategy to support gene therapy products. Herein, we describe the streamlined strategy to validate/qualify the transgene protein and its enzymatic activity assays. The method validation for the I2S quantification in serum and method qualification in tissues was performed to support the mouse GLP toxicological study. Standard curves for I2S quantification ranged from 2.00 to 50.0 μg/mL in serum and 6.25 to 400 ng/mL in the surrogate matrix. Acceptable precision, accuracy, and parallelism in the tissues were demonstrated. To assess the function of the transgene protein, fit-for-purpose method qualification for the I2S enzyme activity in serum was performed. The observed data indicated that the enzymatic activity in serum increased dose-dependently in the lower I2S concentration range. The highest I2S transgene protein was observed in the liver among tissue measured, and its expression level was maintained up to 91 days after the administration of rAAV8 with a codon-optimized human I2S. In conclusion, the multifaceted bioanalytical method for I2S and its enzymatic activity were established to assess gene therapy products in Hunter syndrome., (© 2023. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Heparin, a major anticoagulant drug, comprises a complex mixture of motifs. Heparin is isolated from natural sources while being subjected to a variety of conditions but the detailed effects of these on heparin structure have not been studied in depth. Therefore, the result of exposing heparin to a range of buffered environments, ranging pH values from 7 to 12, and temperatures of 40, 60 and 80 °C were examined. There was no evidence of significant N-desulfation or 6-O-desulfation in glucosamine residues, nor of chain scission, however, stereochemical re-arrangement of α-L-iduronate 2-O-sulfate to α-L-galacturonate residues occurred in 0.1 M phosphate buffer at pH 12/80 °C. The results confirm the relative stability of heparin in environments like those during extraction and purification processes; on the other hand, the sensitivity of heparin to pH 12 in buffered solution at high temperature is highlighted, providing an important insight for heparin manufacturers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder characterized by an accumulation of glycosaminoglycans (GAGs), including heparan sulfate, in the body. Major manifestations involve the central nerve system (CNS), skeletal deformation, and visceral manifestations. About 30% of MPS II is linked with an attenuated type of disease subtype with visceral involvement. In contrast, 70% of MPS II is associated with a severe type of disease subtype with CNS manifestations that are caused by the human iduronate-2-sulfatase (IDS)-Pro86Leu (P86L) mutation, a common missense mutation in MPS II. In this study, we reported a novel Ids-P88L MPS II mouse model, an analogous mutation to human IDS-P86L. In this mouse model, a significant impairment of IDS enzyme activity in the blood with a short lifespan was observed. Consistently, the IDS enzyme activity of the body, as assessed in the liver, kidney, spleen, lung, and heart, was significantly impaired. Conversely, the level of GAG was elevated in the body. A putative biomarker with unestablished nature termed UA-HNAc(1S) (late retention time), one of two UA-HNAc(1S) species with late retention time on reversed-phase separation,is a recently reported MPS II-specific biomarker derived from heparan sulfate with uncharacterized mechanism. Thus, we asked whether this biomarker might be elevated in our mouse model. We found a significant accumulation of this biomarker in the liver, suggesting that hepatic formation could be predominant. Finally, to examine whether gene therapy could enhance IDS enzyme activity in this model, the efficacy of the nuclease-mediated genome correction system was tested. We found a marginal elevation of IDS enzyme activity in the treated group, raising the possibility that the effect of gene correction could be assessed in this mouse model. In conclusion, we established a novel Ids-P88L MPS II mouse model that consistently recapitulates the previously reported phenotype in several mouse models., (© 2023. The Author(s).)

Background: Mucopolysaccharidosis type II is a severe lysosomal storage disease caused by deficient activity of the enzyme iduronate-2-sulfatase. The only medicinal product approved by the US Food and Drug Administration for enzyme replacement therapy, recombinant iduronate-2-sulfatase (idursulfase, Elaprase ® ), is a large molecule that is not able to cross the blood-brain barrier and neutralize progressive damage of the central nervous system caused by the accumulation of glycosaminoglycans. Novel chimeric protein HIR-Fab-IDS is an anti-human insulin receptor Fab fragment fused to recombinant modified iduronate-2-sulfatase. This modification provides a highly selective interaction with the human insulin receptor, which leads to the HIR-Fab-IDS crossing the blood-brain barrier owing to internalization of the hybrid molecule by transcytosis into endothelial cells adjacent to the nervous system by the principle of a 'molecular Trojan horse'., Objectives: In this work, the physicochemical and biological characterization of a blood-brain barrier-penetrating fusion protein, HIR-Fab-IDS, is carried out. HIR-Fab-IDS consists of an anti-human insulin receptor Fab fragment fused to recombinant iduronate-2-sulfatase., Methods: Comprehensive analytical characterization utilizing modern techniques (including surface plasmon resonance and mass spectrometry) was performed using preclinical and clinical batches of HIR-Fab-IDS. Critical quality parameters that determine the therapeutic effect of iduronate-2-sulfatase, as well as IDS enzymatic activity and in vitro cell uptake activity were evaluated in comparison with the marketed IDS product Elaprase ® (IDS RP). In vivo efficiency of HIR-Fab-IDS in reversing mucopolysaccharidosis type II pathology in IDS-deficient mice was also investigated. The affinity of the chimeric molecule for the INSR was also determined by both an enzyme-linked immunosorbent assay and surface plasmon resonance. We also compared the distribution of 125 I-radiolabeled HIR-Fab-IDS and IDS RP in the tissues and brain of cynomolgus monkeys after intravenous administration., Results: The HIR-Fab-IDS primary structure investigation showed no significant post-translational modifications that could affect IDS activity, except for the formylglycine content, which was significantly higher for HIR-Fab-IDS compared with that for IDS RP (~ 76.5 vs ~ 67.7%). Because of this fact, the specific enzyme activity of HIR-Fab-IDS was slightly higher than that of IDS RP (~ 2.73 × 10 6  U/μmol vs ~ 2.16 × 10 6  U/μmol). However, differences were found in the glycosylation patterns of the compared IDS products, causing a minor reduced in vitro cellular uptake of HIR-Fab-IDS by mucopolysaccharidosis type II fibroblasts compared with IDS RP (half-maximal effective concentration ~ 26.0 vs ~ 23.0 nM). The efficacy of HIR-Fab-IDS in IDS-deficient mice has demonstrated a statistically significant reduction in the level of glycosaminoglycans in the urine and tissues of the main organs to the level of healthy animals. The HIR-Fab-IDS has revealed high in vitro affinity for human and monkey insulin receptors, and the radioactively labeled product has been shown to penetrate to all parts of the brain and peripheral tissues after intravenous administration to cynomolgus monkeys., Conclusions: These findings indicate that HIR-Fab-IDS, a novel iduronate-2-sulfatase fusion protein, is a promising candidate for the treatment of central nervous system manifestations in neurological mucopolysaccharidosis type II., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disorder arising from deficiency of iduronate-2-sulfatase (IDS), which results in progressive accumulation of glycosaminoglycans (GAGs) in multiple tissues. Accumulated GAGs are generally measured as the amount of total GAGs. However, we recently demonstrated that GAG accumulation in the brain of MPS II model mice cannot be reliably detected by conventional dye-binding assay measuring total GAGs. Here we developed a novel quantitative method for measurement of disease-specific GAGs based on the analysis of 2-sulfoiduronic acid levels derived from the non-reducing terminal end of the polysaccharides by using recombinant human IDS (rhIDS) and recombinant human iduronidase (rhIDUA). This method was evaluated on GAGs obtained from the liver and brain of MPS II mice. The GAGs were purified from tissue homogenates and then digested with rhIDS and rhIDUA to generate a desulfated iduronic acid from their non-reducing terminal end. HPLC analysis revealed that the generated iduronic acid levels were markedly increased in the liver and cerebrum of the MPS II mice, whereas the uronic acid was not detected in wild-type mice. These results indicate that this assay clearly detects the disease-specific GAGs in tissues from MPS II mice. [ABSTRACT FROM AUTHOR]