Robust LC–MS/MS Methods for Analysis of Heparan Sulfate Levels in CSF and Brain for Application in Studies of MPS IIIA

Makower, Åsa; Arnelöf, Erik; Andersson, Tommy; Edlund, Per-Olof; Gustavsson, Susanne; Janson, Juliette; Gelius, Stefan Svensson; Tjernberg, Agneta

doi:10.4155/bio-2019-0095

Cited by 8 publications

(7 citation statements)

References 30 publications

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“…CM-rhSulfamidase treatment might initially result in a reduction of especially the higher saccharide species thereby producing lower saccharide species. In support of this is the observation that in liver the effect of CM-rhSulfamidase treatment was lower on GlcNS-UA levels as compared with GlcNS-UA-GlcNAc-UA (+1S) levels and a low single dose of CM-rhSulfamidase even resulted in an increase of GlcNS-UA [26]. Thus, enzyme stability and activity, study duration, dose level and frequency and the biomarker might all have contributed to the clear effect on biomarkers in brain in our studies.…”

Section: Discussionmentioning

confidence: 97%

“…For determination of HS levels in brain, liver, spleen, and cerebrospinal fluid (CSF), two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods were developed, one quantitative and one relative [26]. The quantitative method measures individual disaccharides derived from heparinase digestion of HS.…”

Section: Methodsmentioning

confidence: 99%

“…The pellets were washed once with PBS followed by cell lysis by freeze/thaw cycles in 29 mM diethylbarbituric acid, 29 mM sodium acetate and 0.68% w / v sodium chloride, pH 6.5 supplemented with protease and phosphatase inhibitors. Cell debris was removed by centrifugation and the samples were divided into two aliquots, one for HS analysis by LC-MS/MS using the disaccharide biomarker GlcNS-UA [26] and one for rhSulfamidase/CM-rhSulfamidase content analysis.…”

Section: Methodsmentioning

confidence: 99%

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Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

Gustavsson

Sjöström

Tjernberg

et al. 2019

Molecular Genetics and Metabolism Reports

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Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) characterized by severe central nervous system (CNS) degeneration. The disease is caused by mutations in the SGSH gene coding for the lysosomal enzyme sulfamidase. Sulfamidase deficiency leads to accumulation of heparan sulfate (HS), which triggers aberrant cellular function, inflammation and eventually cell death. There is currently no available treatment against MPS IIIA. In the present study, a chemically modified recombinant human sulfamidase (CM-rhSulfamidase) with disrupted glycans showed reduced glycan receptor mediated endocytosis, indicating a non-receptor mediated uptake in MPS IIIA patient fibroblasts. Intracellular enzymatic activity and stability was not affected by chemical modification. After intravenous (i.v.) administration in mice, CM-rhSulfamidase showed a prolonged exposure in plasma and distributed to the brain, present both in vascular profiles and in brain parenchyma. Repeated weekly i.v. administration resulted in a dose- and time-dependent reduction of HS in CNS compartments in a mouse model of MPS IIIA. The reduction in HS was paralleled by improvements in lysosomal pathology and neuroinflammation. Behavioral deficits in the MPS IIIA mouse model were apparent in the domains of exploratory behavior, neuromuscular function, social- and learning abilities. CM-rhSulfamidase treatment improved activity in the open field test, endurance in the wire hanging test, sociability in the three-chamber test, whereas other test parameters trended towards improvements. The unique properties of CM-rhSulfamidase described here strongly support the normalization of clinical symptoms, and this candidate drug is therefore currently undergoing clinical studies evaluating safety and efficacy in patients with MPS IIIA.

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Section: Discussionmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

Gustavsson

Sjöström

Tjernberg

et al. 2019

Molecular Genetics and Metabolism Reports

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“…This advancement has been directly enabled by the technological advances in liquid chromatography mass spectrometry (LC-MS/MS). Over the past decade, LC-MS/MS has become the preferred platform to analyze chemically or enzymatically degraded oligosaccharides from broad classes of GAGs [ 3 , 8 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. Acid hydrolysis of GAGs coupled with LC-MS/MS quantification is a highly sensitive method that has been successfully applied to biological samples from mouse models of MPS and MPS patient samples [ 19 ].…”

Section: Introductionmentioning

confidence: 99%

High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II

Wang

Bhalla

Ullman

et al. 2020

IJMS

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We recently developed a blood–brain barrier (BBB)-penetrating enzyme transport vehicle (ETV) fused to the lysosomal enzyme iduronate 2-sulfatase (ETV:IDS) and demonstrated its ability to reduce glycosaminoglycan (GAG) accumulation in the brains of a mouse model of mucopolysaccharidosis (MPS) II. To accurately quantify GAGs, we developed a plate-based high-throughput enzymatic digestion assay coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS) to simultaneously measure heparan sulfate and dermatan sulfate derived disaccharides in tissue, cerebrospinal fluid (CSF) and individual cell populations isolated from mouse brain. The method offers ultra-high sensitivity enabling quantitation of specific GAG species in as low as 100,000 isolated neurons and a low volume of CSF. With an LOD at 3 ng/mL and LLOQs at 5–10 ng/mL, this method is at least five times more sensitive than previously reported approaches. Our analysis demonstrated that the accumulation of CSF and brain GAGs are in good correlation, supporting the potential use of CSF GAGs as a surrogate biomarker for brain GAGs. The bioanalytical method was qualified through the generation of standard curves in matrix for preclinical studies of CSF, demonstrating the feasibility of this assay for evaluating therapeutic effects of ETV:IDS in future studies and applications in a wide variety of MPS disorders.

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“…Using this chemical modification protocol, repeated i.v. administration of CM-rhSulfamidase reduced biomarkers HS, the disaccharide GlcNS-UA and the tetrasaccharide, GlcNS-UA-GlcNAc-UA (+1S) in brain of MPS IIIA mice [6,12].…”

Section: Introductionmentioning

confidence: 99%

Impact of chemical modification of sulfamidase on distribution to brain interstitial fluid and to CSF after an intravenous administration in awake, freely-moving rats

Janson

Andersson

Bergquist

et al. 2020

Molecular Genetics and Metabolism Reports

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Mucopolysaccharidosis III A (MPS IIIA) is an autosomal recessive lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. The disorder results in accumulation of heparan sulfate, lysosomal enlargement and cellular and organ dysfunction. Patients exhibit progressive neurodegeneration and behavioral problems and no treatment is currently available. Enzyme replacement therapy is explored as potential treatment strategy for MPS IIIA patients and to modify the disease, sulfamidase must reach the brain. The glycans of recombinant human sulfamidase (rhSulfamidase) can be chemically modified to generate CM-rhSulfamidase. The chemical modification reduced the affinity to the cation-independent mannose-6-phosphate receptor with the aim a prolonged higher concentration in circulation and thus at the blood brain barrier. The pharmacokinetic properties in serum and the distribution to brain and to cerebrospinal fluid (CSF) of chemically modified recombinant human sulfamidase (CM-rhSulfamidase) were studied and compared to those of rhSulfamidase, after a single intravenous (i.v.) 30 mg/kg dose in awake, freely-moving male Sprague Dawley rats. Distribution to brain was studied by microdialysis of the interstitial fluid in prefrontal cortex and by repeated intra-individual CSF sampling from the cisterna magna. Push-pull microdialysis facilitated sampling of brain interstitial fluid to determine large molecule concentrations in awake, freely-moving male Sprague Dawley rats. Together with repeated serum and CSF sampling, push-pull microdialysis facilitated determination of CM-rhSulfamidase and rhSulfamidase kinetics after i.v. administration by non-compartments analysis and by a population modelling approach. Chemical modification increased the area under the concentration versus time in serum, CSF and brain interstitial fluid at least 7-fold. The results and the outcome of a population modelling approach of the concentration versus time data indicated that both compounds pass the BBB with an equilibrium established fairly rapid after administration. We suggest that prolonged high serum concentrations facilitated high brain interstitial fluid concentrations, which could be favorable to reach various target cells in the brain.

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Robust LC–MS/MS Methods for Analysis of Heparan Sulfate Levels in CSF and Brain for Application in Studies of MPS IIIA

Cited by 8 publications

References 30 publications

Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

Intravenous delivery of a chemically modified sulfamidase efficiently reduces heparan sulfate storage and brain pathology in mucopolysaccharidosis IIIA mice

High-Throughput Liquid Chromatography–Tandem Mass Spectrometry Quantification of Glycosaminoglycans as Biomarkers of Mucopolysaccharidosis II

Impact of chemical modification of sulfamidase on distribution to brain interstitial fluid and to CSF after an intravenous administration in awake, freely-moving rats

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