Development of a Biosimilar of Agalsidase Beta for the Treatment of Fabry Disease: Preclinical Evaluation.

BACKGROUND AND OBJECTIVE: Fabry disease (FD) is a rare lysosomal storage disorder caused by a deficiency of the enzyme α-galactosidase A (aGal A). Since 2001, two different enzyme replacement therapies have been authorized, with agalsidase beta being used in most parts of the Western world. Currently, biosimilars of several expensive enzyme therapies are under development to improve their accessibility for patients. We present the preclinical results of the development of a biosimilar to agalsidase beta. METHODS: Produced in a Chinese hamster ovary (CHO)-cell system, the biosimilar aGal A Biosidus (AGABIO), was compared with agalsidase beta with respect to amino acid sequence, glycosylation, specific α-galactosidase activity, stability in plasma, and effects on cultured human Fabry fibroblasts and Fabry mice. RESULTS: AGABIO had the same amino acid composition and similar glycosylation, enzymatic activity, and stability as compared with agalsidase beta. After uptake in fibroblasts, α-galactosidase A activity increased in a dose-dependent manner, with maximum uptake observed after 24 h, which remained stable until at least 48 h. Both enzymes were localized to lysosomes. Reduction of accumulated globotriaosylceramide (Gb3) and lysoGb3 in cultured Fabry fibroblasts by AGABIO and agalsidase beta showed comparable dose-response curves. In Fabry knockout mice, after a single injection, both enzymes were rapidly cleared from the plasma and showed equal reductions in tissue and plasma sphingolipids. Repeated dose studies in rats did not raise any safety concerns. Anti-drug antibodies from patients with FD treated with agalsidase beta showed equal neutralization activity toward AGABIO. CONCLUSION: These findings support the biosimilarity of AGABIO in comparison with agalsidase beta. The clinical study phase is currently under development.

Skin biomarkers predict development of atopic dermatitis in infancy.

BACKGROUND: There is currently no insight into biomarkers that can predict the onset of pediatric atopic dermatitis (AD). METHODS: Nested in a prospective birth cohort study that examined the occurrence of physician-diagnosed AD in 300 children, 44 random children with onset of AD in the first year of life were matched on sex and season of birth with 44 children who did not develop AD. Natural moisturizing factor (NMF), corneocyte surface protrusions, cytokines, free sphingoid bases (SBs) of different chain lengths and their ceramides were analyzed from tape strips collected at 2 months of age before onset of AD using liquid chromatography, atomic force microscopy, multiplex immunoassay, and liquid chromatography mass spectrometry, respectively. RESULTS: Significant alterations were observed for four lipid markers, with phytosphingosine ([P]) levels being significantly lower in children who developed AD compared with children who did not (median 240 pmol/mg vs. 540 pmol/mg, p < 0.001). The two groups of children differed in the relative amounts of SB of different chain lengths (C17, C18 and C20). Thymus- and activation-regulated chemokine (TARC/CCL17) was slightly higher in children who developed AD, whereas NMF and corneocyte surface texture were similar. AD severity assessed by the eczema area and severity index (EASI) at disease onset was 4.2 (2.0;7.2). [P] had the highest prediction accuracy among the biomarkers (75.6%), whereas the combination of 5 lipid ratios gave an accuracy of 89.4%. CONCLUSION: This study showed that levels and SB chain length were altered in infants who later developed AD, and that TARC/CCL17 levels were higher.

Children with atopic dermatitis show increased activity of ß-glucocerebrosidase and stratum corneum levels of glucosylcholesterol that are strongly related to the local cytokine milieu.

BACKGROUND: Atopic dermatitis (AD) is characterized by immune dysregulations and an impaired skin barrier, including abnormalities in lipid organization. In the stratum corneum (SC), ß-glucocerebrosidase (GBA) mediates transformation of glucosylceramide (GlcCER) into ceramide (CER) and cholesterol into glucosylcholesterol (GlcChol). Alteration in GBA activity might contribute to skin barrier defects in AD. OBJECTIVES: To investigate GBA activity in the SC of children with AD before and after topical corticosteroid therapy and to compare it with healthy controls; to determine SC levels of GlcCER- and CER-containing hydroxysphingosine base (GlcCER[H] and CER[H], respectively) and GlcChol; and to relate them to disease severity, skin barrier function and the local cytokine milieu. METHODS: Lipid markers and cytokines of innate, T helper 1 and T helper 2 immunity were determined in SC collected from healthy children and from clinically unaffected skin of children with AD, before and after 6 weeks of therapy with topical corticosteroids. AD severity was assessed by Scoring Atopic Dermatitis and skin barrier function by transepidermal water loss (TEWL). RESULTS: Baseline GBA activity and GlcChol levels were increased in children with AD but declined after therapy. CER[H] levels and the CER[H] to GlcCER[H] ratio were increased in AD. GBA activity and GlcChol correlated with TEWL and levels of multiple cytokines, especially interleukin-1α and interleukin-18. GlcChol was strongly associated with disease severity. CONCLUSIONS: We show increased GBA activity and levels of GlcChol in AD. Our data suggest an important role of inflammation in disturbed lipid processing. GBA activity or GlcChol might be useful biomarkers in the monitoring of therapeutic responses in AD. What is already known about this topic? Patients with atopic dermatitis (AD) have a reduced skin barrier, mainly caused by altered lipid organization. The mechanisms underlying these lipid anomalies are not fully understood but likely reflect both genetic abnormalities in AD skin and the local cutaneous inflammatory environment. What does this study add? We show increased activity of the ceramide-generating enzyme ß-glucocerebrosidase in AD. Activity of this enzyme was correlated with the local cytokine milieu and declined after local corticosteroid therapy. We show that glucosylcholesterol levels in the stratum corneum are increased in AD. The function of glucosylcholesterol and the physiological consequences of increased levels are not clear yet; however, its levels were strongly correlated with skin barrier function: high transepidermal water loss strongly correlated with high levels of glucosylcholesterol. What is the translational message? Correction of cutaneous inflammation largely restores alterations in lipid metabolism in the stratum corneum of infants with AD.

An improved functional assay in blood spot to diagnose Barth syndrome using the monolysocardiolipin/cardiolipin ratio.

Barth syndrome is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and neutropenia, caused by deleterious variants in TAFAZZIN. This gene encodes a phospholipid-lysophospholipid transacylase that is required for the remodeling of the mitochondrial phospholipid cardiolipin (CL). Biochemically, individuals with Barth syndrome have a deficiency of mature CL and accumulation of the remodeling intermediate monolysocardiolipin (MLCL). Diagnosis typically relies on mass spectrometric measurement of CL and MLCL in cells or tissues, and we previously described a method in blood spot that uses a specific MLCL/CL ratio as diagnostic biomarker. Here, we describe the evolution of our blood spot assay that is based on the implementation of reversed phase-UHPLC separation followed by full scan high resolution mass spectrometry. In addition to the MLCL/CL ratio, our improved method also generates a complete CL spectrum allowing the interrogation of the CL fatty acid composition, which considerably enhances the diagnostic reliability. This addition negates the need for a confirmatory test in lymphocytes thereby providing a shorter turn-around-time while achieving a more certain test result. As one of the few laboratories that offer this assay, we also evaluated the diagnostic yield and performance from 2006 to 2021 encompassing the use of both the original and improved assay. In this period, we performed 796 diagnostic analyses of which 117 (15%) were characteristic of Barth syndrome. In total, we diagnosed 93 unique individuals with Barth syndrome, including three females, which together amounts to about 40% of all reported individuals with Barth syndrome in the world.

Contributions of amino acid, acylcarnitine and sphingolipid profiles to type 2 diabetes risk among South-Asian Surinamese and Dutch adults.

INTRODUCTION: People of South Asian origin are at high risk of type 2 diabetes (T2D), but the underpinning mechanisms are not fully understood. We determined ethnic differences in acylcarnitine, amino acid and sphingolipid concentrations and determined the associations with T2D. RESEARCH DESIGN AND METHODS: Associations between these metabolites and incident T2D among Dutch and South-Asian Surinamese were determined in participants from the Healthy Life in an Urban Setting (HELIUS) study (Amsterdam, the Netherlands) using Prentice-weighted Cox regression. The HELIUS study includes 95 incident T2D cases and a representative subcohort of 700 people from a cohort of 5977 participants with a mean follow-up of 4 years. RESULTS: Concentrations of acylcarnitines were comparable between both ethnic groups. Amino acid and lactosylceramide concentrations were higher among South-Asian Surinamese than Dutch (eg, isoleucine 65.7 (SD 16.3) vs 60.7 (SD 15.6) µmol/L). Ceramide concentrations were lower among South-Asian Surinamese than Dutch (eg, Cer d18:1 8.48 (SD 2.04) vs 9.08 (SD 2.29) µmol/L). Metabolic dysregulation preceded T2D without evidence for a multiplicative interaction by ethnicity. Most amino acids and (dihydro)ceramides were associated with increased risk (eg, Cer d18:1 HR 2.38, 95% CI 1.81 to 3.12) while acylcarnitines, glycine, glutamine and lactosylceramides were associated with decreased risk for T2D (eg, LacCer d18:2 HR 0.56, 95% CI 0.42 to 0.77). CONCLUSIONS: Overall, these data suggest that the disturbances underlying amino acid and sphingolipid metabolism may be predictive of T2D risk in populations of both South Asian and European background. These observations may be used as starting point to unravel the underlying metabolic disturbances.

Altered Levels of Sphingosine, Sphinganine and Their Ceramides in Atopic Dermatitis Are Related to Skin Barrier Function, Disease Severity and Local Cytokine Milieu.

Dysfunctional skin barrier plays a key role in the pathophysiology of atopic dermatitis (AD), a common inflammatory skin disease. Altered composition of ceramides is regarded as a major cause of skin barrier dysfunction, however it is not clear whether these changes are intrinsic or initiated by inflammation and aberrant immune response in AD. This study investigated the levels of free sphingoid bases (SBs) sphingosine and sphinganine and their ceramides and glucosylceramide in the stratum corneum (SC) and related them to skin barrier function, disease severity and local cytokine milieu. Ceramides were measured in healthy skin, and lesional and non-lesional skin of AD patients by a novel method based on deacylation of ceramides which were subsequently determined as corresponding sphingoid bases by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The cytokine levels were determined by multiplex immunoassay. Atopic skin showed increased levels of most investigated markers, predominantly in lesional skin. The largest difference in respect to healthy skin was found for glucosylceramide with respective median values of 0.23 (IQR 0.18-0.61), 0.56 (IQR 0.32-0.76) and 19.32 (IQR 7.86-27.62) pmol/g protein for healthy, non-lesional and lesional skin. The levels of investigated ceramide markers were correlated with disease severity (scoring atopic dermatitis, SCORAD) and skin barrier function (trans-epidermal water loss, TEWL) and furthermore with cytokines involved in innate, Th-1, and Th-2 immune response. Interestingly, the strongest association with SCORAD was found for sphinganine/sphingosine ratio (r = -0.69, p < 0.001; non-lesional skin), emphasizing the importance of SBs in AD. The highest correlation with TEWL was found for glucosylceramide (r2 = 0.60, p < 0.001), which was investigated for the first time in AD. Findings that the changes in SBs and ceramide levels were predominant in lesional skin and their association with disease severity and cytokine levels suggest an immune-system driven effect. a novel analysis method demonstrates a robust and simple approach that might facilitate wider use of lipid biomarkers in the clinics e.g., to monitor (immune) therapy or dissect disease endotypes.

Inhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis.

BACKGROUND/AIMS: Carnitine palmitoyl-transferase I (CPT I) catalyses the synthesis of long-chain (LC)-acylcarnitines from LC-acyl-CoA esters. It is the rate-limiting enzyme of mitochondrial fatty acid beta-oxidation (FAO) pathway and its activity is regulated by malonyl-CoA. The antiepileptic drug valproic acid (VPA) is a branched chain fatty acid that is activated to the respective CoA ester in the intra- and extra-mitochondrial compartments. This drug has been associated with a clear inhibition of mitochondrial FAO, which motivated our study on its potential effect on hepatic CPT I. METHODS: To investigate the effect of valproyl-CoA (VP-CoA) on CPT I, we performed in vitro studies using control human fibroblasts and rat CPT IA expressed in Saccharomyces cerevisiae. In addition to the wild-type enzyme, two mutant rCPT IAs were studied, one of which showing increased sensitivity towards malonyl-CoA (S24A/Q30A), whereas the other one is insensitive to malonyl-CoA (E3A). RESULTS: We demonstrate that VP-CoA inhibits the CPT I activity in control fibroblasts. Similar results were obtained using rCPT IA WT and S24A/Q30A. Importantly, VP-CoA also inhibited the activity of the rCPT IA E3A. We show that VP-CoA inhibits CPT IA competitively with respect to palmitoyl-CoA, and non-competitively to carnitine. Evidence is provided that VP-CoA interferes at the catalytic domain of CPT IA affecting the sensitivity for malonyl-CoA. CONCLUSIONS: The interference of VP-CoA with CPT IA, a pivotal enzyme in mitochondrial fatty acid beta-oxidation, may be a crucial mechanism in the drug-induced hepatotoxicity and the weight gain frequently observed in patients under VPA therapy.

Adeno-associated virus gene therapy with cholesterol 24-hydroxylase reduces the amyloid pathology before or after the onset of amyloid plaques in mouse models of Alzheimer's disease.

The development of Alzheimer's disease (AD) is closely connected with cholesterol metabolism. Cholesterol increases the production and deposition of amyloid-beta (Abeta) peptides that result in the formation of amyloid plaques, a hallmark of the pathology. In the brain, cholesterol is synthesized in situ but cannot be degraded nor cross the blood-brain barrier. The major exportable form of brain cholesterol is 24S-hydroxycholesterol, an oxysterol generated by the neuronal cholesterol 24-hydroxylase encoded by the CYP46A1 gene. We report that the injection of adeno-associated vector (AAV) encoding CYP46A1 in the cortex and hippocampus of APP23 mice before the onset of amyloid deposits markedly reduces Abeta peptides, amyloid deposits and trimeric oligomers at 12 months of age. The Morris water maze (MWM) procedure also demonstrated improvement of spatial memory at 6 months, before the onset of amyloid deposits. AAV5-wtCYP46A1 vector injection in the cortex and hippocampus of amyloid precursor protein/presenilin 1 (APP/PS) mice after the onset of amyloid deposits also reduced markedly the number of amyloid plaques in the hippocampus, and to a less extent in the cortex, 3 months after the injection. Our data demonstrate that neuronal overexpression of CYP46A1 before or after the onset of amyloid plaques significantly reduces Abeta pathology in mouse models of AD.

Cardiolipin and monolysocardiolipin analysis in fibroblasts, lymphocytes, and tissues using high-performance liquid chromatography-mass spectrometry as a diagnostic test for Barth syndrome.

Barth syndrome (BTHS) is an X-linked recessive disorder caused by mutations in the tafazzin (or TAZ) gene and is clinically characterized by (cardio)myopathy, neutropenia, and growth abnormalities. Biochemical abnormalities include decreased levels of the mitochondrial phospholipid cardiolipin, increased levels of monolysocardiolipin, and a lower degree of unsaturation of the (monolyso)cardiolipin acyl chains. Diagnostic testing for BTHS is routinely performed by TAZ gene sequencing, and recently a BTHS screening method in bloodspots has been developed, but both methods have important limitations. Because a validated confirmatory method is not yet available, we set up and validated a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method for BTHS in cultured fibroblasts, lymphocytes, and skeletal muscle based on cardiolipin, monolysocardiolipin, and the monolysocardiolipin/cardiolipin ratio. In addition, we performed retrospective analysis of 121 muscle samples of patients with myopathy of which mitochondrial origin was presumed, and we identified one patient with cardiolipin abnormalities similar to BTHS patients. Molecular analysis revealed a bona fide mutation in the TAZ gene. We conclude that (monolyso)cardiolipin analysis by HPLC-MS not only is a powerful tool to diagnose patients with clinical signs and symptoms of BTHS but also should be used in patients suffering from mitochondrial myopathies with unknown etiology.

Bloodspot assay using HPLC-tandem mass spectrometry for detection of Barth syndrome.

BACKGROUND: Barth syndrome (BTHS) is a serious X-linked, metabolic, multisystem disorder characterized by cardiomyopathy, neutropenia, myopathy, and growth delay. Because early diagnosis and appropriate treatment are of key importance for the survival of affected boys, we developed a biochemical BTHS screening method based on analysis of the monolysocardiolipin:cardiolipin ratio in bloodspots. METHODS: We performed chloroform/methanol extraction on quarter-inch punches of dried bloodspots on Guthrie cards from BTHS patients and controls. Extracts were dried (60 degrees C, N(2)) and reconstituted in CHCl(3)/methanol/H(2)O [50:45:5 vol/vol/vol, 0.1% NH(3) (25%)]. HPLC-tandem mass spectrometry analysis was performed with a normal-phase HPLC column and multiple reaction monitoring transitions for monolysocardiolipin (MLCL) and cardiolipin (CL) with a total run time of 10 min. The ratio of MLCL and CL was used as screening parameter. RESULTS: All BTHS patients (n = 31) had monolysocardiolipin:cardiolipin ratios >0.40 and all controls (n = 215) had monolysocardiolipin:cardiolipin ratios <0.23. Using a cutoff point of 0.30, a blind test of 206 samples (199 controls, 7 BTHS) had sensitivity and specificity of 100%. Bloodspots could be stored at 4 degrees C or room temperature for >1 year without affecting the test outcome. Three neonatal Guthrie cards of BTHS patients taken 3.6 to 5.8 years previously were correctly identified as positive for BTHS. CONCLUSIONS: HPLC-tandem mass spectrometry analysis of dried bloodspots is an unambiguous screening test for BTHS with potential for rapid screening of neonates suspected of having BTHS, making remote and retrospective diagnosis accessible for a disease that is almost certainly underdiagnosed.