Study design
This is an Investigator Initiated Study supported by a research grant from Sanofi-Genzyme for its design and conduct. The study was sponsored by Manchester University NHS Foundation Trust.
The parent study was a phase 1, open-label, within-patient, repeat-dose, dose-escalation study as previously described 4. In brief, five adult patients (3 males and 2 females) with NPD-B were recruited to the parent study. Informed consent was obtained from all participants before the conduct of any study-related procedures. Participants consented to the storage of samples from the study for use in future ethically approved studies. All 5 patients had hepatosplenomegaly and 4 had thrombocytopenia. 2 of the patients were on stable regimen of lipid-lowering therapy, viz simvastatin 20mg and 40 mg daily respectively and the statin dose was unchanged during the study. Patients received escalating doses (0.1 to 3.0 mg/kg) of olipudase alfa intravenously. The initial dose of 0.1 mg/kg was given on day 1. This was followed 2 weeks later by 0.3 mg/kg. After 2 consecutive doses of 0.3 mg/kg, dose escalation continued at 0.6, 1.0, 2.0 and 3.0 mg/kg, the last dose was maintained until week 26. Patients who experienced adverse events more than mild in severity either stayed on the same dose or received a reduced dose at the next infusion. All patients were successfully escalated to the target dose of 3.0 mg/kg and they all completed the study at week 26.
Samples made available to us from the parent study included pre-ERT serum samples and plasma samples pre- and post-ERT at 4 different time points: day 1, week 8, week 16 and week 26. All the blood samples at these time points were taken 24 hours post-infusion. All study samples were taken in the fasting state. The ERT doses at these times points were 0.1 mg/kg, 1.0 mg/kg, 3.0 mg/kg and 3.0 mg/kg respectively. All samples had been stored at -80°C and were transported to our site on dry ice.
For our study, fifteen age- and sex-matched healthy participants were recruited as a reference group from Manchester University NHS Foundation Trust (Manchester, UK) and University of Manchester (Manchester, UK). Reference subjects had no significant pre-existing medical conditions and were not on any regular medications. Patient Information Sheet was given to all eligible subjects and informed consent was obtained from those who agreed to participate before a single fasting blood sample of 60 ml was obtained from each reference subject.
Laboratory Methods
Total cholesterol, triglycerides, direct high-density lipoprotein cholesterol (HDL-C), small dense low-density lipoprotein cholesterol (sdLDL-C), apolipoprotein A1 (apoA1), and apolipoprotein B100 (apoB100) were measured using an RX Daytona auto-analyzer (Randox Laboratories Ltd, Crumlin, UK). Low-density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation 6.
ApoB-depleted serum prepared and cholesterol efflux assay was conducted as described before 7,8. Briefly, J774A.1 cells were pelleted and incubated for 24 hours with 0.2 µCi of radiolabelled 3H-cholesterol in RPMI 1640 medium with 0.2% BSA at 37°C in a humidified atmosphere containing 5% carbon dioxide. ABCA1 is upregulated using medium containing 0.3 mM C-AMP (8-(4-Chlorophenylthio) adenosine 3′, 5′-cyclic monophosphate sodium salt) for 4 hours. These cells were then incubated with 2.8% apoB-depleted serum using polyethylene glycol (PEG MW8000) for 4 hours. After incubation, the cell media were collected and cells were washed with PBS and dissolved in 0.5 mL 0.2 N NaOH to determine radioactivity. Cellular cholesterol efflux was expressed as the percentage of radioactivity in the medium from the radioactivity in the cells and medium collectively. The intra-assay and inter-assay coefficients of variation were 3.9% and 7.3 % respectively. Cholesterol efflux was calculated using the following formula:
![](https://myfiles.space/user_files/58653_1b1c6aeb34a62c68/58653_custom_files/img1610746154.jpg)
Serum paraoxonase (PON-1) activity was determined using paraoxon (O, O-Diethyl O-(4-nitrophenyl) phosphate) as a substrate (Sigma-Aldrich Company Ltd) using a RX Daytona auto-analyzer (Randox Laboratories Ltd) 9. The intra-assay and inter-assay coefficients of variation were 3.5% and 2.7%, respectively, for the measurement of PON1 activity.
The following tests were done by enzyme linked immunosorbent assay (ELISA) methods: oxLDL 10 and apoB48 11 (Elabscience, Houston, USA.) , intra- and inter-assay coefficients of variation 5.6% and 5.3%, and 4.9% and 7.7% respectively; PCSK9 mass 12 (R&D Systems, Abingdon, UK.), intra-assay coefficient of variation 4.9%; TNF-α 13 (Invitrogen, Fisher Scientific UK Ltd.), intra- and inter-assay coefficients of variation 8.5% and 9.8%.
Statistical Analyses
Statistical analyses were performed using SPSS for Mac (Version 23.0, IBM SPSS Statistics, Armonk, New York, USA) and figures were produced using GraphPad Prism for Mac (Version 7.00, GraphPad Software, La Jolla California, USA). Data were presented as mean and standard deviation for all variables. The independent samples t-test was used for comparison between NPD-B and control groups. A P-value of less than 0.05 was considered to be statistically significant. Changes after ERT is expressed as percentage.