Filgrastim

A total of eleven muscular dystrophy patients were enrolled in this study. Detailed clinical characteristics of all patients are summarized in Table 1. Patients received their current standard treatment which was supplemented only by administration of filgrastim (Neupogen, Amgen) at the following doses: 5 μg/kg of body weight/day for five consecutive days (course 1). Such treatment course was repeated after 1 month (course 2) and after 2 months (course 3).

For mobilization of stem cells to peripheral blood, two sheep received three doses every 24 h of G-CSF (Filgrastim; Amgen, Thousand Oaks, CA, USA) at a dose of 10 μg/kg body weight, subcutaneously (sc). On day 4, they were administered 20 mL of MPB from the jugular vein mixed with heparin 100 IU/mL, also sc.

R-BC154 and BOP were synthesized according to previously published methods21 (link)22 (link). BIO5192 (Tocris Bioscience), AMD3100 (octahydrochloride hydrate salt) (Sigma-Aldrich) and human G-CSF (Filgrastim, Amgen, Thousand Oaks, CA, USA) were used without further purification. For in vivo experiments, lyophilized powders of R-BC154 and BOP sodium salts were dissolved in 10% hydroxypropyl-β-cyclodextrin (HPβCD) in saline (w/v). A stock solution of BIO5192 in DMSO (10 mg ml⁻¹) was diluted to 0.1 mg ml⁻¹ in 10% HPβCD/saline immediately before use.

GCSF-releasing PLLA scaffolds (PLLA/GCSF sample) were prepared by electrospinning as previously described [18 ]. Briefly, GCSF (Filgrastim, 30 MUI/mL, sodium salt, Amgen, Thousand Oaks, CA) was combined to a 13% w/w PLLA (Sigma-Aldrich, St. Louis, MO) polymer solution at a concentration of 250 UI/g, corresponding to the dosage routinely used in literature and in clinical settings [19 (link)]. Procedural parameters [16 (link)] and sterilization techniques [20 ] are described elsewhere.
Pristine PLLA scaffolds (PLLA/CTRL sample) were manufactured using the same experimental conditions to obtain a control for biological experiments.
Mechanical and microstructural characterization of the materials, as well as determination of the drug release profile, have been reported elsewhere [18 ] and performed by means of field emission scanning electron microscopy (FE-SEM), longitudinal uniaxial testing according to Sell et al. with peak stress (PS) and strain at failure (SF) evaluation [21 (link)], and specific ELISA assay for GCSF quantitation.

The activity of HisDapGalNAcT2 was monitored using a glycosyltransferase activity kit (EA001, R&D Systems Europe Ltd., United Kingdom) based on the release of phosphate during transfer of glycosyl residues to an acceptor peptide. UDP-N-acetylgalactosamine was used as donor substrate in combination with the acceptor substrates mucin EA2 (Eurogentec SA Anaspec, Belgium) comprising the peptide sequence PTTDSTTPAPTTK [10 (link)] or Filgrastim (Neupogen®, Amgen Inc.). Prior to use, the elution buffer of the purified HisDapGalNAcT2 (50 mM Tris, 300 mM NaCl, 500 mM imidazole; pH 8) was exchanged by 25 mM Tris, 150 mM NaCl, pH 7.3. The protein sample was stored at 4–8°C for about 3 weeks. Control assays were carried out using purified rhGALNT2 isolated from murine myeloma NS0 cells (7507-GT-020, R&D Systems Europe Ltd., United Kingdom). The enzyme was provided by the manufacturer in 25 mM Tris, 150 mM NaCl, 0.05% w/v Brij-35; pH 7.5 and stored in the freezer at −80°C.
Samples with EA2 as acceptor substrate were desalted, concentrated (ZipTip®_μC18, Millipore, USA) and embedded into a HCCA matrix (α-cyano-4-hydrosycinnamic acid) for subsequent MALDI-TOF-MS analysis. Filgrastim samples were digested with trypsin prior to Electrospray Ionisation Mass Spectrometry (ESI-MS). Both analyses were carried out at the life science center of the University of Hohenheim (Germany).