Modified peptides and their use for treating autoimmune diseases
A technology for autoimmune diseases and immunosuppression, applied in the field of modified peptides, which can solve the problems of peptide instability and inactivity
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[0141] Statistics
[0142] Statistical tests were performed using GraphPad Prism version 5.0. The statistical significance of the difference in urine protein between control and peptide-treated mice was analyzed using a two-way ANOVA test. The survival rate of control and P140 analogue-treated female MRL / lpr mice was analyzed by Kaplan-Meier method, and the significance of the difference was determined by Log-Rank test. For other variables, statistical significance was assessed using Student's t-test. A p-value less than 0.05 was considered significant.
Embodiment 1
[0143] Example 1 Chemical synthesis of peptides.
[0144] P140 peptide and P140(MO) were synthesized by classical fluorenylmethoxycarbonyl (N-[9-fluorenyl]methoxycarbonyl) solid-phase chemistry and purified by reverse-phase high-performance liquid chromatography (HPLC; Neimark and Briand, 1993 ; Monneaux et al., 2003, Eur. J. Immunol. 33, 287-296; Page et al., 2009, PloS ONE 4, e5273). Their homogeneity was checked by analytical HPLC and their identity assessed by LC / MS on a Finnigan LCQ Advantage Max system (Thermo Fischer Scientific). After the reaction was complete, the peptide was purified by HPLC.
[0145] To introduce phosphorylation at the position of the serine residue corresponding to residue 140 of sequence 3, a serine derivative of the Fmoc-Ser(PO(Obz)OH)-OH-type was used. The connection time was increased to 30 min, and a second connection was performed systematically. After unraveling in acid medium, each peptide was precipitated by cold ether, dissolved in wat...
Embodiment 2
[0147] Example 2 Peptide Stability.
[0148] At 37°C, sequence 2 (wherein the serine at position 10 is phosphorylated and the methionine at position 4 is oxidized (P140(MO)) and sequence 1 (wherein the serine at position 10 is oxidized) is determined in 10% (v / v) mannitol solution. Stability of peptides that were phosphorylated (P140). Three concentrations were tested for each peptide: 200, 100 and 50 μg / mL.
[0149] At the indicated times, the integrity of the P140 and P140(M(O) peptides was determined from the peak areas corresponding to the intact peptides by HPLC in saline solution.
[0150] The result is as figure 1 shown.
[0151] Tables 1 and 2 below summarize the results:
[0152] Table 1:
[0153]
[0154] Table 2:
[0155]
[0156] Stability was determined by using HPLC peak surfaces.
[0157] P140M(O) stability remained unchanged (100%, 99.1% and 99.4%) for each concentration tested (50 to 200 μg / ml) over 100 days at 37°C.
[0158] At 37°C, P140 stabilit...
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