Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice

Abstract

Ciliary neurotrophic factor (CNTF) is a neurotrophic cytokine able to induce appetite reduction, weight loss and antidiabetic effects. However, its susceptibility to neutralizing anti-CNTF antibodies in patients hampered its use for treatment of human obesity and diabetes. In addition, CNTF has a very short plasma half-life, which limits its use as a therapeutic agent. Solutions, directed to prolong its in vivo effects, vary from the implantation of encapsulated secreting cells to identification of more active variants or chemical modification of the protein itself. PEGylation is a widely used modification for shielding proteins from circulating antibodies and for increasing their plasma half-life. Here, we have selected DH-CNTF, a CNTF variant which has a 40-fold higher affinity for the CNTF receptor α accompanied by an increased activity in cellular assays. The PEGylated DH-CNTF retained the biological activity of native protein in vitro and showed a significant improvement of pharmacokinetic parameters. In an acute model of glucose tolerance, the PEG-DH-CNTF was able to reduce the glycemia in diet-induced obese animals, with a performance equaled by a 10-fold higher dose of DH-CNTF. In addition, the PEGylated DH-CNTF analog demonstrated a more potent weight loss effect than the unmodified protein, opening to the use of CNTF as weight reducing agent with treatment regimens that can better meet patient compliance thanks to reduced dosing schedules.

Fig 1. Biological activity of CNTF and DH-CNTF.

HepG2 cells were treated with CNTF and DH-CNTF in the absence and presence of different amount of soluble CNTFRα. Results were expressed as a percentage of the maximal CNTF-induced response. Each concentration point was the mean ± S.E. from at least three independent experiments.

Fig 2. SDS-PAGE analysis.

MW markers (lane 1), purified starting material (DH-CNTF, lanes 2 and 3) and PEG-DH-CNTF (lanes 4).

Fig 3. CD analysis of DH-CNTF and PEG-DH-CNTF at different temperatures.

CD spectra were recorded at 20°C, 95°C and after cooling the samples from 95°C to 20°C. Samples were solubilized in water.

Fig 4. BPI chromatograms of LC-MS^E analysis of DH-CNTF (black) and PEG20k-DH-CNTF (red) upon digestion with trypsin.

The peak labelled with an arrow in the figure is the Cys17-containing peptide F3 [–19].

Fig 5. Biological activity of DH-CNTF and PEG-DH-CNTF in HepG2 cells.

Results were expressed as a percentage of the maximal DH-CNTF-induced response and represent the mean ± S.E. from duplicate determinations. Data were from representative experiment repeated several times with comparable results.

Fig 6

Plasma-time profiles in mouse (A) and rat (B) after IV injection of DH-CNTF and PEG-DH-CNTF. Values were the mean ± S.D. of duplicate determination of plasma obtained at different time points from 3 animals per group.

Fig 7. Plasma-time profiles concentration in mouse after SC injection of DH-CNTF and PEG-DH-CNTF.

Fig 8. Effect of short-term administration of DH-CNTF and PEG-DH-CNTF in DIO mice.

A) Body weight effect of 0.2 mg/kg and 1 mg/kg DH-CNTF and PEG-DH-CNTF (0.2 mg CNTF equivalent/kg), after daily SC administration. Body weight is expressed as grams starting before the first treatment and represents the mean ± SEM from 6 animals per group. Control group was treated with vehicle (0.9% saline/0.2 mg/mL endotoxin-free BSA). The arrow indicates the duration of treatment, starting from day 1 to day 5. B) Daily food consumption. Food consumption was calculated as the difference from the quantity proposed the day before and the remaining quantity. Statistical analysis (two-way ANOVA test: post-hoc Tukey’s Multiple Comparison Test) was performed for each group versus vehicle-treated group (*P < 0.05; **P < 0.01, ***P < 0.001).

Fig 9. Effect on insulin and glucose in animal used for the weight loss.

Statistical analysis (ANOVA test: post-hoc Dunnett’s Multiple Comparison Test) was performed for each group versus vehicle-treated group (*P < 0.05; **P < 0.01).

Fig 10. Intraperitoneal glucose tolerance test (IPGTT).

Glucose levels, in DIO ♂ mice after IP administration of 1.5 mg/kg of glucose, were measured at 30 minutes intervals until 2 h.