Intranasal delivery of N-terminal modified leptin-pluronic conjugate for treatment of obesity

Abstract

Leptin is an adipocyte-secreted hormone that is delivered via a specific transport system across the blood-brain barrier (BBB) to the brain where it acts on the hypothalamus receptors to control appetite and thermogenesis. Peripheral resistance to leptin due to its impaired brain delivery prevents therapeutic use of leptin in overweight and moderately obese patients. To address this problem, we modified the N-terminal amine of leptin with Pluronic P85 (LepNP85) and administered this conjugate intranasally using the nose-to-brain (INB) route to bypass the BBB. We compared this conjugate with the native leptin, the N-terminal leptin conjugate with poly(ethylene glycol) (LepNPEG5K), and two conjugates of leptin with Pluronic P85 attached randomly to the lysine amino groups of the hormone. Compared to the random conjugates of leptin with P85, LepNP85 has shown higher affinity upon binding with the leptin receptor, and similarly to native hormone activated hypothalamus receptors after direct injection into brain. After INB delivery, LepNP85 conjugate was transported to the brain and accumulated in the hypothalamus and hippocampus to a greater extent than the native leptin and LepNPEG5K and activated leptin receptors in hypothalamus at lower dose than native leptin. Our work suggests that LepNP85 can access the brain directly after INB delivery and confirms our hypothesis that the improvement in brain accumulation of this conjugate is due to its enhanced brain absorption. In conclusion, the LepNP85 with optimized conjugation chemistry is a promising candidate for treatment of obesity.

Keywords: Leptin; Nose-to-brain delivery; Obesity; Pharmacokinetics; Pluronic block copolymer; Protein-polymer conjugation.

Figure 1. Synthesis, purification, and composition characterization of LepNP85 and LepNPEG5K

(a) MALDI-TOF spectra of reaction mixture. The ratio denotes the molar ratio of leptin monomer to polymer. (b) SEC of leptin conjugates eluted from Superdex75 100/300 GL column in 10% MeOH/0.25 M sodium phosphate, pH 7.5 at 0.5 ml/min. (c) SDS-PAGE of pooled fractions in SEC denoted by elution time. Fraction of 20.3–21.9 min in LepNP85 and fraction of 18.5–21 min in LepNPEG5K were used.

Figure 2

SDS-PAGE of the reaction mixture of leptin and Leptin-2PCA with CHO-P85-OH or with P85-DSP.

Figure 3. Far UV-CD spectra of leptin and leptin conjugates

(a) Leptin 1:1 physical mixture with P85 or PEG5K. (b) Leptin 1:1 conjugates.

Figure 4. RP-HPLC analysis of leptin and leptin conjugates

Leptin and leptin 1:1 conjugates were eluted from Jupiter C4 column (particle diameter 5 μm, pore diameter 300 Å, 4.6 × 100 mm) by gradient elution at 1 ml/min and 25 °C, and monitored by absorption at 220 nm. Mobile phase A: water + 0.1% TFA; mobile phase B: acetonitrile + 0.1% TFA: isopropanol 50:50 (v/v). The elution started from 5% B for 5 min, then linearly increased to 95% B at 1%/min, and stayed at 95% B till 100 min.

Figure 5. Binding affinity of leptin and leptin conjugates to leptin receptor by SPR

(a) Representative sensorgram for native leptin. (b) Representative sensorgram for LepNP85 1:1 conjugates. (c) Representative sensorgram for LepDSSP85 1:1 conjugates. (d) Dissociation constants (K_D) of leptin and leptin 1:1 conjugates. Data are mean ± SD, n= 5~12. *** p < 0.001 and n.s. not significant by One-way ANOVA and post Newman-Keuls Multiple Comparison Test.

Figure 6. Phosphorylation of STAT3 in hypothalamus 30 min after ICV injection of leptin and leptin conjugates

(a) Dose response of leptin and LepNP85 1:1 conjugates. Data are mean ± SEM, n=3. (b) Comparison of leptin and leptin 1:1 conjugates at 100 ng/mouse. Data are mean ± SEM, n= 5~7. ** p < 0.01 and *** p < 0.001 by One-way ANOVA and post Newman-Keuls Multiple Comparison Test.

Figure 7. Serum clearance and unidirectional brain influx rate of leptin and leptin conjugates

In each row, the two compounds were labeled with ¹²⁵I and ¹³¹I respectively and co-injected into CD-1 mice by IV injection. (a, d, g) Serum concentration over time. (b, e, h) Linear regression of serum concentration. (c, f, i) Multiple-time regression analysis for unidirectional brain influx rate. n=1~2/time point, **p<0.01 and ***p<0.001 by two-way ANOVA.

Figure 8. Serum absorption and brain regional distribution of intranasally delivered leptin and LepNP85 1:1 conjugate

Male CD-1 mice were co-injected with ¹²⁵I-LepNP85 and ¹³¹I-leptin. Data are mean ± SEM, n=7/time point, * p < 0.05, ** p < 0.01, and *** p < 0.001 by Two-way ANOVA.

Figure 9. Serum absorption and brain distribution of intranasally delivered 1:1 conjugates of LepNP85 and LepNPEG5K

Male CD-1 mice were co-injected with ¹²⁵I-labeled LepNP85 and ¹³¹I-labeled LepNPEG5K. Data are mean ± SEM, n=7/ time point, * p < 0.05 and *** p < 0.001 by Two-way ANOVA.

Figure 10. Serum absorption and brain regional distribution of intranasally delivered 1:1 conjugates of LepNP85 and LepDSPP85

Male CD-1 mice were co-injected with ¹²⁵I-labeled LepNP85 1:1 conjugates and ¹³¹I-labeled LepDSPP85 1:1 conjugates. Data are mean ± SEM, n=7/time point, ** p < 0.01 and *** p < 0.001 by Two-way ANOVA.

Figure 11. Serum absorption and brain regional distribution of intranasally delivered 1:1 conjugates of LepNP85 with or without cold leptin

Each male CD-1 mouse was injected with ¹²⁵I-labeled LepNP85 with or without 10 μg of cold leptin. Data are mean ± SEM, n=7/time point, * p < 0.05 by Two-way ANOVA.

Figure 12. Brain clearance and serum absorption of leptin and LepNP85 by ICV injection

1 μg of biotin-labeled leptin and LepNP85 in 1 μl of PBS were injected locally into brain by ICV injection. The concentrations of biotin-labeled proteins in brain lysate and plasma were analyzed by ELISA. (a) Blood absorption of biotin-leptin and biotin-LepNP85 after ICV injection. (b) Brain retention of biotin-leptin and biotin-LepNP85 after ICV injection. Data are mean ± SEM, n=4/time point, * p < 0.05 by One-way ANOVA and post Dunnett’s Multiple Comparison Test, and *** p < 0.001 by Two-way ANOVA.

Figure 13. Phosphorylation of STAT3 in hypothalamus 1 h after intranasal injection of leptin and LepNP85

(a) At dose of 5 μg/mouse. n=4–11. The western blotting was repeated twice. (b) At dose of 12 μg/mouse. n= 4~5. Data are mean ± SEM, * p < 0.05 by One-way ANOVA and post Newman-Keuls Multiple Comparison Test.