sFlt Multivalent Conjugates Inhibit Angiogenesis and Improve Half-Life In Vivo

Abstract

Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.

Fig 1. Synthesis of mvsFlt schematic.

mvsFlt bioconjugates were synthesized using a 3-step reaction in which HyA was reacted with EDC and EMCH to create a thiol reactive HyA-EMCH intermediate. sFlt was then treated with 2-iminothiolane and reacted with the HyA-EMCH intermediate for the synthesis of the final mvsFlt bioconjugate.

Fig 2. sFlt and mvsFlt equally inhibit corneal angiogenesis.

A) Schematic depicting methods utilized for carrying out corneal burn model. Mice were treated twice with 5μl of PBS, sFlt or mvsFlt at day 1 and 3 following the chemical burn. B) Representative images of eyes treated with PBS, sFlt and mvsFlt. CD31 positive (green) staining of corneal blood vessels. C) Quantification of corneal angiogenesis at day 10 following treatment. One-way ANOVA gives p value **<0.01 (n.s.- not significant; *p<0.05; **p<0.01). Scale bars correspond to 20 μm.

Fig 3. mvsFlt has longer residence time in the rat vitreous.

A) Schematic depicting methods used to determine the half-life of fluorescently tagged sFlt and mvsFlt in the rat vitreous. The vitreous was injected with 5μl of Alexa Fluor 488-tagged sFlt or mvsFlt. After 0, 4, 12, 24, and 48 hours, the rats were sacrificed and their eyes were enucleated and frozen for analysis. The vitreous was then removed, immersed in RIPA buffer and homogenized for subsequent fluorescence measurements. B) Conjugation to HyA significantly improves residence time of sFlt in the vitreous after 48 hours in comparison to sFlt. Results are expressed as mean ±SD (*p<0.05, **p<0.01, ***p<0.001). * indicates a difference between the mvsFlt and sFlt at the given time point. Two-way ANOVA gives p-value ***<0.001.

Fig 4. mvsFlt inhibits retinal angiogenesis.

A) Schematic showing methods used for carrying out the OIR model. Newborn rat pups were housed in normoxic conditions (21% oxygen, room air) from post-natal day (P) 0–7 to allow for normal retinal vasculature development and then transferred to hyperoxic conditions from P7-P12, which induces vessel pruning. At P13, the pups are transferred back into normoxic conditions and treated with 2 μl of PBS, sFlt or mvsFlt and sacrificed at P17. B) Representative images of retinas treated with PBS, sFlt and mvsFlt. Prior to sacrifice, rat pups were injected with fluorescently-tagged 2 MDa dextran to visualize vessel coverage. Scale bar corresponds to 250 μm. Dashed boxes magnify that portion of tissue (scale bar corresponds to 100 μm). C) Quantified retinal vascularization after 5 days of treatment. Percent retinal vascularization was calculated by comparing the area of vascularization to the total retinal area in the image. One-way ANOVA gives p-value***<0.001 (n.s.-not significant; **p<0.01).

Fig 5. Schematic demonstrating the proposed mechanism of mvsFlt action.

A) sFlt (red, unconjugated) and mvsFlt (red conjugated blue chain of HyA) are injected into a diabetic retina where there is a high concentration of VEGF (green circles). B) After a given time, t, the majority of the sFlt has been cleared from the vitreous and VEGF is thus able to induce blood vessel growth. mvsFlt has a longer residence time in the vitreous and is able to bind and inhibit VEGF over much longer periods of time, leading to prolonged inhibition of retinal angiogenesis.