Anti-Flt1 peptide – Hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy

doi:10.1016/j.biomaterials.2011.01.003

Biomaterials

Volume 32, Issue 11, April 2011, Pages 3115-3123

https://doi.org/10.1016/j.biomaterials.2011.01.003 Get rights and content

Abstract

Anti-angiogenic therapeutics has been investigated extensively for the treatment of retinal and choroidal vascular diseases, and diabetic retinopathy. Anti-Flt1 peptide of GNQWFI is an antagonistic peptide for vascular endothelial growth factor receptor 1 (VEGFR1 or Flt1) inhibiting VEGFR1-mediated endothelial cell migration and tube formation. In this work, anti-Flt1 peptide (GGNQWFI) was chemically conjugated to tetra-n-butyl ammonium modified hyaluronate (HA-TBA) via amide bond formation in dimethyl sulfoxide (DMSO) using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP). The resulting HA – GGNQWFI conjugate self-assembled to form micelle-like nanoparticles in aqueous solution, as confirmed and characterized by transmission electron microscopy (TEM). According to in vitro biological activity tests, HA – GGNQWFI conjugate exhibited a dose-dependent inhibition effect on the binding of Flt1-Fc to VEGF₁₆₅ coated on the well. Furthermore, anti-Flt1 peptide – HA conjugate effectively inhibited retinal choroidal neovascularization (CNV) in laser induced CNV model rats. The retinal vascular permeability and the deformation of retinal vascular structure were also significantly reduced in diabetic retinopathy model rats after treatment with anti-Flt1 peptide – HA conjugate. Pharmacokinetic analysis confirmed the increased mean residence time of anti-Flt1 peptide after conjugation to HA longer than 2 weeks.

Introduction

Retinal neovascularization is a major cause of retinal vascular leakage, which can induce distortion and loss of central vision in patients with retinal diseases. Among them, age-related macular degeneration (AMD) and diabetic retinopathy (DR) are leading causes of blindness all over the world [1], [2], [3]. There are two forms of AMD, dry and wet AMD. Especially, the wet form AMD causes a rapid vision loss and can lead to blindness. Abnormal and fragile blood vessels proliferate in the choroid, which is known as choroidal neovascularization (CNV). The new choroidal vessels leak fluid or blood into the underlying retina causing a significant damage to the retina [2]. Like AMD, DR is also caused by the formation of abnormal blood vessels on the retinal surface [3]. The new fragile blood vessels may leak and bleed into the retina. Under normal conditions, the proliferation of retinal endothelial cells is controlled by the balance between pro-angiogenic factor like vascular endothelial growth factor (VEGF) and anti-angiogenic factor like pigment epithelium derived factor (PEDF) [4]. In both cases of AMD and DR, the expression of VEGF is elevated in the retina leading to angiogenesis and hyperpermeability [5]. Accordingly, the inhibition of VEGF expression and the binding of VEGF to its receptors have been considered as an effective strategy to treat retinal and choroidal vascular diseases, and diabetic retinopathy. Among several anti-angiogenic therapeutics, Ranibizumab (Lucentis^®) and Pegaptanib (Macugen^®) have been approved by the FDA for the treatment of AMD and are currently in clinical trials for diabetic macular edema (DME) [6], [7]. In addition, Bevacizumab (Avastin^®) is being widely used off-label for the treatment of AMD and diabetic retinopathy [8]. Anti-Flt1 peptide is an antagonistic peptide for VEGFR1 (fms-like tyrosine kinase-1 or Flt1) that specifically binds to VEGFR1 inhibiting the interaction of VEGFR1 with a variety of VEGFR1 ligands, such as VEGFA, VEGFB, and placental growth factor (PIGF) [9]. Unlike other monoclonal antibodies or RNA antagonists for VEGFR1, anti-Flt1 peptide is a non-immunogenic hexa-peptide which can be easily synthesized at a low production cost [10]. We previously reported the successful bioconjugation of anti-Flt1 peptide with a sequence of GNQWFI to hyaluronate (HA) [11].

HA is one of the two main structural components of the vitreous in the eye along with collagen [12], [13]. HA was first discovered in bovine vitreous humor in 1934 [14]. In addition to the well-known versatile properties of HA, such as biocompatibility, biodegradability, non-immunogenicity, and non-toxicity, recent studies have shown that HA also plays important biological roles in the regulation of vascular endothelial cell migration [15], proliferation [16], and tube formation [17] depending on its molecular weight. HA has a wide molecular weight range from 1000 to 10,000,000 Da. The native high molecular weight HA in tissues degrades into small molecules during the metabolic pathways through lymphatic system, lymph node, liver, and kidney [18]. There are several kinds of HA receptors, such as CD44, LYVE-1, and HARE [19], [20]. While the half-life of HA is known to be ca. 2.5–5.5 min in plasma [21], it was reported to be ca. 70 d in the vitreous body of eyes [22]. The unique physicochemical properties and various biological functions of HA have led to its wide biomedical applications such as drug delivery [23], [24], arthritis treatment [25], ocular surgery [26], and tissue engineering [27]. In particular, HA has been investigated extensively for target-specific and long-term delivery of bio/pharmaceuticals through various delivery routes [23]. Taking advantage of its viscoelastic and mucoadhesive properties, HA has been exploited as an effective delivery carrier of topical ophthalmic drugs [28]. A number of studies have reported that HA extended the ocular residence time of ophthalmic drugs [29], [30], [31]. In addition, the intraocular injection of HA hydrogel encapsulating gentamicin resulted in the increased half-life in vitrectomized eyes exhibiting an enhanced therapeutic effect on endophthalmitis [32].

In this work, anti-Flt1 peptide – HA conjugates were synthesized and prepared in the form of micelle-like nanoparticles, which were applied as anti-angiogenic therapeutics to the treatment of retinal neovascularization and diabetic retinopathy. After the introduction of glycine to the end of peptide sequence, anti-Flt1 peptide (GGNQWFI) was conjugated to HA-TBA in anhydrous dimethyl sulfoxide (DMSO) using benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) chemistry. The resulting HA – GGNQWFI conjugate self-assembled to form micelles in aqueous solution, which was confirmed and characterized by transmission electron microscopy (TEM). In vitro dose-dependent biological activity of HA – GGNQWFI conjugate was assessed by measuring inhibition effect on the binding of Flt1-Fc to VEGF₁₆₅ coated on the well. With pharmacokinetic analysis, anti-angiogenic effect of HA – GGNQWFI conjugate on retinal choroidal neovascularization (CNV) and diabetic retinopathy was assessed in the laser induced CNV model rats and experimental diabetic rats, respectively.

Section snippets

Materials

Sodium hyaluronate (HA) with a molecular weight of 100 kDa was obtained from Shiseido Co. (Tokyo, Japan). Anti-Flt1 peptide with a sequence of Gly-Gly-Asn-Gln-Trp-Phe-Ile (GGNQWFI) was purchased from Peptron Co. (Daejeon, Korea). Dowex^® 50WX8-40 ion-exchange resin, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), N,N-diisopropyl ethylamine (DIPEA), and streptozotocin were purchased from Sigma–Aldrich (St. Louis, MO). Tetra-n-butyl ammonium hydroxide (TBA-OH) was

Anti-Flt1 peptide – HA conjugate micelles in aqueous solution

Water-insoluble anti-Flt1 peptide (GNQWFI), an effective anti-angiogenic drug, was chemically conjugated to HA in organic solvent for the treatment of retinal neovascularization and diabetic retinopathy. Fig. 1a shows the primary structure of anti-Flt1 peptide. There are two amino acids with a ring structure in anti-Flt1 peptide, tryptophan (W) and phenylalanine (F). They are located close to the C-terminal which is the opposite side of glycine, and their aromatic side chains are relatively

Conclusions

Anti-Flt1 peptide, a water-insoluble anti-angiogenic peptide drug, was successfully conjugated to HA-TBA by the formation of amide linkage in anhydrous DMSO using BOP as a coupling reagent. The resulting anti-Flt1 peptide – HA conjugates self-assembled to form spherical micelle-like nanoparticles in aqueous solution with an average diameter of 234.5 ± 20.6 nm. According to in vitro test, anti-Flt1 peptide – HA conjugate showed a dose-dependent biological activity inhibiting the binding of

Acknowledgment

This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080711). This work was also supported by Mid-career Researcher Program through NRF grant funded by the MEST (No. 2009-0084578).

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¹: These authors contributed equally to this work.

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Anti-Flt1 peptide – Hyaluronate conjugate for the treatment of retinal neovascularization and diabetic retinopathy

Abstract

Introduction

Section snippets

Materials

Anti-Flt1 peptide – HA conjugate micelles in aqueous solution

Conclusions

Acknowledgment

Prog Retin Eye Res

Biomaterials

J Biol Chem

J Invest Dermatol

J Biol Chem

Eur J Cell Biol

J Biol Chem

J Control Release

J Control Release

Biomaterials

Exp Eye Res

J Biol Chem

Age-related macular degeneration - emerging pathogenetic and therapeutic concepts

Ann Med

Age-related macular degeneration (AMD): pathogenesis and therapy

Pharmacol Rep

Diabetic retinopathy

N Engl J Med

Endothelial/pericyte interactions

Circ Res

Upregulation of the vascular endothelial growth factor/vascular endothelial growth factor receptor system in experimental background diabetic retinopathy of the rat

Diabetes