Ocular delivery of proteins and peptides: Challenges and novel formulation approaches

Abstract

The impact of proteins and peptides on the treatment of various conditions including ocular diseases over the past few decades has been advanced by substantial breakthroughs in structural biochemistry, genetic engineering, formulation and delivery approaches. Formulation and delivery of proteins and peptides, such as monoclonal antibodies, aptamers, recombinant proteins and peptides to ocular tissues poses significant challenges owing to their large size, poor permeation and susceptibility to degradation. A wide range of advanced drug delivery systems including polymeric controlled release systems, cell-based delivery and nanowafers are being exploited to overcome the challenges of frequent administration to ocular tissues. The next generation systems integrated with new delivery technologies are anticipated to generate improved efficacy and safety through the expansion of the therapeutic target space. This review will highlight recent advances in formulation and delivery strategies of protein and peptide based biopharmaceuticals. We will also describe the current state of proteins and peptides based ocular therapy and future therapeutic opportunities.

Keywords: AMD; Barriers; Biologics; Biopharmaceuticals; Controlled release; Drug delivery; Eye; Macromolecules; Targeting.

Fig. 1

Numbers of Phase 3 products by technology type for ophthalmic indications (Till Nov., 2015): MIGS (minimally invasive glaucoma surgery); NCE (New chemical entity)

Fig. 2

Number of companies classified by technology as well as global areas for ophthalmology market: This analysis does not include multinational companies, as these entities cannot be defined by a single technology and any one country. Note that the classification “Europe” excludes Scotland to avoid double counting.

Fig. 3

Novel drug R&D venture funding by disease area, 2004–2008 vs 2009–2013

Fig. 4

(A) Effect of intraperitoneally injected therapeutic Vasotide peptide on the vasculature and tuft formation in 19 days old (P19) normal and oxygen-induced retinopathy (OIR) mice in comparison to PBS and control D(CAPAC) peptide; (B) Quantitation of tuft areas in wild-type (WT) mice given treatment groups as eye drops; (C) Cryostat sections of vascular tufts extending from the retina into the vitreous with IB4-stained vessels in red and DAPI (4′,6-diamidino-2-phenylindole) counterstained nuclei in blue at P19. Pathological tuft formation is shown above the dashed lines, and reduced vessel formation within the inner retina is shown below the dashed lines; (D) Paraffin sections showing tuft formation above dashed lines and retinal layers below dashed lines; (E) Diagram of the vasculature in different regions of the retina; (F) Quantitation of percent blood vessel area at 4-mm intervals summed through the full retina on a relative scale; (G) 6-min fluorescein angiograms for monkeys treated with eye drops at 29 days after laser-induced photocoagulation; (H) OCT images from monkeys given eye drops at 29 days after the laser-induced lesion. Yellow arrows indicate CNV complex boundaries; (I) H&E-stained monkey retinas at low (upper row) and high (lower row) magnifications showing eosin red–stained vacuolated fibroblast layer outside of the choroid in the upper row. Red boxes indicate macular region; dashed ovals indicate the RPE and ROS zones 29 days after laser-induced lesioning; (J) Diagram showing vascular differences in the retinas of WT mice and vldlr-null (KO)mice treated with treatment groups. NFL, nerve fiber layer; GCL, the ganglion cell layer; IPL, the inner plexiform layer; INL, the inner nuclear layer, OPL, the outer plexiform layer; ONL, outer nuclear layer; ROS, rod (and cone) outer segments; RPE, the retinal pigment epithelium; CV, choroidal vessels. Reprinted from [25].

Fig. 5

(A) Photomicrographs of retinal sections showing significant reduction in photoreceptor cell death in the murine model of human autosomal recessive retinitis pigmentosa, the rd10 mice at postnatal day (P) 18 after Adalimumab (ADA) treatment in comparison to control (C57Bl6) (TUNEL-stained sections revealing dead photoreceptors and PAR content in DAPI-counterstained sections); (B) Bar graph illustrating the effect of ADA on the number of TUNEL-positive nuclei and nuclear poly (ADP) ribose (PAR)-positive cells; (C) Photomicrographs of retinal sections showing reactive gliosis amelioration by ADA in the rd10 mouse retina at P18 (Iba1-labelling to visualize microglial cells and GFAP content in DAPI-counterstained sections); (D) Bar graphs illustrating the effect of ADA on migration index of microglia, the corrected fluorescence of GFAP content and TNFα gene expression; (E) Topical endoscopic fundal imaging (TEFI) images showing intravitreal administration of infliximab suppresses experimental autoimmune uveitis (EAU) in comparison to vehicle control; (F) combined total disease scores demonstrating the difference in clinical disease progression between treatment groups. In EAU control eyes typical disease progression with signs of raised optic disc, vasculitis and severe inflammation; In infliximab treated eyes, only raised optic disc and initial signs of vasculitis are evident; (G) Graph showing total CD45+ infiltrate numbers from individual eyes. Reprinted from [33, 34].

Fig. 6

Immunofluorescence images from a diabetic mouse (D) after topical administration of GLP-1R agonist, liraglutide (D-lira eye drop) in comparison to vehicle (D-Sham) and a non-diabetic mouse (control, C; db/+). D-lira prevented disruption of the BRB and thus release of VEGF (red) (A), IL-1b (green) (B) and albumin (red) (C), most important players in the pathogenesis of the breakdown of the BRB; (D) Western blotting quantification of proteins from apoptotic (caspase 8, Bax, p53), antiapoptotic (BclxL) and neuroinflammatory (iNOS, FasL) signaling pathways; (E) Retinal concentration of glutamate measured by high-performance liquid chromatography after subcutaneous administration of treatment groups; (F) Comparison of glutamate/aspartate transporter (GLAST) immunofluorescence (red) after topical administration of treatment groups; (G) Quantification of GLAST immunofluorescence in arbitrary units (A.U.). Reprinted from [36].

Fig. 7

Ocular anatomy and tissue barriers. Reprinted and modified from [50].

Fig. 8

(A) Structure of hHyal-1: Stereoscopic representation of a side view. The catalytic and the HyalEGF-like domains are colored light blue and yellow respectively. Disulfide bonds are shown in red. N-linked oligosaccharides are shown as stick models with the atomic color scheme: gray, carbon; red, oxygen; blue, nitrogen; (B) Stereoscopic representation of the active site region of hHyal-1 (gray ribbon) superimposed on that of bvHyal (yellow ribbon). Selected amino acids are colored in the atomic color scheme: red, oxygen; blue, nitrogen; gray (hHyal-1) and yellow (bvHyal), carbon. (C) Molecular surface of the catalytic domain (light blue) and HyalEGF-like (yellow) domains of hHyal-1, illustrating the separation between the HyalEGF-like domain and the active site. A docked tetrasaccharide, inferred from the structure of bvHyal, is shown as a space filling model. Reprinted from [79].

Fig. 9

(A) Functioning of a chemical chaperone: β-lactamase function is restored when aggregation of the target protein is inhibited. This can occur either through stabilization of the native structure (left) or through inhibition of the process of amyloid self-assembly (right). Reprinted from [86]. (B) Schematic summary of human γD-crystallin (a member of crystallin families) polymerization. (i) Crystal structure of human γD-crystallin. (ii) Simulated monomeric aggregation precursor (I2), often referred as N* in the general mechanism of protein aggregation in literature. (iii) Simulated structure of open-ended domain-swapped dimer. (iv) Simulated structure of close-ended domain-swapped dimer. (v) Model of human γD-crystallin hexamer formed via domain swapping. Reprinted from [84].

Fig. 10

Structure of human FcRn in contact with human IgG1 (hIgG1) and human serum albumin (HAS) and FcRn-mediated recycling of IgG and albumin in vascular endothelial cells; IgG and albumin are internalized into vascular endothelial cells through pinocytosis. The pH of the endosome is 6.0, facilitating association with membrane-bound FcRn. The contents of endosomes can be processed in one of two ways: either recycling back to the apical cell membrane or transcytosis from the apical to the basolateral side. In the case of saturated receptors, excess IgG and albumin are degraded by lysosomes. Top, apical side; bottom, basolateral side. Reprinted and modified from [95].

Fig. 11

(A) The log of the equilibrium association constant KA at pH 6.0 are plotted for various engineered anti-VEGF (bevacizumab) variants demonstrating increased binding to human FcRn in contrast to parent bevacizumab native IgG1 antibody; (B) Binding sensorgrams at pH 6.0 and 7.4 of each variant; Log-linear changes in serum concentrations for anti-VEGF (bevacizumab) and anti-EGFR antibodies in cynomolgus monkeys (C) and hFcRn transgenic mice (D) demonstrating antibodies engineered for higher FcRn affinity (Xtend-VEGF and Xtend-EGFR) promotes half-life extension. Reprinted from [96].

Fig. 12

Current and emerging routes for protein and peptide delivery to ocular tissues. Reprinted and modified from [50].

Fig. 13

Schematic of capture and release of microparticles by self-rolling microtubes. Thin film of poly(N-isopropylacrylamide-co-4-acryloylbenzophenone)(poly(NIPAM-ABP)) and polycaprolactone (PCL) with admixed magnetic nanoparticles (i) is able to form self-rolling tube and to encapsulate microparticles at reduced temperature (ii). The particle can be released at elevated temperature when the microtube is unrolled (iii). Reprinted from [146].

Fig. 14

(A) Actuation mechanisms based on the heat generated by an alternating magnetic field (AMF) leading to on-demand pulsatile small molecule release from mesoporous silica nanoparticles (MSNPs): Pseudorotaxane-based nanovalves made of cucurbit[6]uril. Reprinted from [160]; (B) Light-triggered small molecule delivery: Drug delivery through the near-infrared-triggered induction of dehybridization of the DNA conjugated at the surface of gold nanorods. Reprinted from [161].

Fig. 15

(A) Light-triggered drug delivery: Schematic representation of transcription–translation liposomal system for protein production triggered by irradiating caged DNA with light. Reprinted from [174]; (B) Temperature-based actuation mechanisms for liposomal drug delivery: The temperature-triggered unfolding of a leucine zipper peptide inserted in the membrane of a doxorubicin (Dox)-carrying liposome opens a channel through which the drug is released. Reprinted from [175]; (C) Drug-permeable pores can also be created by the temperature-triggered generation of bubbles from the decomposition of encapsulated ammonium bicarbonate. Reprinted from [176].

Fig. 16

(A) Drug delivery from echogenic perfluorocarbon (PFC)-containing nanoemulsions: The delivery mechanism involves a droplet-to-bubble transition under the action of ultrasound, leading to drug transfer from the bubbles to neighbouring cells. Reprinted from [185]; (B) Voltage-responsive vesicles: Structures of polystyrene-β-cyclodextrin (PS-β-CD) and poly(ethylene oxide)-ferrocene (PEO-Fc), and representation of the voltage-responsive controlled assembly and disassembly of PS-β-CD–PEO-Fc supramolecular vesicles. Reprinted from [186]; (C) pH-sensitive nanocarriers for efficient TAT-peptide exposure: Polyhistidine (PHis)-based micelles responding to acidic microenvironments by an efficient TAT-sequence exposure following ionization of the polyhistidine segments. Reprinted from [187].

Fig. 17

(A) Anatomy of a dendrimer: A dendrimer and dendron are represented with solid lines. The colored, broken lines identify the various key regions of the dendrimer. Reprinted from [188]; (B) Generation 4 PAMAM-OH dendrimer. (C) Internally quaternized PAMAM to form QPAMAM-OH dendrimer with inner cationic charges. PAMAM are frequently quaternized by methyl iodide (ICH3) and the terminal surface become very positive allowing the efficient electrostatic binding/loading of negatively charged backbone of siRNA. (D) QPAMAM with different surface modifications, including the addition of acetyl group by direct reaction with acetic anhydride (Ac2O), poly(ethylene glycol) (PEG) and poly-L-lysine (PLL), LHRH peptide. This addition of polymer structures such as PEG and PLL is reported to enhance the surface positive charge and circulation of PAMAM nanoparticles. While, the conjugation to LHRH peptides confers targeting ability in PAMAM based delivery applications. Reprinted from [195].

Fig. 18

Ocular drug delivery nanowafer: (A) Schematic of nanowafer instilled on the cornea. (B) Diffusion of drug molecules into the corneal tissue. (C) Nanowafer on a fingertip. (D) AFM image of a nanowafer demonstrating an array of 500 nm diameter nanoreservoirs. (E) Fluorescence micrograph of a nanowafer filled with doxycycline (scale bar 5 μm). Reprinted from [196].

Fig. 19

(A) Chemical optimization of i, i + 3 hydrocarbon stapling: Top, design and synthesis of SAH-MPER(671–683KKK)(q), in which the N-terminal S5 residue was replaced with R3 to lead to efficient i, i + 3 olefin metathesis under standard reaction conditions. (B) Crystal structure of SAH-MPER(671–683KKK)(q) (shown as a blue ribbon and gray transparent van der Waals surface) bound to 4E10 Fab, at 2.9-Å resolution. (C) 2Fo–Fc electron density map (1σ level) of the antibody-bound SAH-MPER(671–683KKK)(q) peptide. (D) Superposition of the native (green; PDB 2FX7)18 and i, i + 3–stapled (gray) MPER(671–683KKK) peptides, highlighting the similarity of antibody-bound structures, aside from the appended C-terminal lysines and the incorporated staple. Z and X represent R3 and S5, respectively, in the staple (red bar above sequence). Reprinted from [231].

Fig. 20

(A) Hollow microneedle (arrow), 720 μm in length, is shown next to a liquid drop of approximately 50-μL volume from a conventional eye dropper; Scanning electron micrographs of a representative fenestrated TiMN with 200 μm width, 50μm thickness, and 1500μm length: (B) Low magnification image showing the full needle, as well as a portion of its base; (C) Higher magnification image showing the needle tip and fenestrations; (D)Size comparison between a fenestrated TiMN and a conventional 26 gauge hypodermic needle typically used for intravitreal injection; (E) Buckled MN demonstrating graceful, plasticity-based failure mode after mechanical testing; (F) Fluorescence micrographs of PVP/Rhodamine B coated solid and fenestrated TiMNs. Reprinted from [253].