Preclinical

NangioTx Inc. Technology - Proangiogenic V-10 peptide to treat Peripheral Artery Disease – Intermittent Claudication

Peripheral artery disease treatments consist of oral therapies and surgical/endovascular interventions, the former of which work to prevent acute complications from narrowed vessels and dilate the diseased vessels. Surgical bypass grafts and endovascular stenting are not feasible options for many patients due to comorbidities and extent of disease.

We have developed a new method to combat PAD called V-10, a peptide-based material that has been developed through10+ years of research at Rice University. It consists of a novel sequence of 31 amino acids with several domains serving unique purposes and is known as a multi-domain peptide. This peptide is easily produced by solid phase synthesis, in the lab. The peptide is designed to self-assemble to form a nanofibrous matrix that mimics natural extracellular matrix. Our synthetic matrix can be sheared, only to reform, due to the nature of the hydrophobic interactions and hydrogen bonds between peptides. These matrices form a hydrogel in the body. (Figure 1) Addition of domains for peptide cleavage, cell adhesion and angiogenesis (Vascular Endothelial Growth Factor – VEGF – mimic or QK) establish the specific function desired for the treatment of peripheral artery disease and provide significant innovation for intellectual property management. The hydrogel form of the material offers remarkable epitope density, since the bioactive domain is present on each peptide strand in the large polymer. This enables large amounts of target receptor activation, clustering, induction of intracellular signaling and protein production. The resulting cellular sequelae ultimately leads to robust, mature and stable blood vessel growth.

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Additionally, the engineered properties of the material allow it to remain at the site of injection for three weeks, functioning to expose the surrounding tissue to the therapeutic for an extended period. The matrix nature of the material allows blood vessels to stabilize and grow, producing robust, non-leaky vessels by the time the material degrades. This multi-pronged approach ensures maximal effectiveness for clinical improvement.

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V-10 biologically signals and physically supports new blood vessel growth. These new highways for blood flow bypass the narrowed arteries, to return blood flow, reverse tissue atrophy, and prevent the need for amputation in animal models for PAD. V-10 utilizes patent pending “biomimicry” to selectively generate these robust and mature blood vessels. V-10 is delivered by a simple syringe injection and is fast acting, showing preservation of limbs in aged mice in just 7 days, compared to several months in control mice. V-10 is non-toxic to tissue, rapidly infiltrates with cells, biodegrades in 3 weeks, and has better than 1 year room temperature shelf-life based on laboratory studies. V-10 has shown efficacy in a mouse model of PAD. The results we have obtained thus far have shown significantly greater perfusion of the ischemic leg than leading stem-cell based technologies currently undergoing clinical trials (Figure 2).

Comparison to previous strategies and proposed mechanism

A variety of vascular endothelial growth factor (VEGF) mimics have been used previously. Since its identification and isolation in 2005, the VEGF-165 mimic, or QK, which we have attached to our base peptide, activates a host of VEGF receptors. Stemming from this, several groups have conjugated QK to surfaces, PEG hydrogels and self-assembling peptides. These studies affirm QK stimulates VEGF receptor activation, dimerization, and can potentially stimulate tissue regeneration. However, these studies and others to date have not achieved the 3 required criteria for functional angiogenic vessel development: (i) retention of vessels, (ii) stabilization of vessels with pericytes / Smooth Muscle Cells, and (iii) material integration or resorption after 2-3 weeks to prevent hemangiomas. Similar studies with QK and self-assembling amphiphiles achieved similar levels of perfusion for controls, but only achieve modest improvements in their therapeutic groups. In contrast, V-10, shows rapid angiogenesis. Importantly, V-10 allows rapid restoration of blood flow (perfusion) to the tied off leg, to almost 80% of the untreated leg within 28 days. In the design of our study, we assayed the effects that V-10 had on self-assembly, cells and in vivo. We showed that V-10 formed nanofibrous hydrogels and maintain desirable material properties, while stimulating VEGF receptors. Having confirmed cytocompatibility, we demonstrated rapid infiltration by cells and development of stable, perfused vasculature within 7 days, that resorb by 3 weeks. Infiltrating cells result in molecular reorganization of the scaffold, loading scaffolds with necessary vascular support cells, as seen in 3-day histology. The bolus of V-10 then promotes robust angiogenesis into the implant. Due to the lack of a fibrous capsule, communication inside and outside scaffolds is possible. Finally, infiltrating vessels mature with support cells leading to perfused microvessels. The remarkable rate of angiogenesis observed, coupled with excellent hind limb ischemia recovery suggests that V-10 will be a powerful material to help reverse the course of peripheral artery disease.

Angiogenic stimuli present a way for acute and chronic ischemic disease management. In our studies, V-10 forms an injectable, nanofibrous and cytocompatible hydrogel. This material activates VEGFR-1, VEGFR-2 and NP-1. Current competing approaches have failed to promote significant and rapid angiogenesis, instead yielding hemangiomas, neoplasms, or nascent immature vessels. In contrast V-10 scaffolds are rapidly infiltrated without the canonical need for macroporous structure, have no fibrous encapsulation, excellent tissue integration, and have large numerous micro vessels which stain positive for vWF, CD31, & Alpha-SMA and Nestin indicating their mature development (Figure 3). Evaluation in a hind limb ischemia model demonstrates the utility of V-10 in a disease model with localized injections and functional recovery of limbs in old (13 month) mice. A summary of these studies is outlined in Table 1. The results of this study suggest the immense potential for V-10 to be used in therapeutic revascularization of ischemic limbs, post-myocardial infarction, stroke, diabetic foot ulcers and other ischemic tissue diseases.

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Table 1. Outline of studies completed to date, determining efficacy and toxicity, in the laboratory.

Study Experiment Result Repeats/ animals Discussion
Angiogenic receptor binding Upregulation of HUVEC VEGFR1, VEGFR2 and NP-1 receptors determined using PCR  V-10 showed similar binding compared to VEGF 4 repeats Demonstrates a cellular mechanism that is potentiated for angiogenesis
Competitive binding HUVEC receptor binding using fluorescently tagged SLanc, FACS sorted ± VEGF V-10 showed a dose response increase in HUVEC binding and was inhibited by VEGF addition 4 repeats Underscores mechanism of action of V-10 binding to VEGF receptors
In vitro cell assays Culture of hMSCs, HUVEC scratch wound and THP-1 cells hMSCs showed proliferation on V-10 hydrogels, HUVEC showed improved scratch wound healing and THP-1 cell TNF-a and IL-1b levels were not above control 4 repeats V-10 is cytocompatible and does not elicit a proinflammatory response
Subcutaneous implants Normal healthy rats were dosed with 200µL of V-10 subcutaneous Angiogenesis of robust mature CD31+, Nestin+, a-SMA+ vessels within 7 days in scaffolds that biodegraded over 3 weeks 4 repeats / timepoint (t=3 days, 7 days, 14 days, 21 days) Robust and rapid angiogenesis without fibrous encapsulation of rapidly infiltrating cells
Hind limb ischemia Femoral artery ligation in C57BL/6 mice with subsequent treatment of injectable peptide Rapid restoration of blood flow to limb and foot pad with improvement in gait, significantly (p<0.05) greater than controls and similar literature studies 8 repeats for each 8 month and 13- month old mice Establishing preclinical efficacy
In vivo distribution 200µL SC, 50µL IM, 200µL IV injection of fluorescently tagged V-10 into C57BL/6 mice to evaluate degradation, acute thrombotic events, and systemic toxicity SC implants could not be visualized past 2 weeks, while IM implants persisted for 21 days. IV injection localized to the bladder within 10 mins and was excreted within 3 days. No thromboembolic events, adverse events, or gross tissue abnormalities were detected using pathological examination 4 repeats for each route Determining SC/IM toxicity and degradation. Determining potential for thrombotic events (none) and systemic clearance (urine)

 

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