The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Evaluation of therapeutic strategies targeting BCAA catabolism using a systems pharmacology model.

Background: Abnormal branched-chained amino acids (BCAA) accumulation in cardiomyocytes is associated with cardiac remodeling in heart failure. Administration of branched-chain α-keto acid dehydrogenase (BCKD) kinase inhibitor BT2 has been shown to reduce cardiac BCAA levels and demonstrated positive effects on cardiac function in a preclinical setting. The current study is focused on evaluating the impact of BT2 on the systemic and cardiac levels of BCAA and their metabolites as well as activities of BCAA catabolic enzymes using a quantitative systems pharmacology model. Methods: The model is composed of an ordinary differential equation system characterizing BCAA consumption with food, disposal in the proteins, reversible branched-chain-amino-acid aminotransferase (BCAT)-mediated transamination to branched-chain keto-acids (BCKA), followed by BCKD-mediated oxidation. Activity of BCKD is regulated by the balance of BCKDK and protein phosphatase 2Cm (PP2Cm) activities, affected by BT2 treatment. Cardiac BCAA levels are assumed to directly affect left ventricular ejection fraction (LVEF). Biochemical characteristics of the enzymes are taken from the public domains, while plasma and cardiac BCAA and BCKA levels in BT2 treated mice are used to inform the model parameters. Results: The model provides adequate reproduction of the experimental data and predicts synchronous BCAA responses in the systemic and cardiac space, dictated by rapid BCAA equilibration between the tissues. The model-based simulations indicate maximum possible effect of BT2 treatment on BCAA reduction to be 40% corresponding to 12% increase in LVEF. Model sensitivity analysis demonstrates strong impact of BCKDK and PP2Cm activities as well as total BCKD and co-substrate levels (glutamate, ketoglutarate and ATP) on BCAA and BCKA levels. Conclusion: Model based simulations confirms using of plasma measurements as a marker of cardiac BCAA changes under BCKDK inhibition. The proposed model can be used for optimization of preclinical study design for novel compounds targeting BCAA catabolism.

Mathematical Model Predicts that Acceleration of Diabetic Wound Healing is Dependent on Spatial Distribution of VEGF-A mRNA (AZD8601).

INTRODUCTION: Pharmacologic approaches for promoting angiogenesis have been utilized to accelerate healing of chronic wounds in diabetic patients with varying degrees of success. We hypothesize that the distribution of proangiogenic drugs in the wound area critically impacts the rate of closure of diabetic wounds. To evaluate this hypothesis, we developed a mathematical model that predicts how spatial distribution of VEGF-A produced by delivery of a modified mRNA (AZD8601) accelerates diabetic wound healing. METHODS: We modified a previously published model of cutaneous wound healing based on coupled partial differential equations that describe the density of sprouting capillary tips, chemoattractant concentration, and density of blood vessels in a circular wound. Key model parameters identified by a sensitivity analysis were fit to data obtained from an in vivo wound healing study performed in the dorsum of diabetic mice, and a pharmacokinetic model was used to simulate mRNA and VEGF-A distribution following injections with AZD8601. Due to the limited availability of data regarding the spatial distribution of AZD8601 in the wound bed, we performed simulations with perturbations to the location of injections and diffusion coefficient of mRNA to understand the impact of these spatial parameters on wound healing. RESULTS: When simulating injections delivered at the wound border, the model predicted that injections delivered on day 0 were more effective in accelerating wound healing than injections delivered at later time points. When the location of the injection was varied throughout the wound space, the model predicted that healing could be accelerated by delivering injections a distance of 1-2 mm inside the wound bed when compared to injections delivered on the same day at the wound border. Perturbations to the diffusivity of mRNA predicted that restricting diffusion of mRNA delayed wound healing by creating an accumulation of VEGF-A at the wound border. Alternatively, a high mRNA diffusivity had no effect on wound healing compared to a simulation with vehicle injection due to the rapid loss of mRNA at the wound border to surrounding tissue. CONCLUSIONS: These findings highlight the critical need to consider the location of drug delivery and diffusivity of the drug, parameters not typically explored in pre-clinical experiments, when designing and testing drugs for treating diabetic wounds. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00678-9.

Model-Based Analysis Reveals a Sustained and Dose-Dependent Acceleration of Wound Healing by VEGF-A mRNA (AZD8601).

Intradermal delivery of AZD8601, an mRNA designed to produce vascular endothelial growth factor A (VEGF-A), has previously been shown to accelerate cutaneous wound healing in a murine diabetic model. Here, we develop population pharmacokinetic and pharmacodynamic models aiming to quantify the effect of AZD8601 injections on the dynamics of wound healing. A dataset of 584 open wound area measurements from 131 mice was integrated from 3 independent studies encompassing different doses, dosing timepoints, and number of doses. Evaluation of several candidate models showed that wound healing acceleration is not likely driven directly by time-dependent VEGF-A concentration. Instead, we found that administration of AZD8601 induced a sustained acceleration of wound healing depending on the accumulated dose, with a dose producing 50% of the maximal effect of 92 µg. Simulations with this model showed that a single dose of 200 µg AZD8601 can reduce the time to reach 50% wound healing by up to 5 days.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

Design of Selective sPLA2-X Inhibitor (-)-2-{2-[Carbamoyl-6-(trifluoromethoxy)-1H-indol-1-yl]pyridine-2-yl}propanoic Acid.

A lead generation campaign identified indole-based sPLA2-X inhibitors with a promising selectivity profile against other sPLA2 isoforms. Further optimization of sPLA2 selectivity and metabolic stability resulted in the design of (-)-17, a novel, potent, and selective sPLA2-X inhibitor with an exquisite pharmacokinetic profile characterized by high absorption and low clearance, and low toxicological risk. Compound (-)-17 was tested in an ApoE-/- murine model of atherosclerosis to evaluate the effect of reversible, pharmacological sPLA2-X inhibition on atherosclerosis development. Despite being well tolerated and achieving adequate systemic exposure of mechanistic relevance, (-)-17 did not significantly affect circulating lipid and lipoprotein biomarkers and had no effect on coronary function or histological markers of atherosclerosis.

N,N-diacetyl-L-cystine improves endothelial function in atherosclerotic Watanabe heritable hyperlipidaemic rabbits.

N,N-diacetyl-L-cystine (DiNAC), a novel immunomodulator, stimulates contact sensitivity/delayed type hypersensitivity reactions in mice induced by oxazolone and reduces atherosclerosis in Watanabe heritable hyperlipidaemic (WHHL) rabbits. Forty-week-old WHHL rabbits were given DiNAC (3 micromol/kg per day) for 8 weeks, and endothelium-mediated dilatation was investigated in vivo using pulse wave analysis. A significant improvement in endothelial function was found after 3 weeks of treatment, which was further improved after 8 weeks. For experiments on isolated blood vessels, 40-week-old rabbits were treated for 3 weeks. Treatment did not affect plasma lipid levels. At termination, aortic rings from the thoracic and abdominal aorta were contracted with phenylephrine in vitro. Concentration-effect curves to acetylcholine and the calcium ionophore A 23187 were used to measure endothelium-mediated vasodilatation, and nitroprusside to elicit endothelium-independent relaxations. Abdominal aorta relaxations were generally larger than in thoracic aorta. DiNAC improved endothelium-dependent relaxations in the abdominal but not in the thoracic aorta. This effect was independent of the degree of atherosclerosis. It is concluded that DiNAC improved endothelial function in atherosclerotic rabbit arteries in vivo and in vitro, and may represent a new treatment modality for atherosclerosis-related diseases.

Gradient echo MRI characterization of development of atherosclerosis in the abdominal aorta in Watanabe Heritable Hyperlipidemic rabbits.

PURPOSE: The Watanabe Heritable Hyperlipidemic (WHHL) rabbit provides an important model of spontaneous atherosclerosis. With a strain of WHHL rabbits which do not develop abdominal aorta lumen stenosis even with advanced atherosclerosis, we studied the MRI-histology correlation, and the natural progression of atherosclerosis in the abdominal aorta. In addition, intra-reader segmentation repeatability and scan-rescan reproducibility were assessed. METHODS: Two batches of female WHHL rabbits were used. The first batch of 6 rabbits was scanned at 20 weeks old. A second batch of 17 rabbits was scanned at 50 weeks old and then randomly divided into two subgroups: 8 were killed for histologic investigation; 9 were kept alive for follow-up, with repeat scanning a week later to assess scan-rescan reproducibility, and again at 73 weeks old to assess disease progression. MR images were acquired at 4.7 T using a chemical shift selective fat suppression gradient echo with a saturation band suppressing blood signal within the aortic lumen. Five slices per animal were acquired, centered around the renal artery region of the abdominal aorta, with in-plane resolution of 0.195 mm and slice thickness of 3 mm. RESULTS: The coefficient of variation for intra-reader reproducibility for aortic wall thickness measurements was 2.5% for repeat segmentations of the same scans on the same day, but segmentations of these same scans made 8 months later showed a systematic change, suggesting that intra-reader bias as well as increased variability could compromise assessments made over time. Comparative analyses were therefore performed in one postprocessing session. The coefficient of variation for scan-rescan reproducibility for aortic wall thickness was 5.5% for nine pairs of scans acquired a week apart and segmented on the same day. Good MRI-histology correlation was obtained. The MRI-measured mean aortic wall thickness of animals at 20 weeks of age was 76% that of animals at 50 weeks of age (p < 0.001). There was a small increase in aortic wall thickness between 50 and 73 weeks of age, but this was not significant (p > 0.05). The corresponding differences in lumen cross-sectional areas at 20, 50, and 73 weeks of age were not significant. These results were consistent with in-house historical histology data on this strain of rabbits. CONCLUSIONS: High-resolution gradient echo MRI can follow disease progression in the WHHL rabbit spontaneous atherosclerosis disease model.