(530g) Quantification of Hydrogen Sulfide Release for Diagnosis and Treatment of Lower Extremity Ischemia in Peripheral Artery Disease Patients

Background and Relevance. H₂S is an endogenous biomolecule produced in the endothelial cells of blood vessels to regulate vascular function by stimulating endothelial growth, vessel formation, local dilation, blood flow increase, tissue ischemia recovery, and inflammation reduction [1]. Chronic limb threatening ischemia (CLTI) is the most severe stage of lower limb vascular insufficiency known as peripheral artery disease (PAD). It is prevalent among diabetics and smokers while affecting more than 20 million Americans [2]. The bioavailability of endogenous H₂S is found to be lower in this population due to the commonly occurring endothelial dysfunction and consequent deficiency in production of H₂S. In-vitro studies of exogenous H₂S supplements have been shown to stimulate endothelial cell growth, migration, and neovascularization through the action of VEGF leading to more rapid wound healing. Recent small animal studies show that a two-week treatment with systemic delivery of H₂S can ameliorate hindlimb ischemia [3]. However, systemic H₂S delivery comes with high risk of cytotoxicity due to off-target effects including hypotension and hepatotoxicity [4], an issue that can be avoided by precision local-regional H₂S delivery and treatment [5].

Evaluation of Local H₂S Bioavailability and Deficiency in PAD Patients. Recent studies at the UNM vascular physiology and surgery laboratories have shown the correlation between low bioavailability of H₂S, diabetes, and lower limb ischemia. For the first time, measurements of endogenous H₂S have been carried out and reported from an area that matters most for wound healing – i.e., the skin. The device developed by Exhalix, transdermal arterial gasotransmitter sensor, or TAGSâ„¢, non-invasively captures, concentrates, and measures gas-phase H₂S emitted from the surface of the skin. The base unit uses a patent-protected nanoporous gold-Nafion sensing element to electrocatalytically generate the signal for diagnostics [6]. As such, transdermally emitted H₂S can be measured with a limit-of-detection of less than 3 ppb in a few seconds [7]. In an on-going clinical study, TAGSâ„¢ measurements have been made on three different cohorts of human subjects [8]. The leg:arm ratio of H₂S emissions correlated with risk factors for microvascular disease (i.e., high-density lipoprotein levels, estimated glomerular filtration rate, systolic blood pressure, and hemoglobin A1c). Shown on Figure 1, the ratios measured were significantly lower in symptomatic diabetic (DM) subjects being treated for chronic limb-threatening ischemia (n=8, 0.48 ± 0.21) compared with healthy controls (n=5, 1.08 ± 0.30; P=.0001) and with asymptomatic DM subjects (n=4, 0.79 ± 0.08; P=.0086). The asymptomatic DM group ratios were also significantly lower than the healthy controls (P=.0194). Using ratios of leg:arm measurement (17 subjects, 34 ratios), the overall accuracy to identify limbs with severe PAD had an area under the receiver operating curve of 0.93. These results, obtained non-invasively, are consistent with the previous findings that H₂S bioavailability deficiency is linked to poor microvascular health and ischemic condition.

Characterization of In-Situ H₂S Synthesis and Delivery. The patent-protected H₂S synthesis and delivery method described in this presentation and paper is a viable alternative to solution-based or gel-packed precursors (e.g., Na₂S, NaHS, or GYY-4137). The innovative electrolytic method safely synthesizes, delivers, and maintains H₂S within a therapeutic window, thus offering unique characteristics in both shelf-life stability as well as precision in delivery. The core technology employs a controllable, in-situ, real-time process for conversion of a stable metal sulfide compound to H₂S gas for a ‘true sustained delivery’. In a reversible process, as depicted in Figure 2, a potentiometric injection of electrons into the reaction at low voltage (<1 VDC) reduces a thin sulfide coating such as Ag₂S, instantaneously releasing H₂S gas for local and on-demand delivery to tissue:

Ag₂S (solid) + 2 H₂O + 2e^— ⇔ 2 Ag + H₂S (gas) + 2 OH^—. (1)

The dosage can be achieved at a continuous low level (e.g., 10 nmol/hour) or intermittently at large doses (e.g., two discrete doses of 120 nmol each day). To-date, various linear as well as planar source geometry have been developed using a proprietary chemical deposition approach for different applications. Typical dose curves for these sources are shown in Figure 2, in which the correlation between the charge transferred and the H₂S released has been found to be linear. Given a standard curve for each source, the dosage can be controlled predictably in each application. The H₂S generation and release rate constants have been found to be <1 μAh/nmol, thus requiring extremely low power and battery usage. Results on multiple H₂S sources show high control over the dosage amount, with relatively low pulse-to-pulse variability and the ability to deliver as low as 1 nmol ± 0.5 nmol/dose. These sources have been packaged into inexpensive, disposable H₂S delivery systems for use in in-vitro and in-vivo preclinical animal studies.

Quantitative visualization of the linear source shown in Figure 3 indicates uniform production rate along the length of the source in near real-time images captured from the release process using a fluorescent molecular probe, WSP-1 [9]. Figure 3(a) schematically describes the apparatus used to visualize the H₂S emission process from the source. In these experiments, the H₂S source was placed in a WSP-1 containing gel medium within a quartz glass cuvette. Excitation was achieved with a UV LED light at λ_ex~465 nm while scattered light was blocked by a bandpass filter placed in front of the viewing optics. The source delivered 20 nmol/dose of H₂S that recombined with WSP-1 to generate a fluorophore (i.e., 3’-methylfluorescein). The fluorescence re-emission at λ_em~515 nm was then visualized as shown in Figure 3(b) and sequential images were processed with the NIH imageJ software package to obtain a 2-D planar intensity field. These images provided time and spatially resolved source characteristics and information on uniformity in emission from the surface, depth of penetration, and dose-to-dose consistency that is used for optimization of and tailoring the source performance to the end use or application.

Conclusions. This paper discusses different techniques for quantifying surface emission of hydrogen sulfide (H₂S) biomolecule. These techniques are expressly developed to measure the rate of release of enzymatically-generated endogenous H₂S, in addition to evaluation of in-situ synthesis of exogenous H₂S for delivery to an ischemic tissue during healing. While both methodologies are radically different, they share one aspect – they both deal with the elusive H₂S molecule. The final paper and presentation will cover more in-depth detail of the system and performance data on both linear and planar H₂S sources, in addition to the results of the in-vivo physiological studies with rats and the positive impact of in-situ delivery of H₂S on wound healing acceleration.

References:

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[5] B. T. Matheson, D. M. Friedrichsen, B. J. Brooks, J. Giacolone, R. M. Clark and R. Shekarriz, HEALSâ„¢: An Active Hydrogen Sulfide Delivery Technique for Accelerated, Effective Wound Healing, Albuquerque, N.M.: Final Report for NIH/NIGMS Phase I SBIR Grant 1R43GM144027-01., 2022.

[6] A. Shekarriz and D. M. Friedrichsen, "Transdermal Sampling Strip and Method for Analyzing Transdermally Emitted Gases". Patent 10,856,790, 8 December 2020.

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[8] B. Matheson and e. al., "A Novel, Microvascular Evaluation Method and Device for Early Diagnosis of Peripheral Artery Disease (PAD) and Chronic Limb Threatening Ischemia (CLTI) in Individuals with Diabetes," J. Vasc Surg Cases and Innov Tech, vol. 9, no. 2, pp. 1-10, 2023.

[9] Y. Zhou, F. Mazur, K. Liang and R. Chandrawati, "Sensitivity and Selectivity Analysis of Fluorescent Probes for Hydrogen Sulfide Detection," Chem. Asian J., vol. 17, p. e202101399, 2022.

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