The failure of conventional antibiotics to treat numerous antibiotic-resistant infections necessitates the development of new agents — as a natural anti-microbial nitric oxide holds particular promise. To learn more about the power of nitric oxide as a means for treating numerous diseases, including respiratory infections, we spoke to Mark Schoenfisch, PhD, President and Chief Scientific Officer at Vast Therapeutics. Q: Could you provide us with an overview of Vast Therapeutics?
A: Vast Therapeutics is an innovative, preclinical-stage pharmaceutical company committed to helping people suffering from severe respiratory diseases to breathe better and live more fully. Q: Where did you get the inspiration from to pursue nitric oxide as an alternative to antibiotics for the treatment of CF patients? A: During my year tenure as a Professor of Chemistry at the University of North Carolina at Chapel Hill, my long-standing interest in nitric oxide as an antibacterial agent has been grounded in its critical role in many physiological processes in the human body, including our immune response to pathogens.
Lu and colleagues designed NONOate-based chitosan oligosaccharides that were extremely effective at penetrating biofilms and killing P. Another significant class of NO delivery vehicles are nanoparticles Quinn et al. Kafshgari and coworkers devised porous silica-based nanoparticles conjugated to S-nitrosothiols and S-nitrosogluthatione and showed that they have significant antimicrobial activity against E.
Overall, there has been sustained, growing interest in developing NO materials and delivery vehicles capable of harnessing the antimicrobial properties of NO. The examples mentioned above represent only a fraction of such compounds. Despite the development of numerous NO materials, few have been evaluated for therapeutic purposes or have translated to clinical settings Liang et al.
One of the issues is associated with poor control of NO release. Low stability and rapid release of NO make it difficult to deliver NO for extended periods of time, maintain concentrations within desirable ranges, and provide tissue-specific activity.
Traditional materials are loaded with a payload of NO donor that spontaneously dissociates when exposed to water or other conditions. As such, NO dynamics have largely been constrained to rapid accumulation of NO at the onset of delivery followed by progressive decay.
Not only are these dynamics restricted, but they are in stark contrast to the way NO is delivered naturally within phagosomes. During an immune response, NO is delivered for extended periods of time, in which the rates of NO delivery have been suggested to peak hours after phagocytosis Reichner et al.
Recently, our group established a relationship between bolus payload and release kinetics, where at lower payloads faster dissociation rates led to greater antimicrobial activity, while at higher payloads slower dissociation rates were favored Robinson et al.
However, the restricted set of delivery dynamics evaluated and their discordance with the way NO is delivered in physiological environments, raises the question of how this design criterion may impact the development of future NO-based therapeutics. In recent years, the possibility of finely controlling delivery has become achievable with the development of light controlled, photoactivated compounds Sortino, ; Choi et al.
In particular, metal-nitrosyl complexes have gained significant attention, as alternative NO releasing moieties, because of their ability to induce NO release upon exposure to specific wavelengths of light Tfouni et al.
The Mascharak group developed manganese-nitrosyl sol-gel coatings that released NO upon exposure to near infrared light NIR and led to significant reduction of S. Similarly, Evans and colleagues developed manganese-nitrosyl based polymer microparticles that release NO upon exposure to NIR Evans et al. Roveda and coworkers designed polyamidoamine dendrimers modified with ruthenium nitrosyl moieties, which could be activated upon UV irradiation Roveda et al.
In addition to light-activated compounds, enzymatic pro-drug systems represent another methodology to finely tune delivery rates through the control of enzymes or substrates. Jones and colleagues developed a NO probiotic patch in which Lactobacilli fermentation of glucose lead to NO production from nitrite Jones et al. The Zhao group generated a unique methylated galactose NONOate conjugate that was only recognizable by a mutant beta galactosidase enzyme from Thermus thermophilus Hou et al.
NONOate release was restricted to environments containing the selective beta galactosidase and by co-delivering the enzyme and pro-drug, which allowed localization of NO release to specific tissues and reduced systemic toxicity. The capability of precisely controlling NO delivery raises several interesting questions, such as, how delivery dynamics influence the antimicrobial potency of NO; and what is the best way to deliver a given payload of NO?
To begin to address these questions, we used an approach that integrated experiments and computational modeling to assess, analyze, and predict how NO delivery dynamics influence the duration of nitrosative stress in E. Using fed-batch bioreactors, we evaluated four basic modes of delivery, one of which was a traditional bolus delivery, and observed that dosing outcome differed drastically depending on the payload administered.
That data was used to train a computational model of the E. Quantitative analysis of those results suggested that maintaining respiratory inhibition was a major driver of delivery outcome, which was a prediction confirmed by further experimentation.
Finally, with the model as a guide, we constructed delivery regimes capable of maintaining steady state NO concentrations at levels sufficient to inhibit cellular respiration, and this led to dosing schedules that were far more effective than any other tested delivery schemes.
Collectively, the data and analyses presented here demonstrate the importance of dosing dynamics when designing NO-based treatments. In this study, we sought to investigate the impact of delivery dynamics on the antimicrobial potency of NO. Specifically, our system is composed of a fed-batch bioreactor, in which the input flowrates of NONOate and its balance stream NONOate solvent can be programmed and automated using a low flow control system.
We are able to measure and monitor several outputs, such as the concentration of NO and O 2 present in the bioreactor, as well as temperature and culture turbidity. Figure 1. Control of NONOate delivery dynamics. Delivery schedules were programmed using a computer-controlled low flow drip system.
Solid lines represent the mean of three replicates, whereas the lightly shaded areas represent the standard error of the mean. We chose to begin our investigation by evaluating four principle modes of delivery.
In particular, we examined the dynamics of linearly increasing ramp up , linearly decreasing ramp down and constant delivery regimes and compared them to the traditional delivery method, which is a bolus Figure 1B.
In the control case of bolus delivery, both reservoirs were programmed to deliver 10 mM NaOH over 1 h. Figure 1D depicts the differing NO dynamics in cell-free systems for these four modes of delivery. To begin exploring NO detoxification under different delivery schema, aerobic cultures of E.
Our metric of interest to evaluate different delivery modes is NO clearance time t clear , which is the time during which the concentration of NO [NO] is greater than or equal to 0. In contrast, the other delivery schemes failed to reach 0. All four delivery schema produced nitrosative stress, with constant delivery being the most effective dosing scheme with an NO clearance time of 1. This result suggested that the ability of NO to cause nitrosative stress depends both on the payload and the dynamics of how it is delivered.
Figure 2. Delivery outcome is payload dependent. Solid lines represent the mean of three independent experiments, whereas the lightly shaded areas represent the standard error of the mean. To quantitatively explore the relationship between delivery dynamics and antimicrobial efficacy, we trained a kinetic model of NO stress in E. The model was developed in previous studies Robinson and Brynildsen, , , a , b ; Robinson et al.
Specifically, the model was adjusted to comply with fed-batch systems and cellular growth was incorporated and assumed to depend on the availability of aerobic cytochrome oxidases for respiration. Parameter sets were accepted based on Evidence Ratios ER and ensembles of models were generated section Materials and Methods. A complete list of species, reactions, and kinetic parameters can be found in Supplementary Tables S1—S3.
To simulate our microfluidic drip system, continuous NONOate delivery and extracellular species dilution were incorporated into an existing kinetic model of NO metabolism Robinson and Brynildsen, b. The input term had four functional forms, depending on the delivery mode implemented section Materials and Methods and Supplementary Methods.
A volume dependent dilution term was also included to capture dilution of extracellular species, as a result of volume expansion within the bioreactor during operation section Materials and Methods and Supplementary Methods. Previous iterations of the model used in this study did not account for cellular growth but rather focused on the period of NO stress.
This was done because NO is bacteriostatic, and thus under NO stress cells are non-growing. Growth rate was modeled as a 1st order Hill-type function. Under aerobic conditions, the majority of ATP production in E.
A set of 16 uncertain respiratory parameters section Materials and Methods and Supplementary Table S5 , were trained on [O 2 ] and OD data obtained from aerobic, mid-exponential phase E. The ensemble of models could accurately capture O 2 consumption and cell density at all three concentrations of KCN.
Additionally, growth-dependent dilution terms were incorporated into rate equations for cellular species to capture the expansion of intracellular volume that occurs with growth section Materials and Methods. The model predicted that bolus delivery should lead to a t clear of 0. Experimental measurements agreed well with those forward predictions from the model Figure 4.
This confirmed that the model could accurately extrapolate to conditions outside its training data, which gave confidence that it could be used to quantitatively analyze NO stress in E. Figure 3. Cultures of E. Simulations from the ensemble members greatly overlapped, thus resembling a single line. Figure 4. Colored lines represent measured [NO] dynamics bolus- blue; constant-red; ramp down-green; ramp up-pink. The solid lines represent the mean of three independent experiments, whereas the lightly shaded areas represent the standard error of the mean.
We sought to evaluate the dynamics of three of the principle dosing modes by varying an additional parameter, duration of delivery. In addition, simulations revealed that each delivery mode displayed distinct discontinuities when plotting t clear against delivery period. Evaluation of the cumulative NO consumption flux profiles Figures 5D—F , suggested that the discontinuities were associated with failures to inhibit cellular respiration, which led to higher translation rates and ultimately higher concentrations of Hmp Supplementary Figures S3—S5 , which is the main NO detoxification enzyme under aerobic conditions Gardner and Gardner, ; Corker and Poole, ; Robinson and Brynildsen, , b.
Noticeably, the ramp-up delivery mode contains two discontinuities, where the first was due to an initial failure to inhibit cellular respiration which allowed increased translation and Hmp protein expression. This led to cellular NO consumption rates that balanced NO delivery rates. However, near the end of the delivery period, the increasing delivery rates began to exceed cellular consumption, which led to a sudden rise in [NO].
While the second discontinuity, was similarly due to a failure to inhibit cellular respiration, and cellular consumption invariably balanced NO influx throughout delivery. Experiments were performed to assess the accuracy of these predictions, and as depicted by the colored dots in Figures 5A—C , data agreed well with model predictions, including the approximate delivery times that corresponded to the discontinuities.
Figure 5. Relationship between t clear and delivery period. A Constant, B ramp down, C ramp up. Solid lines represent predicted relationship between t clear and delivery period, while dashed lines represent discontinuities in the curves.
Circles represent mean t clear values from at least three experiments and error bars represent the standard error of the mean. The three major NO consumption pathways are autoxidation blue , transport to gas phase red , and cellular consumption yellow. Given the central role of respiratory inhibition in defining the delivery periods at which the principle modes become ineffective or less effective for the first discontinuity of the ramp up mode , we plotted t clear as a function of the duration during which respiration is inhibited.
As depicted in Figure 6 , all of the simulations, regardless of delivery mode, fall onto a single line. This suggested that the duration of NO stress is strongly associated with the ability to achieve and maintain respiratory inhibition.
Figure 6. Duration of respiratory inhibition is a strong predictor of t clear. Solid lines represent the predicted relationship between t clear and delivery period bolus- blue; constant-red; ramp down-green; ramp up-pink , while the lightly shaded lines represent discontinuities in the curves.
B Plot of duration for respiratory inhibition vs. We hypothesized that, for a given payload, a dosing regimen that could raise and maintain NO at concentrations of 1. Using the model, we designed delivery schema capable of maintaining steady state concentrations of NO. Specifically, this was accomplished by constructing composite delivery schemes Figure 7A. First, a bolus was introduced to raise NO to the desired steady state concentration.
The model predicted that the optimal composite dosing regime was achieved by maintaining NO at approximately 2. Model simulations suggested that a bolus payload of 0. Get smart. Sign up for our email newsletter. Sign Up. Support science journalism.
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All tests were performed in duplicate. The colony-forming units were visually counted to determine percentage kill. Conclusion: gNO is bactericidal against various strains of bacteria suspended in saline, including both Gram-positive and Gram-negative organisms, and those that commonly cause nosocomial pneumonia in mechanically ventilated patients.
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