Drugs to Inhibit the NLRP3 Inflammasome: Not Always On Target

The inflammatory response is an essential component of healing after injury.¹ After an acute myocardial infarction (AMI), the inflammatory response to injury, in absence of an invading microbial agent, is referred to as “sterile” inflammation. The activation of the innate immune response is central to sterile inflammation.²

The NACHT, leucine-rich repeat, and pyrin domain (PYD)-containing protein 3 (NLRP3) inflammasome is one of the most studied players in sterile inflammation during AMI. As shown in Figure 1, it consists of 3 different components: a sensor protein belonging to the family of nucleotide-binding oligomerization domain–like receptors (NLRs); a scaffold protein known as apoptosis-associated speck-like protein containing a caspase recruitment domain, or CARD; and caspase-1, the effector enzyme allotted to the processing of pro-interleukin-1β (IL-1β).² Caspase-1 can also drive a highly regulated inflammatory-linked cell death, named pyroptosis.³ The activation of the NLRP3 inflammasome is an extremely tight and time-regulated process which includes 2 distinct phases: a “priming” phase serving as a transcriptional inducer of the main components and a quick “trigger” signal leading to the assembly and therefore the activation of the macromolecular complex.^2,4

FIGURE 1.: Targets for the NLRP3 inhibition. To effectively block the NLRP3 inflammasome, 3 key points are hypothesized to be important. By targeting the ATPase activity of NLRP3, the formation of the inflammasome in response to specific NLRP3 triggers is prevented (1). Alternatively, NLRP3 can be directly inhibited and the consequent oligomerization can be blocked, as already demonstrated by the dapansutrile (OLT1177) compound (2). Another strategy may include the blocking of caspase-1 activity, which would not be able to convert interleukin-1 to its mature, active form (3). This figure was created using Servier Medical Art templates, which are licensed under a Creative Commons Attribution 3.0 Unported License; https://smart.servier.com. ASC, apoptosis-associated speck-like protein containing a caspase recruitment domain, or CARD; ATP, adenosine triphosphate; IL, interleukin; K⁺, potassium; NLRP3, NACHT, leucine-rich repeat, and pyrin domain (PYD)-containing protein 3; P2X7: P2X purinoreceptor 7.

Multiple preclinical investigations have highlighted the beneficial effects of an inhibitory, pharmacological strategy toward the activity of the NLRP3 inflammasome, reducing the extension of the infarcted area and preserving cardiac function after ischemia/reperfusion (I/R) injury (Fig. 1).

In this issue of the Journal, Lu et al present the results of their investigation on the cardioprotective effects of carthamin yellow (CY), a flavonoid compound isolated from safflower showing antiplatelet effects.⁵ Lu et al sought to investigate the effect of CY as a pretreatment in a murine model of I/R injury. CY, given 20 minutes before ischemia, was found to reduce the necrosis and also the infarct size in the hearts of rats exposed ex vivo to 30-minute global ischemia then followed by 1 hour of reperfusion. Similar experiments were then accomplished in an in vivo model of I/R injury where rats were exposed to 30 minutes of ischemia followed by 180 minutes of reperfusion. When given before the I/R injury, CY showed encouraging results reducing the infarct size and preserving the deterioration of the systolic function. To study the mechanism of action, the authors investigated reactive oxygen species (ROS) production, the NLRP3 inflammasome activation, and the release of proinflammatory cytokines, such as IL-1β, IL-6, and tumor necrosis factor-α in H9C2 cells subjected to I/R injury. The administration of CY before I/R injury provided a reduction in the ROS production, therefore highlighting the scavenging abilities of CY while mitigating the inflammatory response, as suggested by lower mRNA levels of IL-1β, IL-6, and tumor necrosis factor-α. The administration of CY was beneficial in lowering caspase-1 activation and the release of IL-1β.

Although the use of CY holds promise, some important issues need to be addressed. First, the clinical perspective of a preadministered drug in the setting of AMI is of questionable translational value. Indeed, AMI onset is classically abrupt and unexpected and the opportunity of pretreatment unlikely. Second, although CY decreased ROS production and the global extent of the damage thus limiting the inflammatory response, it is unclear whether CY possesses the ability to directly inhibit NLRP3 inflammasome activity, and what mechanism(s) this may involve. Indeed, the benefits of CY may be independent of a direct inhibition of the NLRP3 inflammasome activation, but rather indirect, secondary to reduced NLRP3 inflammasome activation due to a reduction in injury. Third, the role of the NLRP3 inflammasome in an ex vivo model is questioned, since in the Langendorff model, the blood is substituted by a crystalloid solution lacking the cellular component of the blood. Therefore, the experiments proposed by Lu et al to explore the anti-inflammatory property of CY as a NLRP3 inhibitor are inadequate and lead to inconclusive results.

The NLRP3 inflammasome was also found to foster the infarct “wavefront” progression after I/R injury in vivo. In mice subjected to surgical coronary ligation for 30 minutes followed by reperfusion at different time points (1, 3, and 24 hours), NLRP3 mRNA and protein levels increased only after 3–6 hours and peaked at 24 hours.⁶ Caspase-1 also showed a similar trend. Therefore, the short follow-up proposed in this study (1-hour time point in the ex vivo model and 3 hours for the in vivo) may have not been able to fully appreciate the effects on the NLRP3 inflammasome and may also inadequately explain the mechanism of action of CY and whether it acutely involves direct inhibition of the NLRP3 inflammasome activity. Looking at NLRP3 inflammasome activation (at 6, 24, and 36 hours) is therefore highly suggested.

Some of these answers could be addressed by investigating a delayed administration of this drug after I/R injury, beyond the phase in which pharmacologic conditions occurs.⁷ Direct NLRP3 inflammasome inhibitors have shown the ability to reduce the extent of the infarcted area when given in an in vivo model of mouse 1 hour after the beginning of the reperfusion.^6–8 This also simulates the clinical setting by which drugs are often administered with some delay during AMI.

Finally, it would also be helpful to perform pharmacokinetic studies to determine serum drug levels, which would help determine the lowest effective dose applicable to murine models. It would also be interesting to determine changes in bioavailability of the drug when administered in different ways (ie, intraperitoneal, parenteral, or oral gavage), as well as to perform a long-term survival study with increasingly high doses to report any possible acute and chronic toxicity.

In conclusion, the article by Lu et al provides insights on the protective mechanisms of CY after AMI, but further mechanistic studies are necessary to investigate the drug's intrinsic anti-inflammatory properties. New investigations addressing the aforementioned limitations are needed before exploring the use of CY in studies in patients with AMI. Nevertheless, the data, as a whole, point once again to an active role of the NRLP3 inflammasome in the tissue response to I/R injury.²