Characterization of the NLRP3 Inflammasome Inhibitor HT-6184 in the Complete Freund's Adjuvant (CFA)-Induced Pain Model

Inflammation plays a crucial role in pain perception. During inflammation, immune cells release pro-inflammatory cytokines that activate pain receptors, resulting in pain. The NLRP3 inflammasome is a critical regulator in this process and a promising non-steroidal analgesic target. NLRP3 is a protein complex that, when activated by stimuli such as ATP or lipopolysaccharide, releases IL-1β and IL-18. Previous studies have shown that IL-1β modulates pain by binding to nociceptors and ion channels. Elevated expression of NLRP3 and other inflammasome components, such as ASC and caspase 1, are associated with various acute and chronic inflammatory conditions, such as osteoarthritis, neuropathic pain, postoperative pain, migraine, and gout. Inhibiting the NLRP3 inflammasome pathway in these conditions alleviates pain. 

Purpose/Objectives:

HT-6184 is a potent small molecule inhibitor of the NLRP3 inflammasome. This compound has recently completed a Phase I first in human clinical trial in healthy volunteers. By targeting the NLRP3 inflammasome pathway, HT-6184 mitigates excessive inflammation and alleviates pain. This study investigates the therapeutic effects of HT-6184 administered orally (PO) and subcutaneously (SC) in the complete Freund's adjuvant (CFA)-induced inflammatory pain model in laboratory rats. The CFA pain model is widely used for studying acute inflammatory pain. This model involves the injection of CFA into the rat's paw, inducing an acute inflammatory response. Previous studies have shown a significant increase in NLRP3, ASC, caspase-1, and IL-1β expression at the inflamed sites, making it an ideal model to test an NLRP3 inhibitor. We also aim to examine the drug's pharmacokinetics at different doses and evaluate the best route of administration in this model. 

Method:

Male Wistar rats (n=69) weighing 250-300 g were used. Thermal hyperalgesia was assessed by measuring paw withdrawal latency to radiant heat, and mechanical allodynia was evaluated using von Frey filaments to measure paw withdrawal thresholds. Basal measurements for each animal were taken before randomization into treatment groups. On day 0, CFA was injected into the right hind paw to induce inflammation. On day 1, animals were treated with vehicle, diclofenac sodium, or HT-6184 (10, or 30 mpk PO and SC). Pain-related behaviors were assessed at 0 hr, 1 hr, 3 hr, 6 hr, 9 hr, and 24 hr post-treatment. Plasma drug concentration analysis was performed on groups treated with HT-6184 at 6-time points (0 hr, 1 hr, 2 hr, 3 hr, 6 hr, 9 hr, 12 hr. 24 hr, 48 hr, and 72 hrs). Statistical analysis was conducted to compare the results from the control and experimental groups. 

Results:

HT-6184 exhibited significant dose-dependent effects in reducing thermal hyperalgesia and mechanical allodynia in this pain model. Both PO and SC 10 mpk and 30 mpk HT-6184 treatments groups showed significant improvement in paw withdrawal threshold and latency at various time points. Furthermore, all HT-6184 groups exhibited a significant effect compared to the control group when analyzing the area under the curve The most pronounced analgesic effects observed were in the group treated with HT-6184 at a dose of 30 mpk subcutaneously with its effect exceeding that of diclofenac. Pharmacokinetic analysis revealed a dose-dependent increase of HT-6184 in the plasma.

Conclusions: This study highlights the potential of HT-6184 as an effective analgesic for treating inflammatory pain. HT-6184 demonstrated dose-dependent effects in reducing thermal hyperalgesia and mechanical allodynia in a pain model induced by CFA. Particularly, the 30 mpk doses of HT-6184 consistently improved paw withdrawal latency and threshold at all time points except 1 hour. These effects were comparable to the reference drug and, in the case of the 30 mpk SC group, even better than diclofenac, indicating the high potency of HT-6184 as an analgesic. Additionally, pharmacokinetic analysis confirmed the dose-dependent presence of HT-6184 in the plasma, supporting its potential therapeutic efficacy. These findings enhance our understanding of HT-6184 as a promising candidate for the treatment of inflammatory pain. With HT-6184 progressing in clinical development, these results provide a solid foundation for its future use in human clinical trials targeting pain relief.

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