Investigation into the Metabolic Limitations of Alpha Conotoxins as Drugs in the Gastrointestinal Tract

Abstract

Peptides isolated from cone snails, known as conotoxins, have potential in treating neuropathic pain. Peptides often lack efficacy as drugs due to poor pharmacokinetic profiles despite displaying high potency and selectivity. Conotoxins present significant sequence diversity, but little information exists about their pharmacokinetic properties. We hypothesize that this diversity will correlate with dynamic pharmacokinetic profiles, with some peptides exhibiting increased stability. In this study, we aimed to gain a basic understanding of the stability of the conotoxins α‐IMI and α‐GI in the stomach and intestines. Both have different primary sequences despite serving as antagonists of the acetylcholine receptor, which is a potential target in cancer, chemotherapy‐induced allodynia and possibly pain associated with Crohn’s disease. In order to study these peptides, novel mass spectrometry (LC‐MS/MS) methods were developed incorporating multiple reaction monitoring scans for each peptide in simulated gastric and intestinal fluids. The quantitation range of our method was 0.5–5 μg/mL, which is 20 times more sensitive than traditional methods. To compare gastrointestinal metabolic stability, each peptide was incubated in simulated gastric fluid and intestinal fluid and their stability was measured using our LC‐MS/MS method at times ranging from 0–90 minutes. Both peptides displayed a high amount of stability in gastric fluid, as no significant loss of either peptide was determined after 90 minutes. In intestinal fluid IMI degraded rapidly (t1/2= ~6 minutes), whereas α‐GI displayed far more stability in the same environment (t1/2= ~257 minutes). α‐GI incorporates a prominent helix in its structure which is likely responsible for its increased stabilization in comparison to IMI. Considering the differences in the metabolic stability of these peptides, further study of conotoxin sequences may result in the identification of stable molecular scaffolds. In the future, we plan to pinpoint what particular features are responsible for metabolic stability or susceptibility across more conotoxins, such as differences in sequence, structure and rigidity and plan to use this information to produce analogs with increased stability within the gastrointestinal tract.Support or Funding InformationSource of Research Support: This work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.