RLS-0071 Moderated Elevated Myeloperoxidase Level and Activity in an Asymptomatic Subject in Clinical Trial RLS-0071-101

Background

The novel, first-in-class, complement-inhibiting, small peptide RLS-0071 was evaluated for safety, tolerability, pharmacokinetics, and pharmacodynamics of single and multiple doses in healthy volunteers. RLS-0071, previously referred to as Peptide Inhibitor of Complement C1 (PIC1), is a dual-targeting peptide being developed for clinical use to moderate humoral and cellular inflammation via inhibition of complement activation and neutrophil effectors [1,2]. This 15-amino acid peptide, originally derived from a region of the astrovirus coat protein, is noted to have limited homology with complement regulatory peptides [1]. RLS-0071 was subsequently discovered to inhibit myeloperoxidase activity in cystic fibrosis sputa ex vivo [3]. Mechanistically, the peptide inhibits myeloperoxidase’s heme ring, which is the enzymatic core, with potency equivalent to ABAH (4-Aminobenzoic acid hydrazide), the criterion standard for myeloperoxidase inhibition, on a molar basis (Figure 1) [3].

Myeloperoxidase contributes to the pathogenesis of certain autoimmune diseases like ANCA-associated vasculitis and chronic inflammatory conditions such as atherosclerosis [4]. In atherosclerosis, elevated levels of myeloperoxidase are associated with an increased risk for future major adverse cardiac events (MACEs) such as myocardial infarction or heart failure [5]. Humans that are otherwise asymptomatic are considered at increased risk of developing future MACEs if they have a plasma myeloperoxidase level of >322 pmol/L [6,7].

Although the subjects in the RLS-0071-101 study were healthy and relatively young, we identified a subject with higher-than-average plasma myeloperoxidase level measured at baseline. During the study, we enrolled 54 subjects and identified 1 individual with an elevated baseline myeloperoxidase level among those that received multiple doses of RLS-0071. While the absolute myeloperoxidase level was not high enough to consider her at risk for MACEs, it was noticeably elevated when compared to the remainder of the study cohort. We postulated that RLS-0071 dosing might lead to changes in myeloperoxidase level, functional activity, or both in that individual (Figure 1).

Case Report

The study was conducted at Altasciences in Canada between January 2021 and August 2021. It was conducted in accordance with the guidelines described in the Declaration of Helsinki and the CIOMS International Ethical and ICH GCP Guidelines. The protocol was reviewed and approved by the local competent Regulatory Authority and Institutional Review Board. The study protocol was approved by Health Canada control number 249562. All participants signed written consent forms.

Subject 1504 was a healthy 21-year-old white woman (weight 61 kg, height 167 cm, and BMI of 21.7) at the time of enrollment in April 2021. Her only medication was an intrauterine contraception device, and she had no underlying chronic medical conditions. Subject 1504 received 9 intravenous infusions of RLS-0071 each at a dose of 10 mg/kg. The only adverse events reported for this subject were mild bruising at the sites of venipuncture. The bruising resolved as expected and was deemed to not be associated with the drug.

Vital signs, including temperature, heart rate, blood pressure, respirations, and oxygen saturations, remained normal throughout the study. The only laboratory abnormalities observed were a mildly low plasma protein concentration on Day 2, 62 g/L, and Day 4, 58 g/L (reference range of 63–80 g/L). The plasma protein returned to the baseline range by Day 6 (Table 1). This pattern of transient, mild hypoproteinemia was seen in most subjects dosed with either active drug or placebo and was attributed to the frequent phlebotomy that occurred during the first several days of the study, needed to characterize their pharmacokinetic profiles. No other lab abnormalities occurred for this subject on blood chemistries, complete blood counts, or coagulation studies. Likewise, she did not develop any antidrug antibodies and her autoantibody panel testing was negative at screening and remained negative through Day 30 post-infusion. The autoantibody panel included: anti-double stranded DNA, antinuclear antibodies, Cardiolipin IgG, Cardiolipin IgM, Ribonucleoprotein antibody, Sjogren SS-A antibody, Sjogren SS-B antibody, and Smith antibody (Table 2). Additional tests that were negative included: HIV, Hepatitis B, Hepatitis C, COVID-19, pregnancy, and drug screen. Urinalysis tests during the study were normal except for trace ketones on Day 1 and Day 2, as well as trace occult blood on Day 2, which resolved by Day 4 (Table 1). The subject was discharged from the Phase 1 unit on Day 6 and was determined to be in good health upon routine follow-up on Day 30.

Blood was collected by venipuncture into K2EDTA tubes (BD) and placed into a wet ice bath. Tubes were centrifuged at 4°C and plasma was recovered, aliquoted, and frozen at −20°C. Frozen plasma samples were shipped on dry ice to ReAlta Life Sciences (Norfolk, VA). To quantify the amount of endogenous myeloperoxidase, plasma was analyzed using a human myeloperoxidase ELISA kit (BMS2038INST, Invitrogen). To determine myeloperoxidase activity in the plasma, subject samples were analyzed using a fluorescence-based myeloperoxidase assay kit (K745-100, BioVision). Approximately 2 μL of the plasma sample was incubated in the kit’s master mix for 20 minutes at 37°C and read at Ex/Em=353/387 nm.

The subject’s plasma samples were measured for myeloperoxidase concentration utilizing a commercial kit as described above. One subject was identified with an elevated myeloperoxidase concentration of 142 pmol/L at baseline (Table 3). Assays were performed in 3 independent experiments for each sample. The plasma myeloperoxidase concentration decreased from baseline by approximately 43% after 8 doses of RLS-0071 (pre-dose 9). The myeloperoxidase level decreased slightly after the 9th dose at end of infusion and 1 hour later. The myeloperoxidase level recovered approximately 26% towards pre-first dose baseline 24 hours after the final dose. In summary, these data demonstrate a mildly elevated baseline plasma myeloperoxidase concentration that decreased after multiple doses of RLS-0071 and then partially recovered to baseline 24 hours after cessation of dosing.

The subject’s plasma samples were also measured for myeloperoxidase activity in a functional myeloperoxidase assay, in which the peroxidase activity of myeloperoxidase was measured using a fluorescence-based myeloperoxidase assay kit (Table 3). The plasma myeloperoxidase activity level decreased from baseline by 49% after 8 doses of RLS-0071 (pre-dose 9). After the 9th dose of RLS-0071, further decreases in activity were measured at end of infusion (26%) and 1 hour later (46%) compared with pre-dose 9 myeloperoxidase activity levels. At 24 hours after the last dose of RLS-0071 (9th dose), the myeloperoxidase activity level had increased to nearly the pre-9th dose level. In summary, these data demonstrate an elevated baseline plasma myeloperoxidase activity level that decreased after multiple doses of RLS-0071, decreased further immediately after dosing, and then partially recovered to pre-9th dose baseline after 24 hours.

Discussion

Although healthy and asymptomatic, the moderate elevation of plasma myeloperoxidase in this subject suggests that she had a subclinical inflammatory process of unknown origin. After 9 doses of RLS-0071 at 10 mg/kg administered intravenously every 8 hours, myeloperoxidase levels and myeloperoxidase activity had both decreased, likely reflecting moderation of the underlying inflammatory process by RLS-0071. After the 9th dose of RLS-0071, further decreases in myeloperoxidase activity were measured at end of infusion and 1 hour after the dose, but myeloperoxidase levels were unchanged. This is consistent with the mechanism of action of RLS-0071, which rapidly inhibits myeloperoxidase enzymatic activity. Thus, after a dose of RLS-0071, the concentration of myeloperoxidase should not change, but activity of circulating myeloperoxidase should be inhibited. The partial return of plasma myeloperoxidase activity 24 hours after the final dose is consistent with the short in-blood half-life of RLS-0071 of approximately 2.7 hours (Table 4). Likewise, the partial return to baseline of plasma myeloperoxidase concentration 24 hours after the last dose is also explained by the short half-life of the compound.

RLS-0071 inhibits extracellular myeloperoxidase activity via the reducing action of the cysteines in the peptide [3], suggesting that RLS-0071 inhibition of myeloperoxidase is likely reversible. The decrease in myeloperoxidase level, as well as activity, suggests that RLS-0071 is inhibiting the underlying inflammatory condition that is leading to the elaboration of extracellular myeloperoxidase. This subject was young and apparently healthy, so the underlying inflammatory condition was occult. However, young and healthy individuals can have elevated plasma myeloperoxidase levels due to underlying unrecognized atheromatous disease and likely other processes that have yet to manifest as illness. It is unclear at this time whether the current initial intravenous formulation of RLS-0071 would be logistically feasible for treatment of chronic conditions like atherosclerosis due to the whole-body half-life of approximately 24 hours. Initial clinical trials for this formulation will focus on the treatment of acute inflammatory conditions in which myeloperoxidase has been shown to play a prominent role in pathogenesis. Development of formulations that are better for treating chronic inflammatory conditions is a future goal.

Taken together, these data show temporal correlations for RLS-0071 decreasing both myeloperoxidase concentration, as well as myeloperoxidase activity level after multiple doses, and suggests that RLS-0071 can have a measurable effect on myeloperoxidase in humans. Although further studies are needed to replicate these findings in other people with elevated myeloperoxidase plasma levels, these results suggest promise for RLS-0071 to moderate markers of underlying inflammatory states and potentially reduce the risk for attendant future major cardiovascular events.

Conclusions

This observation suggests RLS-0071 has therapeutic potential to moderate plasma myeloperoxidase levels and activity and modulate diseases in which myeloperoxidase contributes to pathogenesis.