Early Development of Ocadusertib, a Selective Receptor‐Interacting Serine/Threonine‐Protein Kinase 1 Inhibitor

ABSTRACT

Receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) inhibitors are being investigated for chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis. Ocadusertib is a potent and selective allosteric inhibitor of RIPK1 and is currently being studied in clinical trials. Here, we present data on the development of ocadusertib, including preclinical studies and pharmacokinetic, safety, and target engagement results from phase 1 trials. Preclinically, ocadusertib inhibited RIPK1 enzymatic activity with high potency (half‐maximal inhibitory concentration [IC₅₀] = 12–38 nM) and inhibited necroptotic responses in multiple cell‐based assays. Ocadusertib was highly selective for RIPK1, showing no significant inhibition of more than 100 other kinases representative of the full kinome. In mouse studies, ocadusertib treatment prevented RIPK1‐dependent hypothermia in response to tumor necrosis factor (TNF) challenge and significantly reduced disease severity in a model of chronic proliferative dermatitis. In a first‐in‐human study of ocadusertib in healthy participants, single oral doses exhibited linear pharmacokinetics and dose‐proportional exposure, with a time to maximum concentration of 1–4 h and a half‐life of 13–15 h. Steady state was attained at 4–6 days after multiple once‐daily dose administrations. Ocadusertib was well tolerated, with no deaths, serious adverse events, or significant treatment‐emergent adverse events reported. In a phase 1, double‐blind, randomized, multiple‐dose study in healthy participants, greater than 90% RIPK1 target engagement was achieved at Day 14 with ocadusertib treatment. Taken together, these findings support further assessment of ocadusertib for the treatment of chronic inflammatory diseases.

Study Highlights

• What is the current knowledge on the topic?

  • ○
    Ocadusertib is a potent and selective allosteric inhibitor of receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) that blocks inflammatory cell death downstream of death receptors. RIPK1 inhibitors have shown potential for the treatment of inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis, but thus far, none have been approved.
• What question did this study address?

  • ○
    This study presents data on the development of ocadusertib, including pharmacokinetic, safety, and target engagement results from phase 1 studies in humans.
• What does this study add to our knowledge?

  • ○
    Preclinical studies in cells and animal models demonstrated high degrees of selectivity and activity of ocadusertib. Phase 1 clinical studies showed that ocadusertib is well tolerated in healthy adults, and that robust target engagement is achievable.
• How might this change clinical pharmacology or translational science?

  • ○
    The results support further assessment of ocadusertib for treatment of chronic inflammatory diseases. Results of the phase 2 trial in rheumatoid arthritis (NCT05848258) may support dose selection achieving a high degree of sustained target engagement over the dosing period.

1. Introduction

The receptor‐interacting serine/threonine‐protein kinase 1 (RIPK1) regulates gene transcription and programmed cell death processes, including apoptosis and necroptosis [1]. Since necroptosis is strongly linked to inflammation [2, 3], RIPK1 is tightly regulated to maintain tissue homeostasis [4, 5, 6]. RIPK1 signaling pathways have been linked to the pathogenesis and progression of various diseases, such as rheumatoid arthritis, atherosclerosis, neurodegenerative diseases, and cancer [7, 8, 9, 10].

Through both scaffolding and kinase‐specific functions, RIPK1 mediates signals downstream of a broad range of immune receptors, including tumor necrosis factor (TNF) receptor 1 (TNFR1), Fas, and toll‐like receptor 3 (TLR3) [11, 12]. When RIPK1 is highly ubiquitinated, it acts as a scaffolding protein that enables TNFR1 activation of nuclear factor kappa B (NF‐κB) and mitogen‐activated protein kinase (MAPK) pathways, promoting gene transcription and cell survival [11]. In conditions where caspase‐8 is inactivated or absent, activated RIPK1 autophosphorylates, leading to the recruitment and phosphorylation of RIPK3 and mixed lineage kinase domain‐like protein (MLKL) to drive necroptosis [1]. Alternatively, genetic perturbation of the linear ubiquitination complex LUBAC—via Sharpin deficiency—leads to increased RIPK1 association with the death‐signaling Complex IIb and apoptosis following TNF stimulation [13]. These processes are dependent on the kinase activity of RIPK1.

Small‐molecule inhibitors of RIPK1 are being investigated for the treatment of chronic inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis [14]. Selective RIPK1 inhibition over pan‐inhibition of RIPK1, RIPK2, and RIPK3 unburdens the complexity of mixing different signaling pathways (RIPK2) [15] or the potential safety issues with blocking RIPK3 kinase activity [16]. Ocadusertib (LY3871801/R941552/R552; Figure 1), an allosteric inhibitor of RIPK1, blocks inflammatory cell death driven by necroptosis. Here, we present preclinical data examining ocadusertib's selectivity and potency in cells and mouse models, as well as phase 1 clinical data evaluating its pharmacokinetics (PK), safety, and tolerability in healthy adults.

FIGURE 1.

Chemical structure of ocadusertib. CAS No: 2382811‐41‐6.

2. Materials and Methods

2.1. Cell Studies

2.1.1. Enzymatic Activity Assays in Human U937 Cells

RIPK1, RIPK2, and RIPK3 kinase activities were measured using the ADP‐Glo Kinase Assay (Promega). Human and monkey RIPK1 kinase activities were measured by enzyme‐linked immunosorbent assay (ELISA).

Human U937 cells were preincubated with ocadusertib for 30 min, then stimulated with TNF alone for 10 min or 3 h, or TNF in the presence of a pan‐caspase inhibitor (zVAD) for 3 h. Cells were lysed in sample lysis buffer, and proteins were separated by SDS‐PAGE. Phosphorylated RIPK1, RIPK3, and MLKL were detected by Western blotting using phospho‐specific antibodies.

2.1.2. Necroptotic Response Assays

1. Cell lines: Human U937 cells were preincubated with ocadusertib for 1 h at 37°C with 5% CO₂, then stimulated with 2 ng/mL TNF in the presence of zVAD for 18 h. Primary human T cells, pre‐activated through T‐cell receptor stimulation with anti‐cluster of differentiation (CD)3 and anti‐CD28 for 3–4 days, were preincubated with ocadusertib for 1 h at 37°C with 5% CO₂. For necroptosis assays, cells were stimulated with 5 ng/mL TNF or 25 ng/mL Fas ligand (FasL) in the presence of zVAD with transforming growth factor‐beta‐activated kinase 1 (TAK1) inhibitor (5z‐7‐oxozeaenol) for 16 h. For apoptosis assays, cells were stimulated with TNF in the presence of 5z‐7‐oxozeaenol. Mouse L929 cells were preincubated with ocadusertib for 1 h at 37°C with 5% CO₂, then stimulated with 0.1 ng/mL TNF or 3 μg/mL of the TLR3 agonist poly (I:C) in the presence of zVAD for 18 h. The relative number of viable cells was determined using the CellTiter‐Glo Luminescent Cell Viability Reagent Assay (Promega).

2. Whole blood: Freshly drawn peripheral blood from healthy volunteers was incubated with ocadusertib for 1 h at 37°C with 5% CO₂, then stimulated with 60 ng/mL TNF in the presence of zVAD for 5 h. Serum was collected after centrifugation, and macrophage inflammatory protein (MIP)‐1α, MIP‐1β, and interleukin (IL)‐8 levels were measured by ELISA.

3. Human peripheral blood mononuclear cells: Blood from healthy human donors was collected in heparin cell preparation tubes (CPT), centrifuged to remove red blood cells, and plasma with peripheral blood mononuclear cells (CPT PBMC) was collected. In a 96‐well deep block, CPT PBMC was pretreated with ocadusertib (at 12 concentration points titrated down from 10 μM) at 37°C, then stimulated with 6 ng/mL TNF in the presence of zVAD and birinapant for 4 h at 37°C. Cells were centrifuged, and supernatants were stored at −80°C until analysis using MIP‐1α DuoSet ELISA (R&D Systems). Cell pellets were lysed, and aliquots were stored at −80°C until analysis for RIPK1 target engagement by ELISA.

2.2. Kinase Panel Assays

Inhibition of 105 kinases was tested at an ocadusertib concentration of 10 μM using Z'‐LYTE, Adapta, or LanthaScreen kinase assays (ThermoFisher Scientific). Measurements were performed in duplicate at the Michaelis constant (K _m) for ATP for individual kinases or at 100 μM ATP. Ten‐point titrations against p38α, p38β, p38γ, and p38δ were conducted, starting from ocadusertib 10 μM. The p38α titration was run at the K _m for ATP (500 μM), a higher concentration than that used for the kinase panel (100 μM).

2.3. Target Engagement Assays

Target engagement was assessed by quantitating RIPK1 detection using a pair of antibodies that recognize distinct epitopes, one of which experiences a conformational change resulting in loss of detection following RIPK1 binding by ocadusertib, similar to published methods [17]. Unless otherwise noted, the volume per well was 50 μL, and washes were repeated three times with 200 μL PBST (phosphate‐buffered saline [PBS] with 0.05% Tween‐20, Sigma #P7949). Incubations were performed at room temperature.

Briefly, microplates (Costar #3690) were coated with 3 μg/mL internal mouse immunoglobulin (mIgG1) anti‐RIPK1 antibody (IBA601) in PBS and incubated overnight at 4°C. Plates were washed, then blocked with 2% bovine serum albumin (BSA; Gibco 15260‐037) in PBS (100 μL/well) for 1 h. After washing, samples were added in universal buffer (UB; 2% BSA in PBST) for 1 h. Plates were washed, then 80 ng/mL anti‐RIPK1 D94C12 XP (Cell Signaling #3493) in UB was added for 1 h. Plates were washed, then 110 ng/mL anti‐rabbit‐HRP (Southern Biotech #4049‐05) in UB was added for 45 min. Plates were washed, then 3,3′,5,5′‐tetramethylbenzidine (TMB; Thermo Scientific Pierce #34029) was added for 20 min. The reaction was quenched with 2 N sulfuric acid, plates were shaken, and absorbance read at 450 nm.

Target engagement of recombinant RIPK1 with the inhibitor compound led to reduced anti‐RIPK1 antibody D94C12 XP binding. The percentage of target engagement was calculated using the formula:

$$\begin{array}{c}\text{Percentage}=100-([\text{treated sample}/\text{average baseline or}\hfill \\ \text{untreated control}]\times 100).\hfill \end{array}$$

2.4. Animal Studies

All animal studies were performed in accordance with Rigel Institutional Animal Care and Use Committee (IACUC) guidelines and approved by Rigel institutional review committee.

Mice were housed in microisolator cages and given ad libitum access to rodent chow and water. Temperature was maintained at 72°F ± 5°F, with relative humidity at 35%–70%, and a 12‐h light/dark cycle.

To determine serum cytokine levels, mice were euthanized by CO₂ asphyxiation, and blood was collected via cardiac puncture into serum separator tubes. After clotting, samples were stored on ice and centrifuged to isolate serum, which was stored at −80°C until analysis using the MILLIPLEX Mouse Cytokine/Chemokine Magnetic Bead Panel (EMD Millipore).

To determine circulating compound levels, blood was collected into lithium heparin tubes, stored on ice, and centrifuged to isolate plasma. Plasma samples were stored at −80°C until analysis by liquid chromatography with tandem mass spectrometry (LC–MS/MS).

2.4.1. RIPK1‐Dependent Sepsis Model [18]

Female C57BL/6 mice (11–12 weeks old) were purchased from Envigo (Indianapolis, IN). The day before dosing, mice were randomized, weighed, and assigned to groups (n = 8/group).

Mice received either ocadusertib (5, 15, or 45 mg/kg) or vehicle. Fifteen minutes post‐dosing, mice received an intraperitoneal injection of TNF and zVAD. Body temperatures were measured using a rectal probe thermometer prior to TNF/zVAD administration and every hour thereafter. Three hours post‐challenge, mice were sacrificed, and blood was collected for determination of serum cytokine levels. Separate groups of mice (n = 6/group; n = 3/time point) were included to determine plasma ocadusertib at the time of TNF/zVAD administration. Mice were administered ocadusertib (5, 15, or 45 mg/kg), and blood was collected either 15 min or 3 h and 15 min later.

2.4.2. Sharpin ^cpdm Model of Dermatitis [19]

Female and male Sharpin ^cpdm and littermate control mice (~5 weeks old) were purchased from Jackson Laboratories and assigned to groups (n = 14–20/group). Mice received ocadusertib (10 or 30 mg/kg) or vehicle orally twice daily (BID) for 12 weeks, starting at 5 weeks of age before the development of skin lesions (prophylactic treatment) or at 8 weeks of age after lesion onset (therapeutic treatment). Dermatitis severity scores were evaluated twice weekly [20].

2.5. Statistical Analysis

Statistical analyses for preclinical studies were performed using GraphPad Prism software (San Diego, CA). Statistical significance between groups was assessed using one‐way analysis of variance (ANOVA) with Bonferroni's multiple comparisons, comparing the mean of each group to that of the vehicle group.

3. Human Studies

3.1. Ethics Statement

Clinical studies were conducted in accordance with consensus ethical principles derived from international guidelines, including the Declaration of Helsinki, the Council for International Organizations of Medical Sciences International Ethical Guidelines, applicable International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Good Clinical Practice guidelines, and applicable laws and regulations at the study site. Table S1 lists the Institutional Review Board name, protocol approval number, and date for each study. All participants provided written informed consent prior to enrollment. Table S2 lists full eligibility criteria for each trial.

3.2. Clinical Study Design (EudraCT: 2019‐002520‐32)

A first‐in‐human, phase 1, single‐center study to examine the safety and tolerability of ocadusertib in healthy male and female participants was conducted from September 17, 2019, to September 21, 2020. The study enrolled participants aged 18–55 years and comprised three parts: a single ascending dose cohort (SAD; Part 1A), a multiple ascending dose cohort (MAD; Part 2), and a formulation assessment cohort (Parts 1B and 3). The formulations tested were a lipid administered as an oral solution and two spray‐dried dispersion powders administered as oral suspensions (alternative formulation 1 [ALT1] and alternative formulation 2 [ALT2]). ALT1 was also administered as an oral tablet. For details of formulations, see Supporting Information.

In Part 1A, six participants were enrolled in each dose cohort with five participants randomly assigned to receive ocadusertib and one participant randomly assigned to receive matching placebo. Ocadusertib was administered in one of six escalating single dose levels: lipid solution for the 12 mg, 60 mg, and 180 mg doses; ALT1 suspension for the 300 mg, 750 mg, and 1000 mg doses. Participants were dosed on the morning of Day 1 following an overnight fast (≥ 8 h) and fasted post‐dose (≥ 4 h). In Part 1B, two fasted cohorts of six participants each received a single dose of ocadusertib 180 mg (ALT1 or ALT2 suspension).

In Part 2, eight participants were enrolled in two fasted cohorts, with six participants randomly assigned to receive ocadusertib administered 180 mg or 500 mg once daily (QD), and two participants randomly assigned to receive matching placebo for 14 days.

In Part 3, nine participants were enrolled and randomly assigned to one of three treatment sequences (three participants each). Participants received three treatments administered over three separate dosing occasions, separated by at least 7 days: 125 mg ALT1 suspension following an overnight fast (≥ 8 h); 125 mg ALT1 tablet following an overnight fast (≥ 8 h); and 125 mg ALT1 tablet following a high fat breakfast.

3.3. Clinical Study Design (NCT05960851)

A phase 1, double‐blind, randomized, multiple‐dose study to examine the safety and tolerability of ocadusertib in healthy Asian and non‐Asian participants was conducted from August 9, 2023 to October 25, 2023.

The study consisted of two parts, each involving two cohorts. In Part A, six Japanese and four non‐Asian participants were enrolled in each cohort, with five Japanese and three non‐Asian participants randomly assigned to receive ocadusertib and one Japanese and one non‐Asian participant randomly assigned to receive matching placebo. In Part B, six Chinese participants were enrolled in each cohort, with five participants randomly assigned to receive ocadusertib and one participant randomly assigned to receive placebo. Ocadusertib (125 mg or 375 mg) or placebo was administered orally as spray‐dried dispersion tablets (25 mg/tablet) QD for 14 days.

3.4. Safety Parameters

For both clinical studies, safety evaluation included analysis of adverse events (AEs), laboratory variables, vital signs, electrocardiogram, and physical examination findings. AEs were coded using MedDRA v22.1 or v26.0.

3.5. Pharmacokinetic Parameters

For details of PK sampling and analysis, see Supporting Information.

4. Results

4.1. Potency and Selectivity of Ocadusertib In Vitro

We first assessed the potency and selectivity (target protein and species) of ocadusertib in cell‐based assays (Table S3). Ocadusertib inhibited human RIPK1 kinase activity with a half‐maximal inhibitory concentration (IC₅₀) of 12 and 38 nM, depending on the assay format. Ocadusertib inhibited monkey RIPK1 with similar potency and did not inhibit the closely related kinases RIPK2 and RIPK3.

To trigger RIPK1 activation and necroptosis, human U937 cells were treated with TNF in the presence of zVAD. Ocadusertib inhibited RIPK1 auto‐phosphorylation at serine residue 166 and downstream phosphorylation of RIPK3 and MLKL in a dose‐dependent manner, with a half‐maximal effective concentration (EC₅₀) of approximately 3 nM (Figure 2A).

FIGURE 2.

Ocadusertib inhibits RIPK1 signaling in human cells and whole blood. Increasing concentrations of ocadusertib blocked (A) RIPK1 autophosphorylation and downstream phosphorylation of RIPK3 and MLKL in human U937 cells, and (B) RIPK1‐dependent cytokine release in human peripheral blood. EC₅₀, half‐maximal effective concentration; IL, interleukin; MIP, macrophage inflammatory protein; MLKL, mixed lineage kinase domain‐like protein; pMLKL (S358), MLKL phosphorylated on serine 358; pRIPK1 (S166), RIPK1 phosphorylated on serine 166; pRIPK3 (S227), RIPK3 phosphorylated on serine 227; RIPK1, receptor‐interacting serine/threonine‐protein kinase 1; SD, standard deviation; TNF, tumor necrosis factor; zVAD, pan‐caspase inhibitor.

We examined ocadusertib's ability to protect human and mouse cells from necroptosis induced by TNFR1, Fas, or TLR3 engagement (Table S4). Ocadusertib blocked TNF/zVAD‐induced necroptosis in human U937 cells with an EC₅₀ of 3 nM. In human primary cells, ocadusertib inhibited both TNF‐ and FasL‐dependent necroptosis with similar potency (EC₅₀ = 1 nM) and protected human primary T cells from TNF‐dependent apoptosis with an EC₅₀ of 0.4 nM. Mouse RIPK1 lacks complete homology with human RIPK1 around the active site, which results in some RIPK1 inhibitors being significantly less potent in mouse cells, further complicating animal model studies [21]. However, in mouse L929 cells, ocadusertib inhibited necroptosis induced via TNFR1 or TLR3 (EC₅₀ = 67 and 55 nM, respectively). Similar potency was observed in mouse primary macrophages (EC₅₀ = 85 nM).

To assess the impact of blood components on potency, we measured ocadusertib's effect on RIPK1‐dependent signaling in human peripheral blood. Ocadusertib inhibited TNF/zVAD‐induced chemokine release with an EC₅₀ ranging from 7 to 9 nM (Figure 2B). Its potency was reduced approximately four‐fold in whole blood compared with purified cells, likely due to serum protein binding.

We also assessed ocadusertib's target selectivity against a panel of 105 kinases representing diverse subfamilies of the full kinome (Figure S1). Only two kinases, p38α and RSK2, were inhibited more than 50% by ocadusertib at 10 μM—a concentration 800‐fold higher than the IC₅₀ of RIPK1. For the p38 family of kinases, 10‐point titrations starting at 10 μM ocadusertib showed no significant inhibition of any isoform (Figure S1 inset).

4.2. Efficacy of Ocadusertib In Vivo

We investigated ocadusertib's efficacy in a mouse model of sterile sepsis [18]. Systemic administration of TNF/zVAD induces a shock‐like syndrome in mice, characterized by hypothermia, inflammatory cytokine release, and death. RIPK1 inhibitors can rescue hypothermia in this model.

Compared to animals treated with vehicle alone, mice pretreated with ocadusertib (5, 15, or 45 mg/kg) exhibited reduced hypothermia (85%, 98%, and 98%, respectively) at 3 h post‐TNF/zVAD challenge (Figure 3A). Consistent with this, dose‐dependent suppression of serum IL‐6, IL‐8, MIP‐1β, and MIP‐2 levels was observed, while serum granulocyte colony‐stimulating factor and monocyte chemoattractant protein‐1 were only partially inhibited by ocadusertib (Figure 3B).

FIGURE 3.

Ocadusertib blocks TNF/zVAD‐induced hypothermia and serum cytokines in mice. C57BL/6 female mice were pretreated orally with vehicle or ocadusertib at the indicated doses prior to administration of TNF/zVAD. (A) Body temperature changes and (B) serum cytokine levels are shown at 3 h post‐challenge. Each bar represents the mean body temperature change ± SEM for n = 8 per group, and each dot represents an individual mouse. Percentage of inhibition compared to vehicle column is shown below graphs. Significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. IL, interleukin; G‐CSF, granulocyte colony‐stimulating factor; MCP‐1, monocyte chemoattractant protein‐1; MIP, macrophage inflammatory protein; n, number of animals; SEM, standard error of the mean; TNF, tumor necrosis factor; zVAD, pan‐caspase inhibitor.

We evaluated ocadusertib's efficacy in a Sharpin ^cpdm model of chronic proliferative dermatitis [22]. Sharpin ^cpdm mice develop spontaneous systemic inflammation accompanied by hair loss, scaling, and erythema of the skin of the dorsal neck and chest—a phenotype driven by TNF‐dependent RIPK1‐induced cell death [23]. We determined the effect of ocadusertib administered orally BID to Sharpin ^cpdm mice before the appearance of skin lesions (prophylactic treatment) and after lesion development (therapeutic treatment).

Both prophylactic and therapeutic treatment with ocadusertib decreased the progression and severity of dermatitis. Prophylactic treatment with ocadusertib at 10 mg/kg and 30 mg/kg BID lowered the dermatitis severity scores (85% and 100%, respectively; Figure 4A). Therapeutic treatment with ocadusertib at 30 mg/kg BID lowered the dermatitis severity score 57% (Figure 4B).

FIGURE 4.

Effect of ocadusertib on dermatitis score. Dermatitis severity scores over time in Sharpin ^cpdm mice treated with ocadusertib starting at (A) Week 5 (prophylactic regimen) and (B) Week 8 (therapeutic regimen). Each time point represents mean ± SEM for n = 14 per group. Percentage reductions in dermatitis severity scores compared to the respective vehicle group are shown at Week 17. Significance is denoted as ****p < 0.0001. AU, procedural defined units; BID, twice daily; n, number of animals; SEM, standard error of the mean.

4.3. Dose Rationale for Human Studies

Ocadusertib inhibited RIPK1‐dependent necroptosis in human cells with an average EC₅₀ of 1–3 nM but was approximately 50‐ to 100‐fold less potent in mouse cells (Table S4). The proposed human starting dose was adjusted accordingly based on the species‐related potency shift. In the mouse hypothermia model, doses up to 45 mg/kg were evaluated, and the PK/pharmacodynamic (PD) relationship was examined. A maximal effect at high drug concentration when all receptors were occupied by the drug relationship was observed. Therapeutically relevant effects in humans were expected by maintaining trough levels (within a dosing interval) above the EC₉₀ levels established in the mouse model. The target trough level in humans was 63 ng/mL (0.14 μM) for 90% inhibition.

Based on PK modeling, a first‐in‐human starting single dose of 12 mg predicted a sub‐therapeutic plasma concentration of 0.63 ng/mL at 24 h post‐dose. At this concentration, < 5% target inhibition was predicted based on the in vivo hypothermia mouse model.

The in vivo studies of ocadusertib included 28‐day toxicity studies in rats and monkeys. No dose limiting toxicities were observed at the highest dose level evaluated (1000 mg/kg/day), so that was used as the no observed adverse event level (NOAEL) for the dose selection rationale for first‐in‐human study. None of the doses administered were predicted to give individual exposure greater than the NOAEL exposure levels.

5. Clinical Exploration

5.1. First‐In‐Human Phase 1 Study (EudraCT: 2019‐002520‐32)

5.1.1. Participant Characteristics and Disposition

Baseline characteristics of participants in each part of the study are shown in Table S5. The mean age ranged from approximately 34–43 years, and the majority were male and of White race. There were no clinically relevant differences in demographic details between placebo‐ and ocadusertib‐treated participants or among those receiving different ocadusertib formulations.

Participant disposition in each part of the study is shown in Figure S2. In Part 1A, one participant in the ocadusertib 12 mg cohort was lost to follow‐up and therefore discontinued from the study. All participants completed Parts 1B and 2 of the study. In Part 3, one participant received a single dose of 125 mg ALT1 tablet but was subsequently discontinued from the study per physician's decision.

5.1.2. Safety

Safety events in the first‐in‐human study are summarized in Table S6 and detailed in Table S7. Seventy‐one of 73 participants completed the study, and 63 received ocadusertib. No deaths, serious adverse events (SAEs), severe treatment‐emergent adverse events (TEAEs), TEAEs leading to participant withdrawal, or other significant TEAEs were reported. All TEAEs were mild in severity, except for a moderate headache reported by one participant in Part 1A following administration of a 1000 mg ALT1 suspension. Headache was the most common TEAE (n = 4); no other TEAEs were reported more than once. No clinically significant changes were observed in laboratory assessments (hematology, coagulation, clinical chemistry, or urinalysis), electrocardiogram findings, or vital signs following ocadusertib treatment.

Nine participants (30.0%) in Part 1A and four participants (33.3%) in Part 1B reported TEAEs following dosing with ocadusertib compared with four participants (66.7%) in the placebo group. All TEAEs were resolved before study completion, except for a mild case of calcinosis. Six participants (50.0%) in Part 2 reported at least one TEAE following dosing with ocadusertib; no events were reported in the placebo group. All TEAEs were resolved before study completion. Three participants (33.3%) in Part 3 reported a total of four events; none were considered related to ocadusertib treatment. All TEAEs reported during Part 3 were mild in severity and resolved before study completion.

5.1.3. Pharmacokinetics

PK parameters for the SAD portion of the study are shown in Table 1. The median time to maximum plasma concentration (t _max) was 1 h at the lower dose levels (12–180 mg) when ocadusertib was administered as a lipid formulation. Median t _max occurred slightly later (2–4 h) when ocadusertib was administered as an ALT1 suspension (180–1000 mg). After reaching the maximum plasma concentration (C _max), ocadusertib levels declined in a biphasic manner and remained quantifiable through the final sampling time point at 72 h post‐dose (Figure 5A). Ocadusertib exhibited linear and dose‐proportional PK over the 180–1000 mg dose range. Exposures (area under the curve) for both ALT1 and ALT2 suspensions were generally comparable to those of the lipid formulation at the same dose level (180 mg) across the PK parameters evaluated. The elimination half‐life of ocadusertib was approximately 13–15 h (geometric mean) and appeared consistent across dose levels and formulations.

TABLE 1.

Pharmacokinetic parameters for ocadusertib following single oral doses.

Part 1A							Part 1B
Dose‐level formulation	12 mg Lipid (N = 5)	60 mg Lipid (N = 5)	180 mg Lipid (N = 5)	300 mg ALT1 (N = 5)	750 mg ALT1 (N = 5)	1000 mg ALT1 (N = 5)	180 mg ALT1 (N = 6)	180 mg ALT2 (N = 6)
t max a (h)	1.000 (0.50–1.00)	1.000 (0.50–1.00)	1.000 (0.50–2.00)	2.000 (0.25–4.00)	2.017 (2.00–4.00)	4.000 (2.02–4.00)	4.000 (2.00–4.00)	2.000 (1.00–6.00) [n = 5]
C max (ng/mL)	349 (38.5)	2110 (6.9)	5030 (16.5)	6410 (11.0)	15,900 (28.5)	26,000 (18.7)	4020 (24.2)	4430 (18.0) [n = 5]
AUC0‐tlast (ng⋅h/mL)	4270 (33.5)	21,400 (6.5)	61,400 (19.0)	109,000 (17.3)	272,000 (23.3)	499,000 (16.0)	63,000 (29.4) [n = 5]	71,800 (15.1) [n = 5]
AUC0–∞ (ng⋅h/mL)	4430 (35.3)	22,100 (6.8)	63,200 (19.1)	111,000 (17.8)	282,000 (23.5)	499,000 (16.0)	62,800 (27.5)	73,800 (15.3) [n = 5]
t 1/2 (h)	14.801 (17.0)	14.353 (13.7)	13.775 (14.5)	13.483 (15.7)	15.261 (7.4)	14.533 (12.6)	14.214 (16.1)	13.626 (9.5)

Note: Data are presented as geometric mean (percent coefficient of variation) unless noted otherwise.

Abbreviations: ALT1, alternative formulation 1; ALT2, alternative formulation 2; AUC0‐∞, area under the plasma concentration‐time curve from time 0 to infinity; AUC0‐tlast, area under the plasma concentration‐time curve from time 0 to the last quantifiable time point; C _max, peak observed plasma concentration; Lipid, lipid solution; t _1/2, half‐life; t _max, time to C _max.

^a Median (range).

FIGURE 5.

Ocadusertib pharmacokinetics in the first‐in‐human phase 1 study. (A) Plasma concentration‐time profiles after a single dose; (B) Day 14 plasma concentrations after multiple QD doses; and (C) relative bioavailability. ALT1, alternative formulation 1; Fast, fasted; Lipid, lipid solution; hr., hour; QD, once daily.

PK parameters for the MAD portion of the study are shown in Table S8. Steady state was attained 4–6 days following QD administration of ocadusertib ALT1 suspension at single doses of 180 mg and 500 mg QD for 14 days. On Day 14, the median t _max was 3 h following 180 mg dosing; 4 h following 500 mg dosing (Figure 5B).

PK parameters for Part 3 of the study are shown in Table S9. Under fasted conditions, median t _max following 125 mg dosing was similar between the ALT1 suspension and ALT1 tablet at 1.5–2 h (Figure 5C, Table S9). The C _max of the ALT1 tablet was approximately 34% higher than the ALT1 suspension. ALT1 tablet dosing in the fed state showed similar overall exposures to the same dose in the fasted state, but t _max was delayed and C _max was reduced (Figure 5C, Table S9).

The inter‐subject variability in ocadusertib exposure remained low following single and multiple doses, ranging from 6.0% to 42.7% across all study parts. Comparing ocadusertib doses of 125 mg ALT1 suspension and tablet in the fasted and fed states, the half‐life increased by approximately 2.5 h in the fed state. However, no clinically meaningful food effect was anticipated based on these findings.

5.2. Phase 1 Study in Healthy Asian and Non‐Asian Participants (NCT05960851)

5.2.1. Participant Characteristics and Disposition

The baseline characteristics of participants by cohort are shown in Table S10. The overall mean age across the ethnic groups ranged from 43.4 years in the non‐Asian group to 56.7 years in the Japanese group. In the Japanese cohort, the majority of participants were female (58.3%), while most participants were male in the Chinese (75%) and non‐Asian (62.5%) groups. The mean body mass index was similar across ethnic groups. A total of 32 participants were enrolled, dosed, and completed the study (Figure S2).

5.2.2. Safety

A summary of safety events is presented in Table S11 and detailed in Table S7. QD doses of ocadusertib at 125 mg and 375 mg were well tolerated in all participants. No deaths or SAEs were reported. TEAEs were similar to those in the first‐in‐human phase 1 study, and no clinically meaningful safety findings or trends were identified.

5.2.3. Target Engagement and Ex Vivo Pharmacodynamic Response

To understand the extent of RIPK1 inhibition achievable with daily ocadusertib dosing in humans, we first explored the relationship between target engagement and pharmacodynamic responses in vitro. Necroptosis was induced in whole blood samples from healthy individuals using a cocktail of TNF, zVAD, and birinapant with varying doses of ocadusertib. In this in vitro setting, the minimum concentration of ocadusertib at which inhibition of MIP1α secretion was observed (IC_min) was 0.0265 μM, indicating a high degree of target engagement was necessary to elicit a functional response (Figure 6A). A conservative estimate of the ocadusertib concentration at which maximal inhibitory effect was observed (IC_max; defined as calculated IC₉₉ + 3 SD) was 1.1586 μM, or 532.4 ng/mL (molecular weight of ocadusertib = 459.5 g/mol) (Figure 6A). Notably, this threshold was below the ocadusertib exposures achieved at the 24‐h trough following a single dose of 125 mg (Figure 5C). Similarly, whole blood samples from individuals dosed with ocadusertib (125 mg or 375 mg for 14 consecutive days) demonstrated greater than 90% RIPK1 target engagement in six of seven participants from Part A (Figure 6B). In this study, ocadusertib exposures in individuals dosed with 125 mg or 375 mg daily exceeded the in vitro‐defined IC_max (Figure 6B).

FIGURE 6.

Ocadusertib target engagement in human samples. (A) Percentage of target engagement relative to MIP‐1α secretion from three representative healthy human donor whole blood samples treated with ocadusertib, TNF, zVAD, and birinapant. IC_min = 0.0265 μM; IC_max = calculated IC₉₉ + 3 SD = 1.1586 μM (B) Percentage of RIPK1 target engagement plotted against ocadusertib trough levels at Day 14 of treatment (open diamonds = 125 mg QD; solid circles = 375 mg QD) in the study NCT05960851. Data shown are for ocadusertib‐treated participants with both baseline and Day 14 samples (n = 7) for RIPK1 target engagement. IC_max = 532.4 ng/mL (molecular weight of ocadusertib = 459.5 g/mol). IC_max, ocadusertib concentration at which maximal inhibitory effect was observed; IC_min, minimum ocadusertib concentration at which inhibitory effect was observed; MIP‐1α, macrophage inflammatory protein‐1 alpha; PK, pharmacokinetics; QD, once daily; RIPK1, receptor‐interacting serine/threonine‐protein kinase 1; TNF, tumor necrosis factor; zVAD, pan‐caspase inhibitor.

6. Discussion

Our functional studies in cell culture systems, in vivo animal models, and clinical studies in humans demonstrated that ocadusertib is a potent and highly selective allosteric RIPK1 inhibitor. Ocadusertib also exhibited good target engagement and acceptable safety and tolerability at the doses studied.

In mouse models, ocadusertib prevented RIPK1‐dependent hypothermia in response to TNF challenge and was equally efficacious in preventing genetic chronic proliferative dermatitis. Ocadusertib treatment after disease onset resulted in reduced dermatitis severity compared to untreated animals. The robust activity under both acute and chronic treatment as well as prevention models suggests the potential utility of ocadusertib within the inflammation/reset/homeostasis treatment continuum of autoimmune and inflammatory diseases [24].

In the first‐in‐human study, multiple QD doses of ocadusertib (up to 500 mg for 14 days) were well tolerated in healthy participants. The doses evaluated in the phase 1 study in healthy Asian and non‐Asian participants (125 and 375 mg) were expected to achieve exposures that were previously demonstrated to be safe and tolerable.

The rationale for selecting a QD dosing regimen was based on several considerations, including ocadusertib's extended elimination half‐life in humans, efficacy predictions from human whole blood assays, and an established favorable therapeutic window. The observed half‐life of approximately 14 h supports sustained plasma concentrations with minimal inter‐dose fluctuation, justifying QD administration while also promoting patient compliance. Additionally, efficacy projections from human whole blood assays indicated that target inhibition would be maintained at concentrations achievable and sustainable with such a dosing schedule. These findings were corroborated by target engagement assays, which demonstrated durable high‐level target engagement.

In phase 1 human studies in healthy adults, TEAEs were mild in severity, and no deaths, serious AEs, or study discontinuations due to AEs occurred. Ocadusertib exhibited an excellent PK profile, with a long half‐life, linear PK, dose proportionality, fast and predictable time to steady state, low inter‐subject variability, and no food effects. These features could support the feasibility of QD dosing and are key benefits for dose selection and adjustment in clinical practice.

Other RIPK1 inhibitors have demonstrated promising preclinical activity but have not advanced in clinical development [25, 26, 27]. Based on our exploratory pharmacodynamic studies, one possible explanation is that full inhibition of RIPK1 kinase activity during the dosing interval may require substantially higher exposures than previously achieved. In our study, daily dosing of ocadusertib resulted in robust RIPK1 target engagement by Day 14. Complete inhibition, especially in tissues, which may require sustained exposures not achieved by other inhibitors, could be needed. Ocadusertib appears capable of achieving the necessary exposures to test this hypothesis in the ongoing phase 2 clinical trial of ocadusertib in rheumatoid arthritis patients (NCT05848258).

Author Contributions

All authors wrote the manuscript. S.J.S., J.T.S., V.C.T., A.C., L.Y., and C.L. designed the research. S.J.S., N.C.R., S.H., V.C.T., A.C., L.Y., and C.L. performed the research. S.J.S., N.C.R., S.H., J.T.S., V.C.T., A.C., A.C.V., D.J.D., L.Y., C.L., and E.S.M. analyzed the data.

Funding

This work was funded by Eli Lilly and Company and Rigel Pharmaceuticals.

Conflicts of Interest

J.T. Sims, S. Ho, N.C. Reising, E.P. Hanson, A.C. Vendel, and D.J. Dairaghi are employees and shareholders of Eli Lilly and Company. S.J. Shaw, V.C. Taylor, C. Lamagna, L. Yan, A. Chow, and E.S. Masuda are former employees of Rigel Pharmaceuticals Inc.

Supporting information

Data S1: cts70519‐sup‐0001‐DataS1.docx.

Data S2: cts70519‐sup‐0002‐DataS2.docx.

Shaw S. J., Taylor V. C., Sims J. T., et al., “Early Development of Ocadusertib, a Selective Receptor‐Interacting Serine/Threonine‐Protein Kinase 1 Inhibitor,” Clinical and Translational Science 19, no. 3 (2026): e70519, 10.1111/cts.70519.

Data Availability Statement

Eli Lilly and Company provides access to all individual participant data collected during the trial, after anonymization, with the exception of pharmacokinetic or genetic data. Data are available to request 6 months after the indication studied has been approved in the USA and the European Union and after primary publication acceptance, whichever is later. No expiration date of data requests is currently set once data are made available. Access is provided after a proposal has been approved by an independent review committee identified for this purpose and after receipt of a signed data‐sharing agreement. Data and documents, including the study protocol, statistical analysis plan, clinical study report and blank or annotated case report forms, will be provided in a secure data‐sharing environment. For details on submitting a request, see the instructions provided at www.vivli.org.