Safety and Pharmacokinetics of SRN001, a Novel siRNA Drug Targeting Amphiregulin via the SAMiRNA Platform

Abstract

Introduction

SRN001 is a small interfering RNA (siRNA) drug targeting amphiregulin. It was developed using a novel SAMiRNA^® platform, in which the siRNAs are protected within a self-assembled micelle. This platform eliminates the need for additional excipients to reduce unintended immune reactions. Amphiregulin plays a critical role in tissue repair and immune modulation. By silencing it, SRN001 is expected to be a therapeutic option for various pathologically fibrotic and cancerous diseases. This study aimed to evaluate safety and pharmacokinetics (PK) of SRN001 in humans for the first time.

Methods

A randomized, double-blind, placebo-controlled, single ascending dose, Phase 1 trial was conducted (NCT05984992). Participants randomly received a single intravenous dose of SRN001 (15, 45, 135, or 210 mg) or placebo. Safety was assessed with vital signs, clinical laboratory tests, electrocardiograms, and inflammatory cytokine assays (IL-1β, IL-6, IFN-γ, and TNF-α). Serial blood samples were collected until 168 hours post-dose to quantify plasma antisense siRNA concentrations. Anti-drug antibodies (ADAs) were measured at pre-dose, 15 days, and 29 days post-dose.

Results

Among 25 participants, SRN001 was generally well tolerated. Unlike other approved siRNA drugs, no systemic symptoms or cytokine elevation suggestive of infusion-related reactions (IRRs) were observed in any subjects receiving SRN001, even without premedication. SRN001 exhibited dose-proportional exposure, with the average AUC_inf of 1.35 ug·hour/mL in the 15 mg dose group, achieving the therapeutic exposure in preclinical studies. The average plasma half-life was 0.48 hours. No ADAs were detected.

Conclusion

A single IV administration of SRN001 demonstrated a favorable safety profile and dose-proportional PK in this small first-in-human phase 1 clinical trial. By minimizing the need for excipients, the SAMiRNA platform is anticipated to facilitate the development of safer siRNA drugs in the future. Therapeutic potential of SRN001 will be explored in subsequent clinical trials.

Introduction

Small interfering ribonucleic acid (siRNA) drugs have recently emerged as a powerful modality for gene-specific suppression, with six drugs approved since 2018 (Patisiran, Givosiran, Lumasiran, Inclisiran, Vutrisiran, and Nedosiran).1,2 These agents silence target messenger RNA (mRNA) through RNA interference, leading to reduced protein translation.3 However, naked siRNAs are highly susceptible to degradation by ribonucleases (RNases) and trigger innate immune responses by acting as ligands for Toll-like receptors (TLRs).4–7 To overcome these limitations, the six approved siRNA drugs use various strategies including chemical modification of the oligonucleotide, N-acetyl-D-galactosamine (GalNAc) conjugation, or lipid nanoparticle (LNP) encapsulation.1,2 Although the vulnerability to degradation has been ameliorated, immune-response evasion remains a major challenge.8 Especially, the cationic lipids in its LNP carrier induce additional immune responses, leading to frequent infusion-related reactions (IRRs) that require steroid premedication in Patisiran.9–12

To mitigate the unintended immune reactions, SAMiRNA^® platform, a novel self-assembling micelle-forming supramolecular siRNA conjugate, was developed (Figure 1). 13 The unit structure consists of non-modified siRNAs conjugated with a hydrophilic head at the 3’ end and a hydrophobic tail at the 5’ end. These conjugates generate steric hindrance that prevents interaction with TLRs, thereby enabling immune response evasion.14–16 Also, in an aqueous solution, the unit structure spontaneously assembles into a supramolecular micelle, featuring an external hydrophilic corona and an internal hydrophobic core. The siRNAs within the micelles are protected from RNases, resulting in enhanced stability without the need for additional carrier molecules that could potentially trigger immune responses. The 100 nm-sized micelles preferentially accumulate in the inflamed tissue and tumor microenvironment owing to the enhanced permeability and retention (EPR) effect.17,18 The micelles enter cells via endocytosis and subsequently disassemble into their unit structures as the disulfide bonds in the hydrophobic tails at the 5′ ends are cleaved. The remaining antisense siRNA strand is subsequently loaded onto the argonaute 2 (Ago2) protein, forming the RNA-induced silencing complex (RISC), which suppresses translation of the target gene.5,19

Figure 1.

Synthesis, administration, and mechanism of action of SRN001. Sense DNA containing a hydrophilic head and a hydrophobic tail anneals with antisense RNA to form the structural unit of SRN001. In a buffered solution, these units self-assemble into ~100 nm-sized micelles. Following intravenous administration, SRN001 preferentially accumulates in inflamed tissues via the enhanced permeability and retention (EPR) effect. SRN001 enters fibroblasts through endocytosis, after which the endosome releases the structural unit into the cytoplasm. The unit is then loaded onto the RNA-induced silencing complex (RISC), leading to degradation of amphiregulin mRNA. Inhibition of amphiregulin protein translation subsequently suppresses the differentiation of fibroblasts into myofibroblasts.

Abbreviations: EPR, Enhanced Permeability and Retention; RISC, RNA-Induced Silencing Complex.

SRN001 is a newly developed siRNA drug using SAMiRNA platform targeting amphiregulin. Amphiregulin is a ligand of the epidermal growth factor receptor (EGFR). Chronic dysregulated amphiregulin expression enhances transforming growth factor-beta (TGF-β) signaling, resulting in pathologic fibrosis in idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), and chronic kidney disease (CKD).20–23 In patients with IPF, both tissue-associated and soluble serum amphiregulin concentrations are significantly elevated.22,24–27 Additionally, amphiregulin contributes to tumor microenvironment remodeling via EGFR activation and facilitates immune evasion of the tumor cells by upregulating programmed death-ligand 1 (PD-L1) expression.28–31 Elevated serum amphiregulin concentration is considered as a poor prognostic biomarker indicative of insufficient efficacy of anti-EGFR drugs in multiple cancer types.28–30,32 By silencing amphiregulin, SRN001 is expected to offer a novel therapeutic option for various pathologically fibrotic and cancerous diseases.

Here, we report the first-in-human phase 1 clinical trial of SRN001, assessing its safety and pharmacokinetics (PK) in healthy volunteers.

Material and Methods

Participants

Healthy participants aged 18 to 70 years with a body mass index of 18.0–35.0 kg/m² were enrolled. Subjects were excluded if they had any of the following conditions: a history of clinically significant diseases; surgery within the past 1 year; clinically relevant wounds from invasive procedures such as endoscopy within the past 1 week of screening; significant infection or known ongoing inflammatory process; abnormal laboratory test results for hematologic, renal, or liver function; use of prescribed medications within the past 4 weeks; vaccination within 14 days before dosing; or use of over-the-counter medication within the past 7 days.

The trial was designed and analyzed by the department of clinical pharmacology and therapeutics, Seoul National University College of Medicine and Hospital, and Seoul National University Bundang Hospital (Seoul and Seongnam, Republic of Korea). The trial was conducted at the CMAX Clinical Research (Adelaide, Australia). The study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice (ClinicalTrials.gov registry no. NCT05984992). The study was approved by the Bellberry Human Research Ethics Committee (HREC; an independent ethics committee equivalent to an Institutional Review Board, approval no. 2023-06-684). All procedures were performed after obtaining written informed consent from the subjects.

Study Design

The trial was designed as a randomized, double-blind, placebo-controlled, single ascending dose study (Figure 2). Eligible participants were randomized into each dose group, with 4 participants per cohort receiving SRN001 and 2 receiving placebo. Participants received a single intravenous (IV) SRN001 at 15 mg, 45 mg, 135 mg, or 210 mg, or placebo as randomized. The pharmacologically active dose (PAD) in the IPF mouse model was 5 mg/kg/day, corresponding to a human equivalent dose (HED) of 24 mg/60 kg.33 In a 4-week repeated-dose toxicity study, the no observed adverse effect level (NOAEL) was 300 mg/kg/day for ICR mouse and 100 mg/kg/day for cynomolgus monkey, with corresponding HEDs of 1463 mg/60 kg and 1936 mg/60 kg, respectively. A starting dose of 15 mg was selected based on NOAEL with a 100-fold safety margin, and dose escalation was designed to reach up to 10-fold of the PAD. The investigational product (IP) was administered as an intravenous bolus injection, with the administration duration adjusted across dose cohorts to maintain a consistent injection rate. Cohorts 1 and 2 received manual bolus injections of 15 mg over 0.4 minutes and 45 mg over 1.1 minutes, respectively. Cohorts 3 and 4 received bolus injections of 135 mg over 3.2 minutes and 210 mg over 5 minutes, respectively, using a syringe pump.

Figure 2.

Design of the phase 1 clinical trial. Upward arrows (↑) indicate the timing of procedures. The bold horizontal arrow () indicates the intensive pharmacokinetic sampling period. Downward arrow (↓) indicates drug administration: SRN001 in test group and placebo in control group.

The primary endpoint was the incidence of treatment-emergent adverse events (TEAEs). Any abnormal symptoms or test results observed through the end of the study (29 days post-dose) were recorded as TEAEs. The severity of TEAE was evaluated using Common Terminology Criteria for Adverse Events (CTCAE) version 5.0. Causality to IP was assessed according to the World Health Organization–Uppsala Monitoring Centre (WHO–UMC) criteria. All TEAEs, including those assessed as unlikely related to the investigational product, were included in the analysis. Secondary endpoints included PK parameters calculated from plasma antisense siRNA concentrations obtained through serial blood sampling up to 8 days post-dose. Additionally, the presence of serum anti-drug antibodies (ADAs) was assessed through 29 days post-dose.

Safety Assessment

Safety was monitored through vital signs, physical examinations, inflammatory cytokine profiles, local injection site evaluations, clinical laboratory tests, and 12-lead electrocardiograms.

IRRs are acute immune responses that occur shortly after intravenous drug administration, mediated by cytokine activation. Typical symptoms include fever, chills, chest discomfort, back pain, hypotension, and rash. We measured cytokines including interleukin 1β (IL-1β), IL-6, interferon γ (IFN-γ), and tumor-necrosis factor-α (TNF-α). Cytokine levels were assessed at pre-dose and at 1, 6, and 24 hours post-dose. IL-1β, IL-6, IFN-γ, and TNF-α were analyzed by V-PLEX Proinflammatory Panel 1(4-plex) (Meso Scale Diagnostics, LLC., Maryland, United States), with the lower limit of quantification (LLOQ) value of 2.656, 0.352, 0.296, and 2.875 pg/mL, respectively. The clinically significant levels were 14.4, 27.2, 24.3, and 1.75 pg/mL, respectively.34

Injection site reactions including erythema, warmth, pain, pruritus, swelling, hematoma, or induration, were evaluated at pre-dose and at 1, 3, 9, 12, 24, 36, 48, 168, 336, 504, and 672 hours post-dose. Clinical laboratory tests included routine hematologic, hepatic, and renal function assessments, along with lactate dehydrogenase (LDH) and C-reactive protein (CRP). These tests were conducted at pre-dose and 6, 24, 48, 168, 336, 504, and 672 hours post-dose.

Pharmacokinetic Assessment

PK blood samples were collected at pre-dose and at 0.167, 0.5, 1, 2, 3, 6, 9, 12, 24, 36, 48, and 168 hours post-dose. On each scheduled time, 4 mL of blood was collected in a potassium ethylenediaminetetraacetic acid (K₂-EDTA) tube and centrifuged for 10 minutes at 2000 relative centrifugal force (RCF) at 4°C. The supernatant was separated in eppendorf tubes and stored at −80°C until the analysis.

The antisense siRNA was measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The human plasma samples of 30 µL were purified using a liquid-liquid extraction method with a phenol-chloroform-isoamyl alcohol mixture. Antisense siRNA was extracted using liquid chromatography (LC) with Unison UK-C18 (2.0 × 75 mm, 3 µm) and a mobile phase containing hexafluoro-2-propanol (HFIP). During the preparation process, all supramolecular micelles were fully disassembled into their structural unit, and both the hydrophilic head and hydrophobic tail were degraded, leaving only the antisense siRNA strand. The LC-MS/MS system (SCIEX TO6500+, Ontario, Canada) was operated in electrospray ionization negative mode (ESI-) with multiple reaction monitoring (MRM).35 The standard curves for antisense siRNA were linear over the range of 0.1–100 μg/mL in the plasma samples, with inter-day and intra-day accuracy within ±15% and precision within 15% coefficient of variation. The LLOQ for plasma antisense siRNA concentration was 0.1 ug/mL.

PK parameters were calculated by non-compartmental analysis (NCA) using Phoenix WinNonlin software version 8.3 (Certara, Pennsylvania, USA). The maximum concentration (C_max) was directly observed. The area under the time-concentration curve (AUC) was calculated through non-compartmental analysis using the linear-up log-down method. The AUC_last was calculated from the start of IV administration to the last measurable time. The AUC_inf was obtained by adding the last measurable concentration (C_last) divided by the elimination rate constant (k_el) to AUC_last. The clearance (CL) was calculated by dividing the dose by AUC_inf. The volume of distribution (V_z) was determined by dividing CL by k_el. The half-life (t_1/2) was obtained by dividing ln(2) by k_el.

Dose proportionality of the PK parameters (C_max and AUC_inf) was analyzed using a power model with the following equation:36

The regression slope (β₁) and the intercept (β₀) were estimated with 95% confidence interval (CI) using PROC REG in SAS version 9.4 (SAS Inst., North Carolina, United States). The PK parameters were considered dose-proportional if the 95% CI of the β₁ included 1.

Serum ADA was analyzed from the blood samples collected at pre-dose, 15 days, and 29 days post-dose. On each scheduled time, 4 mL of blood was collected in a serum separator (SST) tube and centrifuged for 10 minutes at 2,000 RCF at 4°C. With 2.5 µL of the supernatant sample, the ADA levels were measured using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) with a pre-coated SRN001 plate (siRNAgen Therapeutics, Daejeon, Republic of Korea). ADA was assessed in three steps. In the screening assay, samples were considered positive if the absorbance at 450 nm exceeded the screening cut point (0.057) in duplicates, with a percentage difference within 30%. In the confirmatory assay, SRN001-specific ADA was confirmed if signal inhibition exceeded the confirmatory cut point (7.8%) under the same variability criteria. In the titration assay, ADA concentration was quantified with a lower limit of quantification (LLOQ) of 348 ng/mL. The method was validated using Anti-SRN001 antibodies (Abclon Co., Ltd., Seoul, Republic of Korea).37

Results

In the first-in-human phase 1 clinical trial, 25 participants were enrolled, and 24 participants completed the trial. A single IV SRN001 15 mg, 45 mg, 135 mg, 210 mg, or a placebo, was administered to 4, 4, 5, 4, and 8 randomized participants, respectively. One participant in the 135 mg dose group received 4.7 mL (141 mg) of SRN001, exceeding the planned volume of 4.5 mL (135 mg) due to a syringe pump malfunction. Data from this participant was analyzed only for safety analysis, while excluded from PK analysis. The participant was replaced. Another participant in the 135 mg dose group adhered to the scheduled visits until day 15, then voluntarily withdrew from the trial. Participants had a mean age of 41.4 years; 44.0% were men; 72.0% were White; 8.0% were American Indian or Alaska native; 12.0% were Asian; 4.0% were Native Hawaiian or another Pacific Islander (Table 1).

Table 1.

Demographic Characteristics

	SRN001				Placebo (N=8)
	15 mg (N=4)	45 mg (N=4)	135 mg (N=5)	210 mg (N=4)
Age (years)	49.5 ± 14.1	48.3 ± 11.0	42.0 ± 20.1	44.8 ± 19.3	32.0 ± 12.1
Sex
Male (%)	1 (25.0)	3 (75.0)	3 (60.0)	1 (25.0)	3 (37.5)
Female with child bearing potential (%)	1 (25.0)	0 (0.0)	1 (20.0)	2 (50.0)	4 (50.0)
Female without child bearing potential (%)	2 (50.0)	1 (25.0)	1 (20.0)	1 (25.0)	1 (12.5)
Race
White (%)	4 (100.0)	3 (75.0)	3 (60.0)	4 (100.0)	4 (50.0)
American Indian or Alaska native (%)	0 (0.0)	0 (0.0)	2 (40.0)	0 (0.0)	0 (0.0)
Asian (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (37.5)
Native Hawaiian or other pacific islander (%)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	1 (12.5)
Other (%)	0 (0.0)	1 (25.0)	0 (0.0)	0 (0.0)	0 (0.0)
Weight (kg)	68.4 ± 3.6	86.7 ± 8.0	90.9 ± 20.0	68.2 ± 15.4	67.0 ± 21.0
Height (cm)	160.1 ± 7.1	177.6 ± 4.8	172.2 ± 12.8	167.6 ± 5.0	165.73 ± 10.0
Body mass index, BMI (kg/m2)	26.7 ± 1.7	27.5 ± 1.7	30.4 ± 4.1	24.2 ± 4.5	24.0 ± 4.9

Notes: All data were presented as the mean ± standard deviation.

Safety Assessment

A single IV administration of SRN001 15–210 mg was generally well tolerated (Table 2). One serious adverse event (SAE) of back pain occurred at 9 days post-dose in the 135 mg dose group. However it was considered a sudden flare-up of a pre-existing condition, which had been fluctuating since a horse fall four years ago. The participant was admitted to another hospital for workup and received physical therapy.

Table 2.

Incidence of Treatment-Emergent Adverse Events

System Organ Class Preferred Term	SRN001					Placebo (n=8)
	15 mg (n=4)	45 mg (n=4)	135 mg (n=5)	210 mg (n=4)	Total (n=17)
Number of subjects with any adverse events	3 (75.0) [11]	2 (50.0) [2]	3 (60.0) [15]	2 (50.0) [5]	10 (58.8) [33]	7 (87.5) [18]
Number of subjects with any serious adverse events			1 (20.0) [1]
Injury, poisoning and procedural complications	2 (50.0) [5]	1 (25.0) [1]	3 (60.0) [7]	1 (25.0) [1]	7 (41.2) [14]	5 (62.5) [7]
Injection site pain			2 (40.0) [4]		2 (11.8) [4]	2 (25.0) [3]
Injection site erythema				2 (50.0) [2]	2 (11.8) [2]	1 (12.5) [1]
Injection site hematoma		1 (25.0) [1]		1 (25.0) [1]	2 (11.8) [2]
Injection site bruising			1 (20.0) [1]		1 (5.9) [1]
Vessel puncture site pain	2 (50.0) [2]				2 (11.8) [2]
Vessel puncture site bruise	1 (25.0) [2]				1 (5.9) [2]	2 (25.0) [2]
Vessel puncture site pruritus	1 (25.0) [1]				1 (5.9) [1]	1 (12.5) [1]
Nervous system disorders	1 (25.0) [1]				1 (5.9) [1]	2 (25.0) [2]
Headache	1 (25.0) [1]				1 (5.9) [1]	1 (12.5) [1]
Somnolence						1 (12.5) [1]
Gastrointestinal disorders	1 (25.0) [2]			1 (25.0) [1]	2 (11.8) [3]	2 (25.0) [5]
Nausea				1 (25.0) [1]	1 (5.9) [1]	1 (12.5) [1]
Abdominal pain	1 (25.0) [1]				1 (5.9) [1]	1 (12.5) [1]
Diarrhoea	1 (25.0) [1]				1 (5.9) [1]	1 (12.5) [1]
Constipation						1 (12.5) [1]
Vomiting						1 (12.5) [1]
Skin and subcutaneous tissue disorders			1 (20.0) [6]		1 (5.9) [6]	2 (25.0) [2]
Rash			1 (20.0) [2]		1 (5.9) [2]
Dysaesthesia			1 (20.0) [1]		1 (5.9) [1]
Pain of skin			1 (20.0) [1]		1 (5.9) [1]
Pruritus			1 (20.0) [1]		1 (5.9) [1]	1 (12.5) [1]
Paraesthesia						1 (12.5) [1]
Skin swelling			1 (20.0) [1]		1 (5.9) [1]
Musculoskeletal and connective tissue disorders	2 (50.0) [3]		1 (20.0) [1]		3 (17.6) [4]
Contusion	1 (25.0) [2]				1 (5.9) [2]
Neck pain	1 (25.0) [1]				1 (5.9) [1]
Back pain			1 (20.0) [1]		1 (5.9) [1]
Cardiac disorders				1 (25.0) [3]	1 (5.9) [3]
Chest discomfort				1 (25.0) [1]	1 (5.9) [1]
Palpitations				1 (25.0) [1]	1 (5.9) [1]
Sinus tachycardia				1 (25.0) [1]	1 (5.9) [1]
Infections and infestations		1 (25.0) [1]			1 (5.9) [1]	1 (12.5) [1]
Chlamydial infection		1 (25.0) [1]			1 (5.9) [1]
Upper respiratory tract infection						1 (12.5) [1]
Respiratory, thoracic and mediastinal disorders			1 (20.0) [1]		1 (5.9) [1]
Productive cough			1 (20.0) [1]		1 (5.9) [1]
General disorders and administration site conditions						1 (12.5) [1]
Fatigue						1 (12.5) [1]

Notes: All data were presented as the number of subjects (percentage of subjects) [number of events].

Adverse events occurred in 10 participants (58.8%) who received SRN001, and 7 participants (87.5%) who received placebo. No apparent relationship was observed between dose and the frequency of adverse events. Local injection site reactions, including pain, erythema, hematoma, and bruising, were the most frequent adverse events. They occurred in 5 participants (29.4%) receiving SRN001 and in 2 participants (25.0%) receiving placebo. No other adverse events were observed across more than one dose cohort.

No symptoms or cytokine elevations suggestive of IRRs were observed in any subjects receiving SRN001, even without premedication. In all participants, IL-1β and TNF-α levels were below LLOQ, while IL-6 and IFN-γ remained below the clinically significant cut-off value up to 24 hours post-dose (Table S1). Furthermore, no clinically significant abnormalities were detected in the clinical laboratory test results of any participants.

One participant in the 210 mg dose group experienced transient chest discomfort while running, palpitations at dawn, and sinus tachycardia on electrocardiogram 6 days post-dose. These symptoms were considered attributable to individual predisposition, a family history of cardiac disorders, and environmental factors rather than to SRN001. One participant in the 15 mg dose group reported mild abdominal pain 1-hour post-dose. However, no other symptoms suggestive of IRRs were observed, and subsequent cytokine analyses showed no increase. One participant in the 15 mg dose group reported neck pain, and one participant in the 135 mg dose group reported back pain. However, in both cases, the symptoms occurred approximately one-week post-dose and developed after physical trauma to the affected area.

One participant in the 135 mg dose group experienced a red lump (rash) on the left 5^th finger; pruritus on the tip of the left 3^rd finger; erythema, swelling, pain, and dysaesthesia on the base of the left 3^rd finger, starting 1 hour post-dose. As the severity of symptoms was mild, the participant did not report the symptoms immediately but did so the following day. Consequently, a single oral antihistamine was administered at 24 hours post-dose. Most symptoms were resolved within 48 hours post-dose, although mild pruritus and dysaesthesia persisted until the Day 15 visit. The participant worked on a horse farm, and at 12 days post-dose, two itchy papules (rash) appeared on the right hand following the arrival of a new horse. The participant did not attend the scheduled visits on days 22 and 29, so symptomatic progress could not be followed up. Given the absence of symptoms suggestive of IRRs or cytokine elevation, as well as the participant’s occupation and the recurrence of similar localized lesions on the contralateral hand following the occupational exposure, the reaction was considered more likely attributable to contact dermatitis caused by environmental allergens. However, a delayed immune response to SRN001 cannot be completely excluded, and further evaluation in subsequent multiple-dose studies will be required to clarify this.

Pharmacokinetic Assessment

The plasma antisense siRNA concentration-time profile exhibited a monoexponential decline, with an average half-life of 0.48 hours (Figure 3). SRN001 demonstrated dose proportionality of AUC_inf across the 15–210 mg dose range (β₁ = 1.0560, 95% CI: 0.9097–1.2023) (Table 3). The average AUC_inf in the 15 mg dose group was 1.35 ug hour/mL, achieving the therapeutic exposure observed in TGF-β transgenic IPF mice at a 5 mg/kg dose administered three times with a 48-hour dosing interval.14 No serum ADAs were detected in this study.

Figure 3.

Plasma antisense siRNA concentration–time pharmacokinetic profile after a single intravenous administration of SRN001 15–210 mg. Filled circles indicate the mean and error bars represent the standard deviation.

Table 3.

Pharmacokinetic Parameters of Antisense siRNA

	Cmax (ug/mL)	AUClast (ug Hour/mL)	AUCinf (ug·Hour/mL)	CL (L/hour)	Vz (L)	t1/2 (Hour)
15 mg (N=4)	1.84 ± 0.38	1.22 ± 0.38	1.35 ± 0.39	12.04 ± 4.23	7.65 ± 1.24	0.46 ± 0.11
45 mg (N=4)	4.85 ± 0.61	2.91 ± 0.66	3.06 ± 0.65	15.20 ± 3.31	8.10 ± 0.84	0.38 ± 0.08
135 mg (N=4)	19.31 ± 4.78	14.04 ± 2.63	14.42 ± 2.75	9.63 ± 1.84	7.76 ± 1.83	0.56 ± 0.10
210 mg (N=4)	31.33 ± 3.16	18.26 ± 4.41	18.51 ± 4.59	11.83 ± 2.65	8.42 ± 1.59	0.5 ± 0.05

Notes: All data were presented as mean ± standard deviation.

Abbreviations: C_max, Maximum concentration; AUC_last, area under the time-concentration curve from dose administration to the last measurable time; AUC_inf, area under the time-concentration curve from dose administration to infinity; CL, clearance; V_z,, volume of distribution; t_1/2, half-life.

Discussion

SRN001 was generally well tolerated, with no cases suggestive of IRRs observed. Many adverse events in siRNA drugs are associated with excipients such as lipids, polymers or surfactants.38–40 They induce unintended immune responses by initiating complement activation-related pseudoallergy (CARPA) and triggering inflammasome activation.41,42 Therefore, IRRs are characteristic immunologic adverse events of siRNA drugs. For example, patisiran has been reported to cause IRRs at an incidence of 16–19%, requiring steroid premedication.12,43 In contrast, SAMiRNA platform eliminated the need for additional excipients. Owing to its unique amphiphilic structure, siRNA is protected within self-assembled micelle without requiring other carrier components. In the mouse peripheral blood mononuclear cells (PBMCs) assay, liposome-formulated siRNA drugs elevated TNF-α, IL-6, and IL-12, while SRN001 induced no such increase.14 In this phase 1 clinical trial, no participants exhibited symptoms or cytokine elevations suggestive of IRRs after SRN001 administration even without any premedication.

A single IV dose of SRN001 ranging from 15 to 210 mg exhibited a dose-proportional exposure with an average plasma half-life of 0.48 hours. The plasma half-life of SRN001 in humans was shorter than that observed in preclinical species, with reported values of 4.3 hours in mice and 2.1 hours in cynomolgus monkeys.44,45 Differences in nuclease activity, endosomal processing, and plasma protein binding affinity may have contributed to these species-dependent PK profiles. Future PK modeling will be conducted to identify factors contributing to the species-dependent differences. In addition, SRN001 was designed to form micelles approximately 100 nm in diameter, favorable for EPR effect, resulting in preferential accumulation to inflammatory tissues and tumor microenvironments.46–48 In wild-type mice, SRN001 showed negligible distribution to peripheral tissues, whereas in the IPF mouse model, Cy5.5-labeled SRN001 showed sustained accumulation within inflamed lung tissue for more than 48 hours.14 Accordingly, differences in PK distribution between healthy volunteers and patients are anticipated and will be assessed in subsequent phase trials.

Suppressing amphiregulin has emerged as a promising therapeutic strategy for fibrotic diseases and tumors. Amphiregulin plays a critical role in tissue repair and immune modulation. During chronic inflammation, regulatory T cells release amphiregulin to suppress excessive inflammatory reactions.49,50 However, persistently dysregulated amphiregulin secretion contributes to pathological fibrosis by promoting fibroblast differentiation.20–23 In the IPF mouse model, SRN001 effectively silenced amphiregulin in the lung tissue, leading to both histopathological and functional improvement.14 The accumulated SRN001 in the inflamed lung tissue entered cells via endocytosis, with CD140-positive fibroblasts exhibiting the highest Cy5.5-labeled signal. SRN001 reduced amphiregulin mRNA expression in lung tissue, demonstrated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). It led to decreased collagen accumulation and improved elasticity and compliance of the respiratory system. The efficacy of SRN001-mediated amphiregulin silencing and its effects on disease progression in humans will be further evaluated in patients in subsequent trials.

A limitation of this study is the small sample size in each dose cohort, reflecting the exploratory nature of this first-in-human phase 1 trial. Accordingly, the statistical interpretation of dose proportionality, as well as the conclusions regarding safety, including the absence of IRRs, should be interpreted cautiously. Another limitation is the lack of pharmacodynamic (PD) assessments to evaluate target engagement. To address these limitations, a multiple-dose clinical trial is currently in progress (NCT07407543). In that study, immune responses, including IRRs and delayed reactions, as well as PD effects, will be further evaluated, with serum amphiregulin levels used as a biomarker. Collectively, these studies will enable PK–PD modeling to establish the translational link between preclinical and clinical findings.

Conclusion

A single IV dose of SRN001 ranging from 15 to 210 mg was generally well tolerated and demonstrated dose-dependent PK profiles in humans. These findings support further evaluation of SRN001 in patient populations. Furthermore, by minimizing the need for excipients, the SAMiRNA platform is anticipated to facilitate future siRNA drug development.

Funding Statement

This study was funded by siRNAgen Therapeutics, Republic of Korea. The sponsor was involved in the study design, conduct of the study, and financial support, but had no role in data analysis, interpretation, or preparation of this paper.

Data Sharing Statement

The authors do not intend to share individual deidentified participant data beyond what is presented in this article. However, clinical trial data, including the protocol and statistical analysis plan, will be available via Email to the corresponding author upon reasonable request. The data will be available from the date of publication and will remain available for a period of 5 years.

Disclosure

The authors report no conflicts of interest in this work.