Abstract
Introduction
This study aimed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple doses of the ketohexokinase inhibitor LY3522348 in healthy participants.
Methods
This first-in-human phase 1 study evaluated LY3522348, a highly selective, oral dual inhibitor of human ketohexokinase (KHK) isoforms C and A. The study was conducted in two parts: a single-ascending dose (SAD) study and a multiple-ascending dose (MAD) study, including a drug–drug interaction analysis with midazolam. Participants in the SAD study received single oral doses of LY3522348 ranging from 5 to 380 mg, while participants in the MAD study received once-daily doses of 50 mg, 120 mg, and 290 mg for 14 days.
Results
A total of 65 healthy participants were included; of these 40 were in the SAD study (placebo = 10; LY3522348: 5 mg = 6; 15 mg = 6; 50 mg = 6; 150 mg = 6; 380 mg = 6) and 25 in the MAD study (placebo = 6; LY3522348: 50 mg = 6; 120 mg = 6; 290 mg = 7). LY3522348 was well tolerated, with the majority of the reported adverse events being mild. PK analysis showed an approximately dose-proportional increase in LY3522348 exposure, and the half-life ranged from 23.7 to 33.8 h. PD analysis indicated a dose-dependent increase in plasma fructose concentrations following the administration of a fructose beverage, supporting the inhibition of fructose metabolism by LY3522348.
Conclusions
LY3522348 demonstrated a favorable safety profile and well-behaved pharmacokinetics following once-daily oral dosing, and effective inhibition of fructose metabolism.
The study was registered on ClinicalTrials.gov (NCT04559568).
Supplementary Information
The online version contains supplementary material available at 10.1007/s13300-025-01752-5.
Keywords: LY3522348, Keto hexokinase inhibitor, MASH, PK/PD
Key Summary Points
| Why carry out this study? |
| Ketohexokinase (KHK) inhibition may decrease hepatic de novo lipogenesis and steatosis in humans, ameliorating the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) and progression to metabolic dysfunction-associated steatohepatitis (MASH) [1]. |
| LY3522348 is a highly selective, oral dual inhibitor of human KHK isoforms C and A (hKHK-C, hKHK-A), and has demonstrated a robust pharmacodynamic response in an in vivo model of fructose metabolism. |
| This first-in-human study was designed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of LY3522348 in healthy participants. |
| What has been learned from the study? |
| LY3522348 was well tolerated, with most adverse events being mild. No serious adverse events or deaths were reported. |
| Pharmacokinetic analysis showed an approximately dose-proportional increase in LY3522348 exposure, with a half-life ranging from 23.7 to 33.8 h. |
| Pharmacodynamics analysis indicated a dose-dependent increase in plasma fructose concentrations following the administration of a fructose beverage, supporting the inhibition of fructose metabolism by LY3522348. |
| LY3522348 demonstrated a favorable safety profile and pharmacokinetics following once-daily oral dosing, and effective inhibition of fructose metabolism. |
Introduction
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a spectrum of fatty liver diseases and a leading cause of liver-related morbidity and mortality [2, 3]. In 2023, the mortality rate for MASLD was estimated to be 12.60 per 1000 person-years for all-cause mortality [3]. The age-adjusted prevalence of MASLD among the adult population in the United States (US) is 32–33% [3, 4]. Approximately 12–14% of people with MASLD develop metabolic dysfunction-associated steatohepatitis (MASH), which is a more aggressive form of MASLD. Approximately 20% of people with MASH may develop progressive fibrosis or cirrhosis. Emerging evidence indicates that dietary fructose causes oxidative and endoplasmic stress, which acts as a pathogenic driver of MASLD/MASH. This, in turn, contributes to the development of type 2 diabetes, obesity, and other metabolic diseases [5–7]. Fructose, delivered in sucrose, and high-fructose corn syrup, has been used in processed foods since the 1970s [8, 9]. This has led to an increase in the average daily fructose consumption in the US, from 37 g in the 1970s to ≥ 50 g in the 2000s [9]. In the liver, the primary site of fructose metabolism, fructose is metabolized by the ATP-dependent enzyme ketohexokinase (KHK) to fructose-1-phosphate [10]. KHK is the rate-limiting enzyme and phosphorylation by KHK is the first committed step of fructose metabolism. There are two isoforms of KHK (KHK-A and KHK-C), which differ by 44 amino acids, KHK-C is predominantly found in the liver, kidney, and small intestine with KHK-A being the predominant form in other tissues [11]. KHK expression and activity are not controlled by hormones or allosteric mechanisms [11, 12].
On a molecular level, various aspects of fructose metabolism set the stage for the liver to develop MASLD. These include fructose’s tendency to increase hepatic de novo lipogenesis (DNL), reduce the oxidation of fatty acids in mitochondria, and initiate inflammatory responses in the liver. Excess fructose intake is known to induce insulin resistance, obesity, and metabolic syndrome, which in turn have been linked to the progression of MASLD [7] by bypassing the requirement for the rate-limiting enzymes (phosphofructokinase and fructose-1,6-bisphosphatase) in glycolysis and gluconeogenesis [12]. Fructose results in the downstream activation of transcription factors (such as carbohydrate-response element-binding protein) that promote and regulate hepatic fatty acid synthesis and DNL [13, 14].
Preclinical and clinical studies have shown that isocaloric fructose restriction also benefits metabolic syndrome, liver steatosis, DNL, and insulin sensitivity [15]. KHK inhibition is expected to mimic the isocaloric fructose restriction profiles and thus presents a potential weight-reduction independent treatment option for patients with MASLD/MASH [16] as reported in previous nonclinical in vivo studies for MASLD/MASH [17–20].
Additionally, individuals with KHK deficiency do not present with negative health phenotypes, despite exhibiting higher plasma levels of fructose than individuals with standard KHK levels, implying that KHK inhibition would not lead to adverse health effects [21–23]. Therefore, KHK inhibition may have the potential to decrease hepatic DNL and steatosis in humans without negative consequences, ameliorating the pathogenesis of MASLD and progression to MASH [1].
LY3522348 is a highly selective, oral dual inhibitor of human KHK isoforms C and A (hKHK-C, hKHK-A) that has demonstrated a robust pharmacodynamic response in an in vivo model of fructose metabolism [11]. Durham et al. presented that LY3522348 was almost equipotent for KHK-C and KHK-A in vitro (hKHK IC50 = 20 ± 8 nM, hKHA IC50 = 24 ± 6 nM, Cell IC50 = 41 ± 14 nM, mKHK-C IC50 = 14 ± 3 nM, and mKHK-A IC50 = 20 ± 7 nM) and achieved a desirable level of preclinical safety in animal studies [11]. This first-in-human study was designed to assess the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of LY3522348 in healthy participants.
Methods
Study Design
This was a phase 1, randomized, double-blind, placebo-controlled, two-part study in healthy participants (November 2020 to August 2021) [Figure S1].
Part 1: a single-ascending dose (SAD) study, and
Part 2: a multiple-ascending dose (MAD) study with a drug–drug interaction analysis.
All participants had screening visits to confirm their eligibility. Screening occurred up to four weeks before enrolment and before randomization to the SAD or MAD study. The study was conducted in compliance with the principles of the Declaration of Helsinki, the Council for International Organizations of Medical Sciences International Ethical Guidelines, and the International Council for Harmonization Good Clinical Practice guidelines. The protocol was reviewed and approved by the Midlands Independent Review Board and the study was registered on ClinicalTrials.gov (NCT04559568). All participants provided written informed consent before participation.
Single-Ascending Dose Study
Six participants were randomly assigned to single-ascending oral doses of LY3522348 and two participants to placebo in five different cohorts (cohort 1: 5 mg; cohort 2: 15 mg; cohort 3: 50 mg; cohort 4: 150 mg; cohort 5: 380 mg; Figure S1). In the SAD study, a sentinel dosing approach was utilized for cohorts 4 and 5. Dose escalation to the next cohort was initiated following a satisfactory review of the safety, tolerability, and PK data from all previous cohorts. PK, PD, and safety assessments were performed at predefined times, including discharge from the investigative site on day 7 and a follow-up visit on day 14 (± 1).
Multiple-Ascending Dose Study
The MAD study was initiated after assessing the safety and tolerability of LY3522348 doses in cohort 4 and PK and PD data in cohort 3 in the SAD study. In the MAD study, six participants were randomly assigned to once daily (QD) oral doses of LY3522348 and two participants to placebo for 14 or 15 days in three different cohorts (cohort 1: 50 mg; cohort 2: 120 mg; cohort 3: 290 mg; Figure S1). PK, PD, and safety assessments were performed at predefined times till discharge from the investigative site on day 20 for cohort 1 and 2 or day 21 for cohort 3 and during a follow-up visit on day 28 (± 2). In addition, as an extrapolatory study to assess the potential impact of LY3522348 treatment on CYP3A metabolism, participants in cohort 3 received a single oral dose of 200 μg midazolam, a CYP3A substrate, on day – 1 and within 15 min of LY3522348 or placebo dosing on day 15. Figure S1 provides details on the study duration, doses, dosing strategy, participant admission, and treatment.
Study Population
Healthy men and women aged 18–65 years who were not of childbearing potential, had a body mass index between ≥ 18.5 kg/m2 and ≤ 40 kg/m2, had a stable weight for 1 month prior to screening and enrollment, and had safety laboratory test results within the normal reference range for the population were considered for inclusion. Participants were excluded if they had an abnormality in the 12-lead electrocardiograms (ECG) at screening that may confound ECG analysis, had a blood pressure of > 160/90 mmHg and pulse rate < 50 or > 100 bpm, had a history of fructosuria, or used strong inducers or inhibitors of CYP3A or over-the-counter or prescription medication including herbal medications (St. John’s wort and vitamin/mineral supplements) within 14 days prior to dosing/start date (Figure S1).
Analyses
Demographics and Participant Disposition
Participant disposition and demographic variables such as age, sex, race, ethnicity, body weight, height, and body mass index were reported.
Safety Analyses
Safety was evaluated throughout the study period by monitoring for adverse events (AEs), physical examination, clinical safety laboratory assessments, vital sign measurements, and 12-lead ECG.
Pharmacokinetic Analysis
LY3522348 plasma and urine concentrations were determined using liquid chromatography-tandem mass spectrometry (LC–MS) at PPD Laboratories, WI, USA, and Q2 Solutions, Ithaca, NY, USA, respectively.
In the SAD cohorts, drug plasma concentrations were quantified from pre-dose to 144 h post-dose. In the MAD cohorts, drug plasma concentrations were quantified following the day 1 dose (pre-dose to 24-h post-dose), prior to the day 7 dose, and following the last dose (pre-dose to 144 h post-dose). Plasma PK parameters calculated included area under the concentration–time curve (AUC) from time zero to infinity (AUC(0–inf)), AUC over one dosing interval (AUCτ), maximum observed drug concentration (Cmax), time of Cmax (tmax), apparent volume of distribution during the terminal phase after extravascular administration (Vz/F), half-life associated with the terminal rate constant in noncompartmental analysis (t1/2), apparent total body clearance calculated after extravascular administration (CL/F) and accumulation ratio based on AUCτ and Cmax. The dose proportionality of LY3522348 PK was assessed separately for the SAD and MAD study, using day 14 PK data in the MAD assessment.
Plasma concentrations of midazolam and its metabolite, 1ʹ-hydroxymidazolam, were assayed on days − 1 (pre-dose to 24-h post-dose) and day 15 (pre-dose to 24-h post-dose) using validated LC–MS methods at Q2 Solutions, Ithaca, NY, USA. The impact of LY3522348 treatment on AUC(0–inf), Cmax, and tmax was evaluated.
Pharmacodynamic Analysis
A fructose tolerance test (FTT) was performed on day 1 in the SAD study and days 1 and 14 in the MAD study in which a fructose beverage was served with a low fructose meal (20 min, 6 h, 12 h post LY3522348 or placebo dose). Fructose plasma concentrations were measured following fructose beverage administration and these concentrations were used to calculate a fructose AUC(0–24).
Statistical Analysis
All analyses were performed using SAS® Version 9.4. For continuous data, mean, standard deviation (SD), median, minimum, maximum, and number of participants were reported. For log-normal data, such as AUC and Cmax, geometric mean, and geometric coefficient of variation were reported. For categorical data, frequency and percentages were presented.
PK parameter estimates were calculated using standard noncompartmental analysis methods using Phoenix WinNonlin Version 8.1.1. To estimate ratios of dose-normalized geometric means and corresponding 90% CIs, log-transformed LY3522348 PK parameters were evaluated using a power model (log-dose acted as an explanatory variable). The estimated ratio of dose-normalized geometric means of PK parameters between the highest and lowest doses was used to assess dose-proportionality. Renal clearance of LY3522348 was calculated as the amount excreted over 24 h (Ae[0–24]) divided by the plasma AUC over the same 24 h in the SAD study only.
In the SAD study, log-transformed FTT and biomarker parameters (low- and high-density lipoproteins, cholesterol, and triglycerides) were analyzed in an ANOVA model, with treatment as a fixed effect. In the MAD study, log-transformed FTT parameters were analyzed using a mixed model with repeated measures, with treatment, day, and treatment-by-day interaction as fixed effects and participants included as a random effect. The difference in the least square (LS) means between LY3522348 and placebo, along with the 90% CIs, were back-transformed to produce the ratio of geometric means and the CIs comparing LY3522348 to placebo by day. In the analysis of the effect of LY3522348 on midazolam and 1ʹ-hydroxymidazolam, PK parameters were assessed using an ANOVA model (the day was included as a fixed effect). The ratio of geometric means and corresponding CIs, along with the p value were also reported. Wilcoxon signed-rank test was used to analyze the tmax nonparametrically. The median of the differences comparing day 15 to day − 1 and the corresponding 90% CIs were presented alongside the p value. p values were not adjusted for multiplicity considering the phase and exploratory nature of the study. Missing data was not presented in the final analysis.
Results
Demographics and Participant Disposition
A total of 65 healthy participants were included; of these 40 were in the SAD study (placebo = 10; LY3522348: 5 mg = 6; 15 mg = 6; 50 mg = 6; 150 mg = 6; 380 mg = 6) and 25 in the MAD study (placebo = 4; placebo [placebo + midazolam] = 2; LY3522348: 50 mg = 6; 120 mg = 6; 290 mg [LY3522348 + midazolam] = 7; Table 1). Twenty-three participants completed the MAD study. 1 participant in the 120 mg group was discontinued from the study due to the physician’s decision after dosing on day 8, due to a positive COVID-19 test. One participant from the 290 mg group who discontinued after dosing on day 5 due to inability to swallow capsules is not included in the analysis. Table 1 presents the baseline demographics and clinical characteristics of the participants included in the SAD and the MAD studies.
Table 1.
Baseline demographic characteristics of participants in the SAD and MAD study
| SAD study | |||||||
|---|---|---|---|---|---|---|---|
| Placebo | 5 mg | 15 mg | 50 mg | 150 mg | 380 mg | Overall | |
| n = 10 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | n = 40 | |
| Age, years | 50.2 (11.5) | 53.5 (7.6) | 48.8 (9.6) | 56.0 (9.7) | 57.0 (8.4) | 31.0 (3.0) | 49.5 (11.9) |
| Sex, n (%) | |||||||
| Female | 3 (30.0%) | 4 (66.7%) | 5 (83.3%) | 3 (50.0%) | 4 (66.7%) | 0 (0.0%) | 19 (47.5%) |
| Male | 7 (70%) | 2 (33.3%) | 1 (16.7%) | 3 (50%) | 2 (33.3%) | 6 (100%) | 21 (52.5%) |
| Ethnicity, n (%) | |||||||
| Not Hispanic or Latino | 8 (80.0%) | 4 (66.7%) | 4 (66.7%) | 5 (83.3%) | 4 (66.7%) | 2 (33.3%) | 27 (67.5%) |
| Hispanic or Latino | 2 (20.0%) | 2 (33.3%) | 2 (33.3%) | 1 (16.7%) | 2 (33.3%) | 4 (66.7%) | 13 (32.5%) |
| Race, n (%) | |||||||
| Black or African American | 5 (50.0%) | 1 (16.7%) | 3 (50.0%) | 1 (16.7%) | 2 (33.3%) | 2 (33.3%) | 14 (35.0%) |
| White | 5 (50.0%) | 5 (83.3%) | 3 (50.0%) | 5 (83.3%) | 4 (66.7%) | 4 (66.7%) | 26 (65.0%) |
| Weight (kg) | 94.3 (20.2) | 83.1 (27.7) | 73.8 (14.7) | 90.9 (22.5) | 85.7 (18.7) | 80.8 (12.5) | 85.7 (20.0) |
| Height (cm) | 172.2 (9.2) | 169.9 (7.7) | 164.8 (8.0) | 174.0 (8.3) | 169.7 (5.5) | 172.3 (5.7) | 170.3 (7.8) |
| Body mass index (kg/m2) | 31.7 (5.5) | 28.3 (7.0) | 27.2 (5.3) | 29.8 (6.0) | 29.6 (5.2) | 27.2 (4.0) | 29.3 (5.8) |
| MAD study | ||||||
|---|---|---|---|---|---|---|
| Placebo | Placeboa | 50 mg | 120 mg | 290 mga | Overall | |
| n = 4 | n = 2 | n = 6 | n = 6b | n = 7c | n = 25 | |
| Age, years | 47.5 (11.0) | 57.0 (NC) | 43.0 (8.7) | 36.3 (4.1) | 45.0 (12.8) | 43.8 (10.3) |
| Sex | ||||||
| Female | 2 (50.0%) | 1 (50.0%) | 2 (33.3%) | 1 (16.7%) | 1 (14.3%) | 7 (28.0%) |
| Male | 2 (50.0%) | 1 (50.0%) | 4 (66.7%) | 5 (83.3%) | 6 (85.7%) | 18 (72.0%) |
| Ethnicity | ||||||
| Not Hispanic or Latino | 1 (25.0%) | 1 (50.0%) | 2 (33.3%) | 5 (83.3%) | 6 (85.7%) | 15 (60.0%) |
| Hispanic or Latino | 3 (75.0%) | 1 (50.0%) | 4 (66.7%) | 1 (16.7%) | 1 (14.3%) | 10 (40.0%) |
| Race | ||||||
| Black or African American | 1 (25.0%) | 1 (50.0%) | 2 (33.3%) | 4 (66.7%) | 4 (57.1%) | 12 (48.0%) |
| White | 3 (75.0%) | 1 (50.0%) | 4 (66.7%) | 2 (33.3%) | 3 (42.9%) | 13 (52.0%) |
| Weight (kg) | 82.1 (6.5) | 83.9 (NC) | 74.3 (13.7) | 91.2 (17.3) | 83.5 (17.2) | 83.0 (15.4) |
| Height (cm) | 168.6 (10.0) | 163.8 (NC) | 170.5 (12.4) | 175.0 (3.5) | 175.7 (11.1) | 172.2 (9.6) |
| Body mass index (kg/m2) | 29.0 (1.9) | 31.4 (NC) | 25.4 (2.0) | 29.7 (5.2) | 26.9 (4.4) | 27.9(4.4) |
Values are mean (SD) unless otherwise specified
n number of participants, NC not calculated, SD standard deviation, SAD single-ascending dose, MAD multiple-ascending dose
a200 ug midazolam was administered on day − 1 and day 15
bN = 5. One participant who discontinued after dosing on day 8 due to a positive Coronavirus disease 2019 test is not included in the analysis
cN = 6. One participant who discontinued after dosing on day 5 due to inability to swallow capsules is not included in the analysis
Safety Analyses
No deaths, other serious adverse events, or AEs leading to treatment or study discontinuation were reported during this study.
Safety in SAD Study
Overall, nine treatment-emergent AEs (TEAEs) were reported in the SAD study. Eight TEAEs were reported for eight participants who received LY3522348, and one TEAE was reported for one participant who received a placebo (Table 2). All TEAEs reported in the SAD study were mild, except for one event of presyncope in the 15 mg LY3522348 group. The investigator considered one TEAE of nausea and one of myalgia to be related to LY3522348. TEAEs in the SAD study were not associated with increasing the LY3522348 dose. No clinically significant differences in vital signs or trends in ECG parameters were noted in systolic or diastolic blood pressure, but there was an apparent decrease in supine pulse rate in the 380 mg treatment group compared to placebo and lower doses of LY3522348.
Table 2.
Treatment-emergent adverse events in healthy participants receiving single or multiple doses of LY3522348 or placebo
| SAD study | |||||||
|---|---|---|---|---|---|---|---|
| Parameters | Placebo | 5 mg | 15 mg | 50 mg | 150 mg | 380 mg | Overall |
| n = 10 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | n = 40 | |
| Any TEAE, n (%) | 1 (10) | 2 (33.3) | 1 (16.7) | 1 (16.7) | 3 (50.0) | 1 (16.7) | 9 (22.5) |
| Medical device site dermatitis | – | 2 | – | – | – | – | 2 |
| COVID-19 | 1 | – | – | – | – | – | 1 |
| Musculoskeletal chest pain | – | – | – | – | – | 1 | 1 |
| Myalgia | – | – | – | 1 | – | – | 1 |
| Nausea | – | – | – | – | 1 | – | 1 |
| Presyncope | – | – | 1 | – | – | – | 1 |
| Rectal hemorrhage | – | – | – | – | 1 | – | 1 |
| Rhinitis allergic | – | – | – | – | 1 | – | 1 |
| MAD study | |||||
|---|---|---|---|---|---|
| Placebo | 50 mg | 120 mg | 290 mga | Overall | |
| n = 6 | n = 6 | n = 6 | n = 7 | n = 25 | |
| Any TEAE, n (%) | – | – | 3 (50.0) | 1 (14.3) | 4 (16.0) |
| Headache | – | – | 3 | – | 3 |
| Blurred vision | – | – | 2 | – | 2 |
| Abdominal distension | – | – | 1 | 1 | |
| Asymptomatic COVID-19 | – | – | 1 | – | 1 |
| Constipation | – | – | 1 | – | 1 |
| Decreased appetite | – | – | 1 | 1 | |
| Dysuria | – | – | 1 | – | 1 |
n number of participants studied, TEAE treatment-emergent adverse event, SAD single-ascending dose, MAD multiple-ascending dose
a200 μg midazolam was administered on day − 1 and day 15; Adverse events with a change of severity are only counted one time at the highest severity
Safety in MAD Study
Overall, eight TEAEs were reported for three participants in the 120 mg treatment group (Table 2). All TEAEs reported in the MAD study were mild. Among the TEAE events, three events of headache and one event each of constipation and decreased appetite were considered to be possibly related to LY3522348 by the investigator. TEAEs in the MAD study were not associated with increasing the LY3522348 dose. On days 1, 7, and 14, among all the cohorts in the MAD study, mean supine systolic blood pressure was comparable to the readings at baseline. However, on day 14, a dose-related decrease in supine diastolic blood pressure was noted in participants in the three cohorts who were administered LY3522348 with or without midazolam. On days 1 and 7, no drug-related trends in ECG parameters were observed in participants administered LY3522348 up to 290 mg compared to placebo.
Pharmacokinetic Analysis
SAD Study
Figure 1a and b present the plasma LY3522348 concentration–time profiles observed in the SAD study and Table 3 presents the noncompartmental PK analysis summary statistics. LY3522348 was absorbed with a median tmax ranging from 3.0 to 7.0 h post-dose and concentrations subsequently declined among all the cohorts with geometric mean t1/2 ranging from 23.7 to 33.8 h. A dose-proportional increase in LY3522348 AUC(0–inf) and Cmax was observed between 5 to 380 mg doses (Table S1). The fraction of the dose excreted unchanged between time zero- and 24-h post-dose (Fe[0–24]) ranged from 3.76 to 6.32% and renal clearance ranged from 1.03 to 1.71 l/h (Table 3).
Fig. 1.
The mean (SD) linear and semi-logarithmic LY3522348 plasma concentration–time profiles in the SAD study (a and b, respectively) and following the last dose in the MAD study (c and d, respectively). *200 μg midazolam was coadministered on day − 1 and day 15. h hours, MAD multiple-ascending dose, SAD single-ascending dose, SD standard deviation
Table 3.
LY3522348 pharmacokinetic parameters in the SAD study
| Parameter | 5 mg | 15 mg | 50 mg | 150 mg | 380 mg |
|---|---|---|---|---|---|
| (n = 6) | (n = 6) | (n = 6) | (n = 6) | (n = 6) | |
| AUC (0–inf) (μg h/ml) | 0.35 (29%) | 1.29 (30%) | 3.43 (20%) | 9.59 (38%) | 25.8 (24%) |
| Cmax (μg/ml) | 0.00964 (17%) | 0.0399 (23%) | 0.113 (23%) | 0.323 (39%) | 0.897 (18%) |
| tmax (h)a | 3.03 (3.0–6.0) | 6.0 (4.0–10.0) | 6.0 (3.0–8.0) | 4.08 (4.0–8.0) | 7.00 (3.0–10.2) |
| t1/2 (h)b | 33.8 (25.7–45.6) | 25.5 (20.8–29.7) | 23.7 (16.5–28.8) | 24.2 (19.6–26.3) | 25.9 (22.0–34.2) |
| CL/F (l/h) | 14.3 (29%) | 11.6 (30%) | 14.6 (20%) | 15.6 (38%) | 14.7 (24%) |
| Vz/F (l) | 698 (9%) | 426 (21%) | 500 (23%) | 545 (31%) | 549 (24%) |
| Ae [0–24] (mg) | – | – | 1.88 (42%) | 7.66 (12%) | 24.0 (31%) |
| Fe [0–24] (%) | – | – | 3.76 (42%) | 5.10 (12%) | 6.32 (31%) |
| CLr (l/h) | – | – | 1.03 (41%) | 1.47 (25%) | 1.71 (38%) |
Geometric mean (CV%) are presented unless otherwise noted
Ae[0–24] amount of drug excreted unchanged between time zero and 24-h post-dose, AUC(0–inf) area under the concentration–time curve from time zero to infinity, CL/F apparent total body clearance of drug calculated after extravascular administration, CLr renal clearance, Cmax maximum observed drug concentration, CV coefficient of variation, Fe[0–24] fraction of dose excreted unchanged between time 0 and 24-h post-dose, n number of participants, SAD single-ascending dose, t1/2 half-life associated with the terminal rate constant in noncompartmental analysis, tmax time of maximum observed drug concentration, Vz/F apparent volume of distribution during the terminal phase after extravascular administration
aMedian (minimum–maximum)
bGeometric mean (minimum–maximum)
MAD Study
Figure 1c and d present the plasma LY3522348 concentration–time profiles following the last dose of LY3522348 (day 14 in cohorts 1 and 2 and day 15 in cohort 3) and Table 4 presents the noncompartmental PK analysis summary statistics. Following the last dose, LY3522348 was absorbed with a median tmax ranging from 3.1 to 4.0 h and concentrations subsequently declined with a geometric mean t1/2 ranging from 25.3 to 26.8 h. An approximately dose-proportional increase in AUCτ and Cmax was observed following the day 14 dose over the dose range from 50 to 290 mg (Table S1). Evaluation of the impact of LY3522348 on midazolam and 1’-hydroxymidazolam PK is presented in Table S2.
Table 4.
LY3522348 plasma pharmacokinetic parameters in the MAD study
| Parameter | Day 1 | ||
|---|---|---|---|
| 50 mg | 120 mg | 290 mga | |
| n = 6 | n = 6 | n = 7 | |
| AUCτ (μg h/ml) | 1.77 (25%) | 5.05 (18%) | 11.1 (25%) |
| Cmax (μg/ml) | 0.109 (29%) | 0.330 (15%) | 0.690 (13%) |
| tmax (h)b | 6.00 (4.00–8.00) | 6.00 (3.00–8.00) | 6.00 (0.75–6.00) |
| Parameter | Day 14 (cohorts 1 and 2) or day 15 (cohort 3) | ||
|---|---|---|---|
| 50 mg | 120 mg | 290 mga | |
| n = 6 | n = 5 | n = 6 | |
| AUCτ (μg h/ml) | 3.24 (24%) | 7.92 (21%) | 21.8 (18%) |
| Cmax (μg/ml) | 0.188 (25%) | 0.464 (15%) | 1.37 (12%) |
| tmax (h)b | 4.00 (3.00–8.00) | 4.00 (0.75–6.00) | 3.08 (3.00–6.00) |
| t1/2 (h)c | 25.3 (23.2–28.8) | 26.0 (22.7–29.2) | 26.8 (20.7–32.4) |
| CL/F (l/h) | 15.4 (24%) | 15.2 (21%) | 13.3 (18%) |
| Vz/F (l) | 563 (17%) | 568 (16%) | 513 (20%) |
| RA AUCτ | 1.83 (7%) | 1.60 (15%) | 1.96 (19%) |
| RA Cmax | 1.72 (12%) | 1.40 (18%) | 1.99 (10%) |
Geometric mean (CV%) are presented unless otherwise noted
AUCτ area under the concentration–time curve during one dosing interval, CL/F apparent total body clearance of drug calculated after extravascular administration, Cmax maximum observed drug concentration, CV coefficient of variation, MAD multiple-ascending dose, n number of participants, RA accumulation ratio, t1/2 half-life associated with the terminal rate constant in noncompartmental analysis, tmax time of maximum observed drug concentration, Vz/F apparent volume of distribution during the terminal phase after extravascular administration
a200 μg midazolam was administered on day − 1 and day 15
bMedian (minimum–maximum)
cGeometric mean (minimum–maximum)
Pharmacodynamic Analysis
SAD Study
Figure 2a presents the fructose plasma concentration–time profile following an FTT with the administration of a placebo or LY3522348. Following LY3522348 treatment, a dose-dependent increase in fructose AUC(0–24) was observed over the dose range (Table 5). Fructose AUC(0–24) in the various dosing cohorts, relative to placebo, is presented in Fig. 3a with fructose AUC(0–24) 8.27-fold higher in the 380 mg LY3522348 cohort relative to placebo.
Fig. 2.
The mean (SD) fructose plasma concentration–time profiles during a fructose tolerance test on a day 1 in the SAD study, b day 1 in the MAD study, and c day 14 in the MAD study. *200 μg midazolam was coadministered on day − 1 and day 15. The 13-h post-dose fructose concentration data was missing for all participants in the 50 mg MAD cohort on day 14 and thus this timepoint is not plotted. h hours, MAD multiple-ascending dose, SAD single-ascending dose, SD standard deviation
Table 5.
Fructose AUC(0–24) during a fructose tolerance test in the SAD and MAD study
| SAD study | ||||||
|---|---|---|---|---|---|---|
| Parameter | Placebo | 5 mg | 15 mg | 50 mg | 150 mg | 380 mg |
| n = 10 | n = 6 | n = 6 | n = 6 | n = 6 | n = 6 | |
| AUC(0–24) | 126 (102, 155) | 170 (129, 224) | 224 (170, 295) | 239 (182, 314) | 410 (312, 539) | 1039 (790, 1367) |
| MAD study | ||||
|---|---|---|---|---|
| Day 1 | ||||
| Placebo | 50 mg | 120 mg | 290 mga | |
| n = 4 | n = 6 | n = 6 | n = 6 | |
| AUC(0–24) | 215 (171, 272) | 339 (278, 414) | 510 (419, 622) | 912 (748, 1112) |
| Day 14 | ||||
|---|---|---|---|---|
| n = 6 | n = 5 | n = 6 | ||
| AUC(0–24) | 203 (158, 260) | – | 640 (490, 836) | 1339 (1044, 1717) |
Fructose AUC(0–24) are presented as geometric least squares mean (90% CI) in units of μg h/ml
AUC(0–24) area under the concentration–time curve from time zero to 24 h, CI confidence interval, MAD multiple-ascending dose, n number of participants, SAD single-ascending dose
Fig. 3.
Ratio of fructose AUC(0–24) geometric least squares mean (90% CI) [p value] (LY3522348:placebo) during a fructose tolerance test on a day 1 in the SAD study and b day 1 and day 14 in the MAD study. *200 μg midazolam was coadministered on day − 1 and day 15. The 13-h post-dose fructose concentration data was missing for all participants in the 50 mg MAD cohort on day 14 and thus this AUC(0–24) was not calculated and is not included in the analyses. Vertical dotted line corresponds to a geometric least squares means ratio of 1. AUC(0–24) area under the concentration–time curve from time zero to 24 h, CI confidence interval, SAD single-ascending dose, MAD multiple-ascending dose
MAD Study
Figure 2b and c present the fructose concentration–time profile following an FTT with administration of placebo or LY3522348 on day 1 and day 14. A dose-dependent increase in fructose AUC(0–24) was observed over the dose range on both day 1 and day 14 (Table 5). Fructose AUC(0–24) in the various cohorts, relative to placebo, is presented in Fig. 3b with fructose AUC(0–24) increasing 6.60-fold on day 14 in the 290 mg LY3522348 cohort.
No significant treatment effects on concentrations of cholesterol, HDL cholesterol, LDL cholesterol, or triglycerides were observed following exposure to LY3522348 in the SAD study and MAD studies.
Discussion
We performed a first-in-human phase 1 trial of LY3522348 in healthy participants to evaluate its safety, tolerability, PK, and PD effects.
Following administration of single and multiple doses, LY3522348 was well tolerated, and all AEs reported were mild in severity. None of the AEs led to discontinuation, and no serious AEs or deaths were reported. The PK analyses indicated an approximately dose-proportional increase in LY3522348 exposure, with a half-life ranging from 23.7 to 33.8 h, following both single and multiple doses. The PD analysis demonstrated a dose-dependent increase in plasma fructose concentrations following the administration of fructose, supporting the inhibition of fructose metabolism by LY3522348.
Exposure to sustained or excessive fructose can result in metabolic syndrome, fat accumulation, and fatty liver. Studies on liver-specific fructokinase knockout mice have shown that excessive catabolism of fructose in the liver can lead to metabolic syndrome, elevate triglycerides, increase harmful LDL cholesterol, and promote insulin resistance [24]. These factors may contribute to liver inflammation and the progression of MASLD to MASH. The high reactivity of fructose and its metabolites likely contribute to the formation of intracellular advanced glycation end-products (AGEs) and associated vascular complications [25]. However, the evidence remains unconvincing. In addition, since we are inhibiting fructose metabolism by blocking KHK activity, the metabolites would not be present, so any role those play in AGE formation would be void. Finally, while KHK inhibition may lead to an increase in circulating fructose, our clinical data reveals that the increase is acute, where likely cleared through renal excretion [16].
Although inhibiting KHK cannot control fructose intake, it can inhibit fructose metabolism via KHK and instead facilitate the metabolism of fructose via hexokinase or excretion via the urine. In a recent human and murine analysis, Shepherd et al. detailed the role of KHK inhibition on fructose metabolism. They reported the benefit of KHK inhibition to improve steatosis, fibrosis, and inflammation in patients with NAFLD [26]. In a randomized placebo-controlled phase 1 study, SAD of PF-06835919 (another KHK inhibitor) demonstrated a dose-dependent increase in plasma fructose levels in the healthy human study participants. Available literature has established that dietary fructose has been associated with high levels of cholesterol and triglycerides in healthy participants [27, 28]. In a study with healthy participants, triglyceride levels were 1.48 (95% CI 1.00–2.19; p = 0.045) times higher in high fructose consumers than those for moderate fructose consumers [28]. Another study investigating the effects of KHK inhibition on MASLD induced by fructose reported that KHK inhibition reduced hepatic steatosis by decreasing liver fat accumulation and enhancing fatty acid oxidation. However, this path of inhibition showed limited efficacy as KHK inhibition did not fully prevent fructolysis and led to some adverse effects like impaired glucose tolerance [16, 29].
The inhibition of KHK has shown promise in the treatment of MASLD/MASH due to its role in reducing fructose metabolism and subsequent hepatic fat accumulation. Recent advancements in therapeutic approaches, such as GLP-1 receptor agonists and dual agonists, have demonstrated superior efficacy in improving metabolic parameters. These treatments not only target multiple pathways involved in MASLD/MASH but also offer additional benefits such as weight reduction and improved insulin sensitivity. Consequently, while KHK inhibition remains a valuable area of research, the efficacy of the emerging mechanism of action may be compared to LY3522348 in assessing the therapeutic potential of KHK inhibition in MASLD/MASH. Future studies in humans are required to further corroborate the idea that inhibition of fructose metabolism may prove to be an effective therapeutic strategy for the treatment of metabolic disorders like MASLD/MASH.
The current study was limited by the small number of healthy participants in each cohort.
In conclusion, the data presented here provides a safe and tolerated dose range of LY3522348 as well as well-behaved pharmacokinetics and dose-dependent pharmacological target engagement of LY3522348 in fructose metabolism in humans. LY3522348 showed effective inhibition of fructose metabolism and improvements in metabolic parameters. Drug developers may further use the insights gained from PK and PD analyses in human beings to optimize candidate molecules.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
The authors would like to acknowledge Era Seth, an employee of Eli Lilly Services India Pvt. Ltd, for providing medical writing support.
Author Contributions
All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. Tsuyoshi Fukuda: Design of the work, acquisition of data, analysis of data, interpretation of data for the work, and drafting of the work. Brian R Thompson: Design of the work, analysis of data, interpretation of data for the work, drafting of the work, and critical review of the work for important intellectual content. Bram Brouwers: Conception and design of the work, interpretation of data for the work, and critical review of the work for important intellectual content. Hui-Rong Qian: Design of the work, analysis of data, interpretation of data for the work, and critical review of the work for important intellectual content. Wei Wang: acquisition of data and critical review of the work for important intellectual content. Bridget L Morse: Conception, design of the work, analysis of data, interpretation of data for the work, and critical review of the work for important intellectual content. Elizabeth Smith LaBell: Analysis of data and critical review of the work for important intellectual content. Timothy B. Durham: Conception, design of the work, and critical review of the work for important intellectual content. Manige Konig: Conception and critical review of the work for important intellectual content. Axel Haupt: Conception, interpretation of data for the work, and critical review of the work for important intellectual content. Charles T Benson: Conception and design of the work, interpretation of data for the work, and critical review of the work for important intellectual content. James MacKrell: Conception and design of the work, interpretation of data for the work, drafting of the work, and critical review of the work for important intellectual content.
Funding
The study was funded by Eli Lilly and Company, Indianapolis, United States, and sponsored by Clinpharmacology. All other support for the manuscript, including writing and the journal’s Rapid Service Fee, was funded by Eli Lilly and Company, Indianapolis, USA.
Data Availability
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 six months after the indication studied has been approved in the US and EU 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, blank or annotated case report forms for the study (NCT04559568) will be provided in a secure data-sharing environment. For details on submitting a request, see the instructions provided at http://www.vivli.org.
Declarations
Conflict of Interest
Jim MacKrell, Bram Brouwers, Timothy B Durham, Wei Wang, Charles T Benson, Axel Haupt, Bridget Morse, Brian Thompson, Hui-Rong Qian, and Elizabeth Smith LaBell are the employees and stakeholders of Eli Lilly and Company. Manige Konig and Tsuyoshi Fukuda were employees of Eli Lilly and Company at the time of this study.
Ethical Approval
The study was conducted in compliance with the principles of the Declaration of Helsinki, the Council for International Organizations of Medical Sciences International Ethical Guidelines, and the International Council for Harmonization Good Clinical Practice guidelines. The protocol was reviewed and approved by the Midlands Independent Review Board and the study was registered on ClinicalTrials.gov (NCT04559568). All participants provided written informed consent before participation.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
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 six months after the indication studied has been approved in the US and EU 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, blank or annotated case report forms for the study (NCT04559568) will be provided in a secure data-sharing environment. For details on submitting a request, see the instructions provided at http://www.vivli.org.