Preclinical Evaluation of the Oral Toxicity, Genotoxicity, and Safety Pharmacology of LPM4870108, a Novel Potent Tropomyosin Receptor Kinase Inhibitor

ABSTRACT

Tropomyosin receptor kinase (Trk) inhibitors are an essential class of anticancer drugs treating NTRK gene fusions‐positive cancer. However, the potential for the emergence of on‐target resistance suggests newer Trk inhibitors with low drug resistance risk are needed. LPM4870108 is a novel Trk inhibitor with robust anticancer efficacy in preclinical studies. To support future clinical development, this study systematically evaluated the acute and subacute (4‐week) toxicity, toxicokinetic, genotoxic, and safety pharmacology of LPM4870108. The acute toxicity study revealed the maximum tolerated dose of LPM4870108 was 300 mg/kg, whereas subacute studies determined its STD₁₀ in rats was 10 mg/kg/day. The toxicological effects of LPM4870108 were consistent with its pharmacodynamic efficacies as a Trk inhibitor, including corneal inflammation, splenic lymphocytopenia, hepatocyte vacuolar degeneration, scab formation, and increased food consumption and body weight. These changes were partially or fully recovered after 4 weeks of recovery. In rats treated with 10 or 20 mg/kg/day, 2/30, or 6/30 rats died or were moribund, and the primary organs affected by treatment‐related toxicity included the eyes, liver, and skin. Rat toxicokinetic findings were consistent with a dose‐dependent effect of LPM4870108. There was no evidence of LPM4870108‐related genotoxicity, nor did it affect respiratory function or neurobehavioral activity in rats or blood pressure or electrocardiogram results in rhesus monkeys. The IC₅₀ of LPM4870108 for hERG current inhibition was 18.2 μM. Together, these results demonstrate that LPM4870108 exhibits a satisfactory safety profile which is appropriate for further clinical development.

Preclinical evaluation of the oral toxicity, genotoxicity, and safety pharmacology of LPM4870108.

Abbreviations

2‐AF

2‐aminofluorene

A/G

albumin/globulin

ALB

albumin

AMY‐P

amylas‐P

APTT

activated partial thromboplastin time

AST

aspartate aminotransferase

AUC

area under the curve

BIL

bilirubin

BLO

occult blood content

CHOL

cholesterol

CK

creatine kinase

CLA

appearance

C _max

maximum concentration

CMC

carboxy‐methylcellulose

COL

color

EOS

eosinophils

FIB

fibrinogen

GLP

Good Laboratory Practice

GLU

levels of glucose

HPLC

high‐performance liquid chromatography

IACUC

Institutional Animal Care and Use Committee

KET

ketone bodies

K–W

Kruskal–Wallis

LDH

lactate dehydrogenase

LYM

lymphocyte

MCH

mean corpuscular hemoglobin

MCV

mean corpuscular volume

MONO

monocyte

MTD

maximum tolerable dose

M–W

Mann–Whitney

Mydrin‐P

tropicamide eye drops

NEU

neutrophil

NIT

nitrite

NMPA

National Medical Products Administration

PRO

protein

PT

prothrombin time

RBC

red blood cell

RET

reticulocyte

SD

standard deviation

SG

specific gravity

T _max

time at maximum concentration

Trk

tropomyosin receptor kinase

URO

urobilinogen

WBC

white blood cell

1. Introduction

NTRK gene fusions involving the NTRK1, NTRK2, and NTRK3 genes, which respectively encode TrkA, TrkB, and TrkC, have been established as important drivers of oncogenesis in a range of pediatric and adult cancers [1, 2]. Although such NTRK gene fusions are relatively rare in common solid tumors, presenting in less than 1% of cases, they are detected in over 90% of secretory breast carcinoma, mammary analog secretory carcinoma, and rare pediatric tumor types [3, 4, 5].

Targeted inhibitors of Trk can effectively treat patients whose tumors harbor NTRK gene fusions [1]. Accordingly, entrectinib and larotrectinib, which are first‐generation Trk inhibitors, were granted landmark regulatory approval as antagonistic targeted treatments for cancers harboring such gene fusions in 2018 and 2019 [1, 6]. The use of these drugs was associated with rapid and durable responses in individuals with both locally advanced or metastatic diseases [3, 7, 8]. Given the potential emergence of on‐target resistance to these first‐generation Trk inhibitors; however, next‐generation inhibitors including repotrectinib and selitrectinib (LOXO‐195) have been designed. These inhibitors are intended to overcome resistance resulting from de novo mutations in the kinase domains of Trk proteins, including mutations resulting in amino acid substitutions at gatekeeper or solvent front residues [1, 9, 10, 11].

LPM4870108 (Figure 1) is a next‐generation candidate Trk inhibitor currently undergoing preclinical development that has shown satisfactory efficacy against abrogating tumor growth in animal cancer models by suppressing Trk activity [12]. In vitro pharmacodynamic analyses showed that LPM4870108 can suppress TrkA, TrkA^G595R, TrkA^G667C, and TrkC activity in the low nanomolar range, supporting its designation as a Trk inhibitor [12]. This inhibitor was developed by Luye Pharma as a candidate therapy for tumors positive for NTRK gene fusion and exhibiting acquired resistance to larotrectinib and/or other Trk inhibitors.

FIGURE 1.

Chemical structure of LPM4870108.

The present study was designed to comprehensively assess the safety profiles of LPM4870108 through a range of approaches, including an acute toxicity study, a 4‐week subacute toxicity study, and analyses of genotoxicity and pharmacological safety. The goal of this study was to provide solid evidence regarding the safety profile of LPM4870108 and to enable further drug development efforts.

2. Materials and Methods

2.1. Chemicals and Animals

LPM4870108 (purity > 94.5%) was provided by Shandong Luye Natural Medicine Research and Development Co. Ltd. (Yantai, China). Compound concentration, homogeneity, and stability over a 24‐h interval were confirmed before dosing. Reverse‐phase high‐performance liquid chromatography (HPLC) was used to determine the compound's purity.

Sprague–Dawley rats (males: 210–250 g, females: 160–200 g) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). NIH mice (males and females, 25–32 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangdong, China). An equal number of 3–5‐year‐old male and female rhesus monkeys (3.2–5.5 kg) from Hengshu Bio‐Technology Co. Ltd. (Sichuan, China) were used for this study. To ensure that these animals were in good health for research use, rodents were monitored for 7 days and monkeys were monitored for 31 days before the initiation of the studies. Rats were randomized into treatment groups using PRISTIMA 7.2.0 such that the mean body weights in each group were similar. All animals were housed with free access to food and water under controlled conditions (room temperature of 21.5°C–25.5°C, relative humidity of 45%–70%, 12‐h light/dark cycle).

Good Laboratory Practice (GLP) principles were strictly followed throughout this study, including those from the CFDA (now renamed as National Medical Products Administration, NMPA) (September 18, 2017), those from the OECD (ENV/MC/CHEM(98)17), and the FDA GLP principles for Nonclinical Laboratory Studies (21CFR58). All animal protocols described here received Institutional Animal Care and Use Committee (IACUC) approval.

2.2. Dose Preparation

Experiments were performed at WestChina‐Frontier Pharma Tech Co. Ltd. (Chengdu, Sichuan, China). A 10 mg/kg/day dose in mice was selected for antitumor activity, which was equivalent to a rat dose of 5.0 mg/kg/day [12]. The same dosing regimens used in the 4‐week toxicity study were also used for the toxicokinetic study, which included a control group (n = 4 animals/sex) and 2.5, 5, 10, and 20 mg/kg/day treatment groups (n = 8 animals/sex). Animals in each of these treatment groups were separated into two subgroups, one of which was administered a single dose of LPM4870108 while the other was administered consecutive doses over the 4‐week period. These two subgroups of animals were respectively used for the collection of blood after the first dose and after the 4‐week dose [13].

2.3. Acute Toxicity Study

An acute oral toxicity study [14, 15] was performed as a means of identifying the maximum tolerable dose (MTD) for LPM4870108. In this study, rats were randomized into four groups (n = 5/sex/group). These rats were treated (oral gavage) with vehicle (0.5% w/v carboxymethylcellulose (CMC) and 1% Tween‐80 mixed with saline) or one dose of LPM4870108 at 100, 300, or 1000 mg/kg. These rats were then closely monitored for 14 days for mortality, clinical signs, changes in food intake, body weight, gross morphological abnormalities, and clinical pathology (including clinical chemistry and hematology).

2.4. Subacute Oral Toxicity Study

2.4.1. Study Design

A total of 222 rats (n = 111/sex) were randomly assigned to a control group (n = 38, including 8 for toxicokinetic analyses) and four experimental groups (n = 46 each, including 16 for toxicokinetic analyses) with an equal number of males and females. Animals in the experimental groups were orally dosed (5 mL/kg) with LPM4870108 once per day at 2.5, 5, 10, or 20 mg/kg/day for 4 weeks, followed by a 4‐week recovery period. At the end of the 4‐week treatment period, 20 rats from each group (n = 10/sex) were euthanized with sodium pentobarbital (60 mg/kg), whereas the remaining rats were given a 4‐week recovery period, after which they were euthanized to evaluate the potential reversibility or belated incidence of treatment‐associated toxicities.

2.4.2. Clinical Observations

Rats were assessed twice per day for apparent changes in respiration, glandular secretions, stool consistency, fur or skin condition, behavior, mucous membranes, mental state, genitals, death, or any other potential evidence of toxicity. Body weight was measured twice per week during the 4‐week treatment period and once per week during the 4‐week recovery period. Measures of 24‐h food consumption were taken once per week. At the end of each 4‐week period, the ophthalmic status of enrolled rats was assessed with a binocular indirect ophthalmoscope. The eyes of all animals were treated with Mydrin‐P (tropicamide eye drops), after which the lens, iris, cornea, fundus, and posterior/anterior chamber were evaluated.

2.4.3. Clinical Testing

After fasting for 12 h, abdominal aortic blood samples were collected with and without anticoagulants from rats after the 4‐week dosing and recovery periods. These samples were then used for hematological and serum biochemistry analyses as reported previously [16]. Samples of urine were also obtained after the 4‐week dosing and recovery periods and assessed with a Siemens Clinitek Atlas automated urine analyzer, which evaluated sample appearance (CLA), color (COL), pH, specific gravity (SG), white blood cell (WBC), occult blood content (BLO), and levels of glucose (GLU), ketone bodies (KET), bilirubin (BIL), protein (PRO), urobilinogen (URO), and nitrite (NIT).

2.4.4. Major Organ and Bone Marrow Cytology Analyses

After the 4‐week dosing and recovery periods, organ weight, gross pathology, and histopathology analyses were conducted as in prior reports [13]. A pathologist performed all pathological analyses in a blinded fashion.

For cytological analyses, bone marrow smears from the sternum of rats after each 4‐week period were subjected to Wright–Giemsa staining and imaged under a light microscope. These samples were assessed for cellular morphology and evidence of hyperplasia, and the total numbers and frequencies of erythrocytes, monocytes, lymphocytes, megakaryocytes, and granulocytes therein were recorded.

2.4.5. Toxicokinetic Analyses

Heparinized tubes were used to collect jugular vein blood samples at baseline (0 h) and at 0.5, 1, 2, 4, 6, 8, 12, and 24 h following dosing on Days 1 and 28 of this study [17]. Mean plasma drug concentrations at all time points were used to conduct toxicokinetic analyses of parameters including the maximum serum concentrations after doses 1 and 2 (C _max1 and C _max2), the time at which these respective maximum concentrations were observed (T _max1 and T _max2), and the area under the time‐concentration curve from time 0 to time t (AUC_{0–t h}).

2.5. Genotoxicity Studies

2.5.1. Ames Test

Histidine‐auxotrophic Salmonella typhimurium (TA97a, TA98, TA100, TA102, and TA1535) strains were purchased from Molecular Toxicology Inc. (Boone, NC, USA). The genotoxic effects of LPM4870108 were evaluated with or without the rat liver‐derived S9 in vitro metabolic activation system (WestChina‐Frontier PharmaTech Co. Ltd., China). DMSO was used to resuspend LPM4870108, and a series of LPM4870108 concentrations were then established (50, 150, 500, 1500, or 5000 μg/plate; 0.1 mL/plate) and combined with 0.1 mL of bacterial solution, including the S9 mixture (0.5 mL) as appropriate. In the negative control group, 0.1 mL of DMSO was added to each plate, whereas in the positive control group, 0.1 mL of an appropriate strain‐dependent positive control solution was added as follows: trinitrofluorenone (0.2 μg/plate) for TA97a and TA98 in the absence of the S9 mixture; sodium azide (SA; 1.5 μg/plate) for TA100 and TA1535; mitomycin (MMC; 0.5 μg/plate) for TA102; 2‐Aminofluorene (2‐AF; 10 μg/plate) for TA97a, TA98, and TA100 in the presence of the S9 mixture; danthron (Dan; 50 μg/plate) for TA102; and cyclophosphamide (CPA; 200 μg/plate) for TA1535. These solutions were combined with 2 mL of culture medium at 55°C. For each group, plates were prepared in triplicate and incubated (37°C ± 2°C) for 48 h, after which colonies were counted.

2.5.2. Mouse Bone Marrow Micronucleus Formation Test

This study was conducted using 50 specific pathogen‐free NIH mice (n = 25/sex) [18]. These mice were assigned to groups (n = 5/sex/group), including a vehicle control group, a positive control group (CPA, 40 mg/kg), and low, intermediate, and high LPM4870108 dose groups (20, 60, and 200 mg/kg/day, respectively). To prepare LPM4870108 solution, 0.5% CMC‐Na with 1% Tween‐80 was used as vehicle, and mice were dosed (p.o.) (10 mL/kg) twice per day for 3 days. CPA was administered once on the third day. At 24 h following the final treatment, mice were euthanized and bone marrow was isolated from femurs for smearing, fixation, Giemsa staining, and examination via microscopy. For each mouse, 4000 polychromatic erythrocytes (PCEs) were analyzed to calculate the micronucleated polychromatic erythrocyte frequency (MNPCE, ‰), also known as micronucleated ratio (MR, ‰). Additionally, 500 PCEs were measured to record the normochromatic erythrocyte (NCE) count. The PCE/(PCE + NCE) ratio was used to assess cytotoxicity.

2.5.3. In Vitro Chromosomal Aberration Test

CHL cells (Shanghai Institutes for Biological Sciences, Shanghai, China) were plated in 50 mL flasks (5 × 10⁴/mL) followed by a 26 h incubation period. These tests were performed with or without the rat liver‐derived S9 mixture using the following conditions: (1) 4 h treatment with metabolic activation (+S9, 4 h), (2) 4 h treatment without metabolic activation (−S9, 4 h), and (3) 24 h treatment without metabolic activation (−S9, 24 h). CPA and MMC (Sigma‐Aldrich, USA) served as positive controls with and without metabolic activation, respectively. DMSO (0.1 mL) was instead used to treat cells in the solvent control group. Results were recorded as previously reported [19].

2.6. Safety Pharmacology Study

2.6.1. Functional Observational Battery (FOB) Test in Rats

A FOB approach was used to examine the neurobehavioral impacts of LPM4870108 in SD rats by comparing the pre‐dosing time points and data collected at 1, 4, and 24 h post‐dosing [20]. Rats were randomized into equally sized groups (n = 5/sex/group), including a control group and three LPM4870108 groups (10, 30, and 100 mg/kg). These rats were orally dosed (10 mL/kg) with a single dose of vehicle (0.5% w/v CMC with 1% Tween‐80) or LPM4870108. FOB parameters analyzed included open field, hand‐held, and cage‐based observations, as well as the monitoring of elicited behaviors, body temperature, and grip performance.

2.6.2. Analyses of Rat Respiratory Function

SD rats were randomized into equally sized groups (n = 5/sex/group), including a control group and three LPM4870108 groups (10, 30, and 100 mg/kg). These rats were orally dosed (10 mL/kg) with a single dose of vehicle (0.5% w/v CMC with 1% Tween‐80) or LPM4870108, and then the tidal volume, respiratory rate, and minute volume were measured before dosing and at 1, 4, and 24 h post‐dosing with a respiratory DSI whole body plethysmography system (Data Sciences International, USA).

2.6.3. Cardiovascular Safety Test in Monkeys

For this study, four experimental conditions were tested using the same 6 rhesus monkeys (3 males, 3 females), including a control condition and three LPM4870108 doses (5, 10, and 20 mg/kg). These animals underwent ECG and blood pressure monitoring. Dosing was separated into four phases using a 6 × 4 modified Latin square design. These animals were orally administered vehicle (0.5% w/v carboxymethylcellulose with 1% Tween‐80) or appropriate LPM4870108 doses. Data were collected from pre‐dosing to a minimum of 24 h after the final dose with an EMKA noninvasive telemetry system (Emka Technologies, France). All the data, including systolic and diastolic pressure, heart rate, mean arterial pressure, RR interval, PR interval, QRS duration, QT interval, and corrected QT interval, were analyzed with the EcgAUTO software (Data Sciences International, USA).

2.6.4. Effect on hERG Potassium Channel Activity

In order to assess the effect of LPM4870108 exposure on the current activity of the hERG potassium channels, CHO cells expressing hERG were utilized. Using a whole‐cell patch clamp approach, the effects of a range of LPM4870108 concentrations (0, 0.16, 0.49, 1.48, 4.44, 13.33, and 40.00 μM) or cisapride (0.30 μM; positive control) on hERG current activity were assessed using these CHO‐hERG cells. Testing was performed with a minimum of 3 cells per condition as previously reported [21].

2.7. Statistical Analyses

Quantitative and qualitative data were reported as means ± standard deviation (SD) and frequencies, respectively. The LEVENE test was used to assess all quantitative data, and one‐way ANOVAs were used to compare those data exhibiting variance homogeneity (p > 0.05), Kruskal–Wallis (K–W) H tests were used. Dunnett's test was utilized for paired comparisons between the control and experimental groups when ANOVA results were significant (p ≤ 0.05). Similarly, Mann–Whitney (M–W) U tests were used to compare groups in cases where K–W test results were significant (p ≤ 0.05). K–W tests were used to analyze all ordinal data, whereas non‐ordinal and binomial data were evaluated with Fisher's exact probability test.

PRISTIMA 7.2.0 was used to analyze body weight, serum biochemistry, hematology, organ weight, and organ/body and organ/brain weight ratios, whereas Stata/IC 15.0 was used for all other analyses.

2.8. Nomenclature of Targets and Ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY [22], and are permanently archived in the Concise Guide to PHARMACOLOGY 2023/24 [23].

3. Results

3.1. Acute Toxicity Study

No noticeable findings were observed with the rats in the 100 and 300 mg/kg LPM4870108 groups as compared to the vehicle treatment group. For the 1000 mg/kg dose group, three rats (1/5 males; 2/5 females) were found dead on Day 2, and mildly reduced activity was noted with other rats. On Days 3–8, 2/7 rats (1 male; 1 female) exhibited decreased activity, and 3/7 rats (2 males; 1 female) exhibited gait instability. Body weight gain in this treatment group was significantly reduced on Day 10, whereas food intake was significantly increased on Days 7 and 13 as compared to the vehicle treatment group. At the end of the study (Day 15), clinical pathological examination found increased RET% and RET, as well as reduced NEU% in female rats in the 1000 mg/kg group. No macroscopic abnormalities were detected in any treatment group on necropsy. Given these results, the MTD for LPM4870108 in SD rats was established as 300 mg/kg.

3.2. A 4‐Week Subacute Oral Toxicity Study

3.2.1. Mortality and Clinical Condition

On Days 12–27, a total of six rats in the 20 mg/kg/day group (4/15 males; 2/15 females) and two rats in the 10 mg/kg/day group (2/15 males) died or were moribund. Beginning on Day 7, the abovementioned rats began to develop progressive symptoms including corneal opacity, pale skin on the limbs and ears, increased mobility, and scabbing beneath the jaw. The remaining male and female rats in the 20 and 10 mg/kg/day groups also exhibited dose‐dependent abnormalities including corneal opacity, increased eye secretions, hyperactivity and/or body rotation, and the formation of scabs on the jaw, neck, or face. At the end of the recovery period, all of these symptoms disappeared with the exception of persistent corneal opacity in some rats in the 20 mg/kg/day group.

As compared to the control group, significant body weight gain was observed for all male and female LPM4870108‐treated rats beginning on Day 3 (p ≤ 0.05) (Tables 1 and 2), and the body weights returned to normal level (as compared to the control group) at the end of the recovery period in male rats. Male and female rats in all LPM4870108 treatment groups exhibited increased food consumption as compared to controls (p ≤ 0.05) (Tables 3 and 4). On Day 10 of the recovery period, males in the 20 mg/kg/day group and females in the 5 and 2.5 mg/kg/day groups exhibited a significant increase in food intake relative to controls (p ≤ 0.05).

TABLE 1.

Body weights in male rats (g).

Detection time	Control	2.5 mg/kg/day	5 mg/kg/day	10 mg/kg/day	20 mg/kg/day
Pretest	231.0 ± 7.8	225.4 ± 7.8	227.5 ± 7.8	226.7 ± 8.3	230.6 ± 9.0
Day 3	255.0 ± 7.5	266.3 ± 10.9*	268.9 ± 10.9*	272.2 ± 11.1*	275.4 ± 12.0*
Day 7	295.2 ± 10.7	332.2 ± 14.4*	340.2 ± 20.9*	348.5 ± 15.5*	355.8 ± 22.4*
Day 10	323.5 ± 12.7	375.9 ± 16.0*	375.4 ± 23.2*	389.8 ± 22.0*	401.6 ± 23.2*
Day 14	355.8 ± 17.4	417.0 ± 20.1*	414.4 ± 27.5*	430.4 ± 26.7*	434.9 ± 26.0*
Day 17	378.3 ± 18.7	441.6 ± 23.8*	450.2 ± 33.3*	457.4 ± 36.9*	465.6 ± 28.8*
Day 21	395.6 ± 22.1	453.9 ± 28.7*	453.2 ± 30.3*	464.2 ± 27.3*	457.7 ± 26.4*
Day 24	409.7 ± 21.6	452.0 ± 29.7*	447.1 ± 27.8*	458.3 ± 28.4*	442.4 ± 23.7*
Day 28	397.2 ± 31.7	451.4 ± 37.2*	436.9 ± 38.5*	455.0 ± 27.9*	435.6 ± 27.3*
Recovery 3	419.8 ± 33.1	470.7 ± 40.1	475.3 ± 28.1	451.6 ± 29.9	448.6 ± 37.9
Recovery 10	439.5 ± 27.2	480.9 ± 38.8	484.4 ± 37.2	464.8 ± 36.3	460.7 ± 35.7
Recovery 17	487.7 ± 28.7	511.8 ± 40.8	518.8 ± 44.3	508.2 ± 27.8	465.4 ± 35.5
Recovery 24	523.4 ± 32.2	540.9 ± 45.8	547.6 ± 48.6	535.6 ± 27.3	495.6 ± 38.9

Note: Changes in male rats on body weights during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). At the end of treatment period, two and four rats died or moribund in the 10 and 20 mg/kg/day group, respectively on Day 12 to Day 26. Date presented as mean values ± SD.

^* p ≤ 0.05 compare with control group.

TABLE 2.

Body weights in female rats (g).

Detection time	Control	2.5 mg/kg/day	5 mg/kg/day	10 mg/kg/day	20 mg/kg/day
Pretest	185.3 ± 6.1	182.5 ± 8.1	184.8 ± 8.7	185.7 ± 9.2	182.8 ± 8.8
Day 3	198.2 ± 8.0	213.9 ± 9.5*	219.2 ± 12.4*	223.0 ± 10.2*	220.2 ± 11.0*
Day 7	214.3 ± 9.6	263.3 ± 11.5*	275.2 ± 17.6*	284.4 ± 19.0*	287.3 ± 16.4*
Day 10	225.6 ± 11.7	300.8 ± 15.7*	314.8 ± 17.8*	322.0 ± 29.4*	330.0 ± 24.0*
Day 14	237.7 ± 13.0	333.6 ± 18.9*	349.5 ± 20.5*	351.0 ± 41.5*	358.7 ± 30.1*
Day 17	244.5 ± 13.9	356.8 ± 15.5*	376.7 ± 20.6*	367.6 ± 48.2*	371.9 ± 26.1*
Day 21	251.6 ± 11.0	358.0 ± 18.9*	375.2 ± 24.2*	368.7 ± 51.5*	361.2 ± 32.3*
Day 24	259.5 ± 12.1	347.0 ± 17.5*	362.6 ± 18.6*	346.5 ± 43.9*	341.4 ± 23.3*
Day 28	245.7 ± 20.5	348.6 ± 20.2*	358.5 ± 20.2*	340.6 ± 47.3*	331.8 ± 38.9*
Recovery 3	267.1 ± 9.9	338.0 ± 11.4*	349.5 ± 13.8*	336.1 ± 49.6	340.9 ± 23.3*
Recovery 10	280.8 ± 13.5	328.8 ± 15.4*	325.6 ± 17.0*	326.2 ± 41.4*	314.9 ± 9.2
Recovery 17	282.0 ± 8.8	324.2 ± 15.8	317.2 ± 22.2	323.8 ± 39.7	311.1 ± 20.4
Recovery 24	285.5 ± 14.6	321.3 ± 13.1*	312.2 ± 15.1	329.3 ± 30.5*	312.9 ± 19.7

Note: Changes in female rats on body weights during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). At the end of treatment period, two rats died in the 20 mg/kg/day group on Day 19 and Day 27, respectively. Date presented as mean values ± SD.

^* p ≤ 0.05 compare with control group.

TABLE 3.

Food consumption in male rats (g).

Detection time	Control	2.5 mg/kg/day	5 mg/kg/day	10 mg/kg/day	20 mg/kg/day
Day 3	25.5 ± 1.9	33.1 ± 4.2*	36.5 ± 2.8*	38.5 ± 2.2*	39.5 ± 3.8*
Day 10	28.5 ± 2.2	37.1 ± 2.4*	37.1 ± 1.6*	37.6 ± 1.8*	37.4 ± 1.6*
Day 17	29.4 ± 2.9	46.1 ± 0.6*	45.6 ± 1.2*	45.4 ± 6.6*	47.2 ± 1.4*
Day 24	33.4 ± 2.7	42.0 ± 1.6*	41.4 ± 1.0*	43.0 ± 7.5*	44.9 ± 2.1*
Recovery 3	34.9 ± 6.0	34.6 ± 3.0	39.5 ± 3.7	28.9 ± 6.8	30.5 ± 12.7
Recovery 10	39.2 ± 4.9	35.7 ± 2.3	43.4 ± 2.5	33.7 ± 5.2	27.9 ± 6.6*
Recovery 17	27.4 ± 8.8	21.3 ± 12.2	30.9 ± 13.8	29.3 ± 8.6	19.1 ± 4.7
Recovery 24	29.5 ± 3.9	28.8 ± 1.1	31.5 ± 7.6	28.9 ± 2.2	28.4 ± 4.7

Note: Changes in male rats on food consumption during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). At the end of treatment period, two and four rats died or moribund in the 10 and 20 mg/kg/day group, respectively on Day 12 to Day 26. Date presented as mean values ± SD.

^* p ≤ 0.05 compare with control group.

TABLE 4.

Food consumption in female rats (g).

Detection time	Control	2.5 mg/kg/day	5 mg/kg/day	10 mg/kg/day	20 mg/kg/day
Day 3	18.1 ± 1.7	28.7 ± 2.2*	30.9 ± 2.8*	32.6 ± 2.8*	31.6 ± 3.3*
Day 10	19.0 ± 1.7	35.5 ± 1.2*	36.2 ± 2.3*	37.9 ± 1.9*	37.6 ± 1.8*
Day 17	20.4 ± 1.9	38.0 ± 1.2*	39.5 ± 0.5*	38.3 ± 0.9*	39.2 ± 0.8*
Day 24	20.5 ± 2.7	37.5 ± 1.1*	37.5 ± 1.6*	36.2 ± 1.0*	37.4 ± 1.0*
Recovery 3	20.1 ± 3.4	14.2 ± 3.0	20.3 ± 8.0	17.8 ± 8.6	14.0 ± 15.2
Recovery 10	23.7 ± 4.5	14.5 ± 2.3*	11.1 ± 0.8*	15.3 ± 4.8	16.0 ± 4.9
Recovery 17	13.5 ± 5.0	15.6 ± 4.9	17.1 ± 7.0	13.8 ± 5.0	16.7 ± 6.4
Recovery 24	17.1 ± 4.0	14.6 ± 1.2	12.3 ± 3.3	18.5 ± 5.2	17.4 ± 6.0

Note: Changes in female rats on food consumption during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). At the end of treatment period, two rats died in the 20 mg/kg/day group on Day 19 and Day 27, respectively. Date presented as mean values ± SD.

^* p ≤ 0.05 compare with control group.

3.2.2. Ophthalmological Findings

At the end of the 4‐week drug treatment period, ocular symptoms including corneal opacity, ulceration, neovascularization, and edema were evident in 12/28 surviving rats in the 10 mg/kg/day group (5 female; 7 male) and 23/26 rats in the 20 mg/kg/day group (13 female; 10 male). In addition, corneal opacity, neovascularization, and other symptoms were noted in 2/30 rats in the 2.5 mg/kg/day group (1 female; 1 male) and 3/30 rats in the 5 mg/kg/day group (3 male). At the end of the ensuing recovery period, persistent symptoms including corneal opacity and neovascularization were noted in 4/10 (1 female; 3 male), 6/10 (2 female; 4 male), 8/10 (4 female; 4 male), and 6/9 (3 female; 3 male) rats in the 2.5, 5, 10, or 20 mg/kg/day LPM4870108 treatment groups, respectively.

3.2.3. Hematology, Serum Biochemistry and Urine Analyses

Hematological findings for rats included in this study are presented in Tables S1 and S2. In the 10 and 20 mg/kg/day groups, male rats exhibited significantly increased MONO% values and significantly lower APTT values (p ≤ 0.05). As compared to control animals at the end of the treatment period, LPM4870108‐treated rats exhibited increased MCV, MONO, and/or MCH values and decreased RBC counts (p ≤ 0.05). Rats in the 2.5 and 20 mg/kg/day groups exhibited NEU and NEU% increases. The FIB levels in rats of the 5, 10, and 20 mg/kg/day groups were increased, and the LYM% values were reduced in the 2.5, 10, and 20 mg/kg/day groups, along with a decrease in the HGB levels in the 20 mg/kg/day group (p ≤ 0.05). Similarly, female rats exhibited significant increases in NEU%, NEU, MONO%, MONO, MCV, and FIB values at the end of the treatment period in all groups along with reduced LYM%, MCHC, and APTT levels. In addition, the MCH and PT values were increased after treatment in the 10 and 20 mg/kg/day groups, the EOS and EOS% values were elevated in the 2.5 mg/kg/day group, the RBC levels were reduced in the 20 mg/kg/day group, and the WBC levels were elevated in the 5 and 20 mg/kg/day groups (p ≤ 0.05). These hematological parameters did not differ significantly in male or female rats when comparing LPM4870108‐treated and control rats with the exception of EOS%, which was increased in female rats in the 20 mg/kg/day group (p ≤ 0.05).

At the end of the treatment period, male rats in all LPM4870108 treatment groups exhibited reduced A/G ratios and elevated Na⁺ levels as compared to controls (p ≤ 0.05) (Tables S3 and S4). At this same time point, rats in the 10 and 20 mg/kg/day groups exhibited reduced ALB levels while those in the 2.5 and 5 mg/kg/day groups showed lower CK and LDH levels (p ≤ 0.05). Moreover, rats in the 5 mg/kg/day group showed reduced AST level and elevated CHOL value (p ≤ 0.05). At the end of the recovery period, the LDH and/or CK values in the 2.5, 5, and 10 mg/kg/day groups remained lower than the control group (p ≤ 0.05). Similarly, female rats treated with various LPM4870108 doses exhibited lower ALB and A/G levels and higher AST, UREA, CHOL, GLU, AMY‐P, Na⁺, and Cl⁻ levels as compared to controls at the end of the treatment period (p ≤ 0.05). At the end of the recovery period, the urea concentration remained lower in female rats in the 5 mg/kg/day group as compared to the control group (p ≤ 0.05).

Urinalysis revealed no evidence of abnormal changes related to LPM4870108 treatment (data not shown), with all pH, specific gravity, and volume parameters within normal ranges.

3.2.4. Major Organ and Bone Marrow Cytology Analyses

Terminal organ weight and organ/body weight ratio values were calculated for all rats enrolled in this study (Tables S5 and S6). Male or female rats in several of the LPM4870108 treatment groups showed increases in absolute and/or relative (to body or brain) weight for organs including the heart, kidneys, liver, and spleen (p ≤ 0.05). Females in several LPM4870108 treatment groups exhibited increases in the absolute and relative (to brain weight) thymic and ovarian weight, together with reductions in relative (to body weight) brain, adrenal, and uterine weight (p ≤ 0.05). Male rats treated with LPM4870108 also showed increases in adrenal/brain weight ratio while their epididymis/body and brain/body weight ratios were reduced as compared to controls (p ≤ 0.05). In all cases, these changes partially or fully reverted to normal levels at the end of the recovery period.

Major gross pathology findings for this study are presented in Table 5. After the 4‐week treatment period, male and female rats in all LPM4870108 treatment groups showed cutaneous scabbing. Moreover, males and females in several of the LPM4870108 treatment groups exhibited splenic enlargement, femoral swelling, grayish‐white eye coloration, and swollen liver. At the end of the recovery period, cutaneous scabbing and cervical lymph node enlargement were only evident in one female rat in the 10 mg/kg/day group. Over the course of the treatment, eight rats in the 10 and 20 mg/kg/day groups died or were found to be moribund, and the major gross pathology findings exhibited by these rats included splenic enlargement, eye discoloration, and swollen liver.

TABLE 5.

Results of gross observation in scheduled necropsy of male (M) and female (F) rats.

	Incidence
	Control		2.5 mg/kg/day		5 mg/kg/day		10 mg/kg/day		20 mg/kg/day
	M	F	M	F	M	F	M	F	M	F
End of treatment period
Skin
Scab	0/10	0/10	1/10	0/10	2/10	3/10	2/8	4/10	2/6	3/9
Damage	0/10	0/10	1/10	0/10	0/10	0/10	0/8	0/10	0/6	0/9
Spleen
Enlargement	0/10	0/10	0/10	0/10	0/10	0/10	1/8	0/10	0/6	0/9
Liver
Sallowness	0/10	0/10	0/10	0/10	1/10	0/10	1/8	0/10	0/6	1/9
Eyes
Turn gray	0/10	0/10	0/10	0/10	0/10	0/10	2/8	2/10	1/6	4/9
Turn dull red	0/10	0/10	0/10	0/10	0/10	0/10	0/8	1/10	0/6	1/9
Femur
Swell	0/10	0/10	0/10	0/10	0/10	0/10	1/8	0/10	0/6	1/9
End of recovery period
Skin
Scab	0/5	0/5	0/5	0/5	0/5	0/5	0/5	1/5	0/5	0/4
Damage	0/5	0/5	0/5	0/5	0/5	0/5	0/5	1/5	0/5	0/4
Cervical lymph node
Enlargement	0/5	0/5	0/5	0/5	0/5	0/5	0/5	1/5	0/5	0/4

Note: Changes in male and female rats on main gross pathology parameters during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). Six rats (4/10 of male and 2/10 of female) and two rats (2/15 of male) died or moribund in the 20 and 10 mg/kg/day group, respectively on Day 12 to Day 27.

Necropsy was performed with two and six rats in the 10 and 20 mg/kg/day groups, respectively. Microscopic analyses of samples from these rats revealed evidence of toxic damage to the liver (necrocytosis, vacuolar degeneration, hypertrophy), spleen (decreased lymphocytes), and eyeballs (mixed cell infiltration). Major treatment‐related histopathological findings for all rats are presented in Table 6 and Figure 2. Hepatocyte vacuolar degeneration, hepatocellular necrosis, and hypertrophy were observed in all treatment groups, including the control group (Figure 2A–D, Table 6). Splenic lymphopenia was observed for rats in all LPM4870108 treatment groups (Figure 2E,F). Corneal and anterior chamber inflammation was observed in both male and female rats in the 10 and 20 mg/kg/day groups (Figure 2G–I). Skin ulceration, scabbing, and subcutaneous inflammation were detected in male and female rats from all LPM4870108‐treated groups (Figure 2J–L).

TABLE 6.

Selected histopathology observation in rats.

		Incidence
		Control		2.5 mg/kg/day		5 mg/kg/day		10 mg/kg/day		20 mg/kg/day
		M	F	M	F	M	F	M	F	M	F
End of treatment period	N	10	10	10	10	10	10	8	10	6	9
Liver
Vacuolar degeneration	Grade: 1	2	4	3	5	4	1	0	3	2	1
	2	0	5	0	2	1	4	2	2	0	2
	3	0	0	0	1	0	3	1	2	0	2
	4	0	0	0	0	0	0	0	1	0	4
	Total	2	9	3	8	5	8	3	8	2	9
Hepatocellular necrosis	Grade: 1	1	1	0	0	0	0	4	1	4	2
	Total	1	1	0	0	0	0	4	1	4	2
Hepatocellular hypertrophy	Grade: 1	0	0	0	2	0	4	2	2	0	3
	2	0	0	0	2	0	2	0	2	1	0
	Total	0	0	0	4	0	6	2	4	1	3
Spleen	Grade: 1	0	0	3	1	7	6	5	6	3	4
Lymphopenia	2	0	0	0	0	0	1	1	0	0	1
	3	0	0	0	0	0	0	0	0	0	1
	Total	0	0	3	1	7	7	6	6	3	6
Eyes	Grade: 1	0	0	0	0	0	0	0	0	1	0
Corneal cell inflammation	2	0	0	0	0	0	0	1	0	0	0
	3	0	0	0	0	0	0	0	0	0	4
	4	0	0	0	0	0	0	0	0	0	2
	5	0	0	0	0	0	0	0	2	0	0
	Total	0	0	0	0	0	0	1	2	1	6
Anterior chamber cell inflammation	2	0	0	0	0	0	0	0	0	0	2
	3	0	0	0	0	0	0	0	2	0	1
	Total	0	0	0	0	0	0	0	2	0	3
Ulceration	Grade: 1	0	0	0	0	0	0	0	1	0	0
	2	0	0	0	0	0	0	0	0	0	2
	3	0	0	0	0	0	0	0	1	0	2
	4	0	0	0	0	0	0	0	1	0	0
	Total	0	0	0	0	0	0	0	3	0	4
Corneal epithelial segregation	Grade: 1	0	0	0	0	0	0	0	0	2	1
	2	0	0	0	0	0	0	2	2	0	1
	5	0	0	0	0	0	0	0	0	0	1
	Total	0	0	0	0	0	0	2	2	2	3
Skin	Grade: 1	0	0	1	0	0	0	1	1	1	0
Ulceration	2	0	0	0	0	0	1	0	0	0	0
	3	0	0	1	0	1	1	0	2	1	2
	4	0	0	0	0	0	0	1	0	0	0
	Total	0	0	2	0	1	2	2	3	2	2
Scab	Grade: 1	0	0	2	0	0	1	1	1	0	0
	2	0	0	0	0	2	1	1	1	1	1
	3	0	0	0	0	0	1	0	1	1	1
	4	0	0	0	0	0	0	0	1	0	0
	Total	0	0	2	0	2	3	2	4	2	2
Epidermal hyperplasia	Grade: 1	0	0	1	0	0	2	0	1	1	0
	2	0	0	1	0	1	1	0	1	1	0
	3	0	0	0	0	1	0	1	2	0	1
	Total	0	0	2	0	2	3	1	4	2	1
Subcutaneous inflammation	Grade: 1	0	0	1	0	1	1	2	1	2	1
	2	0	0	0	0	0	2	0	2	0	1
	3	0	0	1	0	1	0	0	1	0	0
	Total:	0	0	2	0	2	3	2	4	2	2
End of recovery period	N	5	5	5	5	5	5	5	5	5	4
Liver
Vacuolar degeneration	Grade: 1	1	0	0	0	0	0	1	2	1	2
	2	0	1	0	0	1	1	0	0	0	0
	3	0	0	0	0	0	0	0	2	1	0
	4	0	0	0	0	0	1	0	0	0	0
	Total	1	1	0	0	1	2	1	4	2	2
Eyes	Grade: 1	0	0	0	0	0	0	0	0	1	0
Corneal cell inflammation	Total	0	0	0	0	0	0	0	0	1	0

Note: Changes in male and female rats on main histopathology findings during the LPM4870108 administration period (n = 10) and following a 4‐week recovery period (n = 5). Six rats (4/10 of male and 2/10 of female) and two rats (2/15 of male) died or moribund in the 20 and 10 mg/kg/day group, respectively on Day 12 to Day 27.

FIGURE 2.

Histological sections of liver (A–D, 200×), spleen (E, F, 200×), eye (G–I, 200×), and skin (J–L, 100×) with H&E staining. Liver sections are shown from the control (A), 20 mg/kg/day (B, D), and 10 mg/kg/day (C) groups. Spleen sections are shown from the control (E) and 20 mg/kg/day (F) groups. Eye sections are shown from the control (G) and 20 mg/kg/day (H, I) groups. Skin tissue sections are shown from the control (J) and 10 mg/kg/day (K, L) groups.

No LPM4870108‐treated rats showed any biologically significant changes in bone marrow parameters compared to control animals (data not shown).

3.2.5. Toxicokinetics

The toxicokinetic parameters determined in this study are presented in Table 7. Plasma concentrations of LPM4870108 initially rose and generally peaked at 1–2 h post‐dosing. The C _max1, C _max2, and AUC_{0–24 h} values on Day 1 and Day 28 of the treatment period increased in a dose‐dependent manner. No evidence of LPM4870108 accumulation was observed over the course of the 4‐week treatment period, and female rats exhibited higher LPM4870108 plasma exposure than males.

TABLE 7.

Plasma LPM4870108 exposure in female and male rats.

		2.5 mg/kg/day		5 mg/kg/day		10 mg/kg/day		20 mg/kg/day
		Day 1	Day 28	Day 1	Day 28	Day 1	Day 28	Day 1	Day 28
AUC0–24 h (h*ng/mL)	F	3210 ± 611	3320 ± 926	5900 ± 826	7610 ± 1920	13 100 ± 1790	17 200 ± 3130	26 600 ± 2560	27 700 ± 6690
	M	981 ± 334	1160 ± 258	1890 ± 464	2400 ± 1030	4300 ± 1120	7080 ± 688	8200 ± 2700	11 900 ± 2240
C max1 (ng/mL)	F	348 ± 87	259 ± 54.2	618 ± 63.7	605 ± 214	1550 ± 325	1140 ± 355	2670 ± 559	2210 ± 701
	M	133 ± 36.9	159 ± 72.6	225 ± 49.8	322 ± 117	430 ± 113	752 ± 155	860 ± 135	1540 ± 277
C max2 (ng/mL)	F	252 ± 72.3	383 ± 89.6	292 ± 12	818 ± 144	832 ± 240	1970 ± 240	1510 ± 145	3410 ± 945
	M	92.4 ± 24.6	159 ± 25.3	195 ± 28.3	354 ± 169	375 ± 174	1060 ± 96.3	617 ± 237	1710 ± 437
AUC0–6 h (h*ng/mL)	F	1210 ± 81.8	1140 ± 238	2410 ± 371	2540 ± 796	5840 ± 1000	5250 ± 1290	10 500 ± 1650	9760 ± 1980
	M	328 ± 102	588 ± 198	665 ± 130	1040 ± 193	1330 ± 419	2700 ± 706	2570 ± 862	5220 ± 585
AUC6–24 h (h*ng/mL)	F	2000 ± 540	2180 ± 737	3500 ± 602	5060 ± 1230	7250 ± 1650	11 900 ± 2040	16 100 ± 925	18 000 ± 6520
	M	705 ± 213	691 ± 152	1280 ± 317	1480 ± 853	2960 ± 799	4380 ± 516	5630 ± 1970	6680 ± 1860
T max1 (h)	F	1.38 ± 0.75	2.5 ± 2.38	1.5 ± 0.577	1.13 ± 0.629	1.75 ± 0.5	1.25 ± 0.5	1.25 ± 0.5	1.67 ± 0.577
	M	0.875 ± 0.25	0.75 ± 0.289	1 ± 0	0.75 ± 0.289	1.13 ± 0.629	0.625 ± 0.25	1 ± 0	1.25 ± 0.5
T max2 (h)	F	2 ± 0	2 ± 0	5 ± 2	2 ± 0	1.5 ± 1	2 ± 0	3 ± 2	2 ± 0
	M	2 ± 0	2 ± 0	2 ± 0	2 ± 0	4 ± 2.31	2 ± 0	7 ± 7.57	2 ± 0

Note: Changes in male and female rats on toxicokinetic in the Day 1 (n = 8) and Day 28 (n = 8) after the LPM4870108 administration. Date presented as mean values ± SD.

3.3. Genotoxicity Analyses

3.3.1. Ames Test

At the drug concentration levels of 50–5000 μg/plate, LPM4870108 treatment did not result in any increase in the frequency of revertant Salmonella typhimurium colonies in the presence or absence of S9 as compared to the vehicle control group (Table S7) whereas the positive control group exhibited significantly more revertant colonies (p ≤ 0.05). A second Ames test yielded comparable results (data not shown). Thus, these findings demonstrated that LPM4870108 was not genotoxic within the study dose range.

3.3.2. Bone Marrow Micronucleus Formation Test

During this study, no mice exhibited any clinical abnormalities or mortality. Relative to the mice in the negative control group, micronucleated PCE counts in mice of the positive control group were significantly elevated (p ≤ 0.05) (Table S8). Moreover, as compared to the negative control group, an increase in MR (‰) was observed among females in the 200 mg/kg LPM4870108 group, which was within the range of our historical control data (p ≤ 0.05) thus, it was not statistically significant. The PCE/(PCE + NCE) ratio was significantly reduced in mice treated with 60 mg/kg (males) and 200 mg/kg (males and females) LPM4870108 as compared to mice of the negative control group, suggesting that these doses were associated with myelosuppressive activity.

3.3.3. In Vitro Chromosomal Aberration Test

In this analysis, the frequencies of metaphase cells exhibiting abnormal chromosomes were recorded (Table S9). Relative to the vehicle control, LPM4870108 treatment did not significantly increase the chromosomal aberration ratio irrespective of whether or not S9 was included (p > 0.05), whereas the frequency of metaphase cells exhibiting abnormal chromosomes was significantly higher in the positive control group (p ≤ 0.05). Therefore, LPM4870108 exhibited a negative response in this chromosomal aberration assay.

3.4. Safety Pharmacology Study

3.4.1. Neurofunctional FOB Test in Rats

The results of neurofunctional analyses in rats are presented in Tables 8 and 9. A decrease in motor activity was evident in 3/10 rats in the 100 mg/kg/day LPM4870108 group at 4 h post‐dosing. Moreover, a reduction in rectal temperature was evident at 4 h post‐dosing in the 10, 30, and 100 mg/kg/day groups (p ≤ 0.05). No other significant anomalous neurofunctional results were observed in LPM4870108‐treated or control rats at the end of the treatment or recovery periods, including the evaluation of elicited behaviors in the rats and open field, cage‐based, and hand‐held observations.

TABLE 8.

Selected parameters of male and female rats in standard FOBs.

Detection time	Motor activity	Control		10 mg/kg/day		30 mg/kg/day		100 mg/kg/day
		n	%	n	%	n	%	n	%
Predose	Free motor activity	10	100.0	10	100.0	10	100.0	10	100.0
1 h postdose	Free motor activity	10	100.0	10	100.0	10	100.0	10	100.0
4 h postdose	Free motor activity	10	100.0	10	100.0	9	90.0	7	70.0
	Decreased motor activity	0	0.0	0	0.0	1	10.0	3	30.0
24 h postdose	Free motor activity	10	100.0	10	100.0	10	100.0	10	100.0

Note: Changes in male and female rats on FOB parameters during the LPM4870108 administration period (n = 10).

TABLE 9.

Rectal temperature parameter of rats (°C).

Detection time	Control		10 mg/kg/day		30 mg/kg/day		100 mg/kg/day
	n	Mean ± SD	n	Mean ± SD	n	Mean ± SD	n	Mean ± SD
Predose	10	38.3 ± 0.2	10	38.4 ± 0.2	10	38.4 ± 0.2	10	38.4 ± 0.1
1 h postdose	10	38.3 ± 0.2	10	38.5 ± 0.2	10	38.4 ± 0.2	10	38.4 ± 0.1
4 h postdose	10	38.3 ± 0.2	10	38.0 ± 0.4*	10	37.9 ± 0.4*	10	37.7 ± 0.5*
24 h postdose	10	38.4 ± 0.1	10	38.4 ± 0.1	10	38.4 ± 0.2	10	38.4 ± 0.2

Note: Changes in SD rats on rectal temperature parameters during the LPM4870108 administration period (n = 10).

^* p ≤ 0.05 compare with control group.

3.4.2. Analyses of Rat Respiratory Function

The rats in all groups were generally in good health, exhibited normal spontaneous movement, and did not show any other abnormalities. None of these rats were moribund. Moreover, no abnormalities were noted in the respiratory function of these rats as measured by the tidal volume, respiratory rate, and minute volume at 1, 4, or 24 h post‐dosing.

3.4.3. Cardiovascular Safety Test in Monkeys

Over the course of this safety study using conscious rhesus monkeys, all animals exhibited good general health with normal spontaneous movement and no evidence of abnormal reactions. Sinus arrhythmia in Lead‐II ECG was detected in monkeys treated with LPM4870108 at 2, 6, 8, 10, and 24 h post‐dosing. No abnormalities in any other cardiovascular parameters were observed in LPM4870108‐treated monkeys within 24 h post‐dosing.

3.4.4. hERG Potassium Channel Activity

When assessing the effects of LPM4870108 on hERG channel activity in CHO cells, the IC₅₀ for this drug was determined to be 18.2 μM (Table S10).

4. Discussion

NTRK gene fusions result in the transcription of potentially oncogenic chimeric Trk proteins exhibiting enhanced or constitutively active kinase activity [24]. These gene fusions and other NTRK gene abnormalities have recently been discovered as promising targets for anticancer therapy [25, 26]. In our prior study, the pharmacological effects of a novel Trk inhibitor LPM4870108 were characterized. After the selection through chemical and pharmacokinetic optimization, LPM4870108 was determined to be a lead compound with excellent activity against wild‐type and mutated Trk both in vitro and in vivo [12]. LPM4870108 is thus a promising structurally differentiated asset well‐suited for further development in a range of clinical anticancer applications [12]. To facilitate such clinical developments, the present study performed a series of acute toxicity, subacute toxicity, genotoxicity, and pharmacological safety studies of LPM4870108 in order to identify major toxicological effects associated with the pharmacodynamic properties of this novel Trk inhibitor.

In the acute toxicity study, three of the rats in the 1000 mg/kg group were found dead, and other animals in this group showed altered body weight, food intake, activity levels, and hematological parameters. Based on these results, the MTD for LPM4870108 was established as 300 mg/kg.

Over the course of the 4‐week subacute toxicity study, 4/15 males and 2/15 females in the 20 mg/kg/day group and 2/15 males in the 10 mg/kg/day group died or were found moribund. The primary gross pathology and microscopic pathology results from these rats revealed pathological lesions of the liver, spleen, skin, and eyes. Based on these experimental results, the STD₁₀ for LPM4870108 in rats was established as 10 mg/kg/day. This value will be used to support IND filings and the design of an initial reference dose of 1 mg/kg/day in future first‐in‐human Phase I trials. When performing first‐in‐human Phase I trials, the selection of a drug dose of 1/10th of the rodent STD₁₀ when converted according to the body surface area generally yields a good safety profile conducive to efficient dose‐escalation [27]. Additionally, in a 28‐day repeated dose oral toxicity study of LPM4870108 in rhesus monkeys, the highest non‐severely toxic dose (HNSTD) of LPM4870108 was defined as 20 mg/kg/day, indicating that LPM4870108 exhibited a safety profile in treated monkeys [28].

All animals treated with LPM4870108 in this 4‐week study exhibited significant increases in body weight and food intake, both of which can serve as indicators of adverse drug‐related effects [16]. However, in this case, the observed changes in LPM4870108‐treated rats are the expected result of the pharmacodynamic activity of LPM4870108. It is well known that the signaling via the brain‐derived neurotrophic factor (BDNF)‐TrkB pathway is an important regulating mechanism of eating behavior and body weight [29, 30], and mice exhibiting heterozygous BDNF expression show pronounced obesity that can be reversed via intraventricular BDNF infusion [29]. Moreover, mice exhibiting reduced TrkB expression to ~25% of those in wild‐type animals exhibit hyperphagic behavior and excessively high levels of weight gain when fed a high‐fat diet [31].

Ocular examinations of both male and female rats in the 10 and 20 mg/kg/day groups revealed evidence of corneal and anterior chamber inflammation. Ophthalmoscopy also showed evidence of various symptoms including corneal opacity, edema, ulceration, and neovascularization in these LPM4870108‐treated rats. Recent studies have explored MEK inhibitor‐associated ocular side effects and find that trametinib and dabrafenib, two MEK inhibitors, are associated with adverse effects of the ocular system that include chorioretinopathy and retinal vein occlusion [32]. Because Trk inhibition leads to the suppression of its downstream MEK signaling, the observed ocular inflammation in LPM4870108‐treated rats may be a direct result of LPM4870108‐mediated Trk inhibition‐induced MEK signaling suppression.

Any change in organ weight could indicate impaired organ functionality [33]. Changes in absolute or relative organ weight were observed in the LPM4870108‐treated rats over the course of the 4‐week subacute toxicity study. Other than the liver and the spleen, none of the changes in other organs exhibited any apparent dose‐ or time‐dependency, and they were not apparent in both male and female rats. Moreover, although increases in absolute and relative kidney and heart weight were observed in these rats, these increases did not correspond to any microscopic changes and, as such, they were not regarded as being of toxicological importance. Gross pathology and histopathology analyses revealed that LPM4870108‐treated rats harbored skin lesions in the form of scabbing, ulceration, and subcutaneous inflammation, as well as splenic lymphopenia and hepatic evidence of hypertrophy, hepatocellular necrosis, and hepatocyte vacuolar degeneration. LPM4870108 tissue distribution analyses indicated that this drug accumulated at higher levels in the liver (data not shown). Given this and the evidence of liver functional impairment in LPM4870108‐treated rats, it was determined that LPM4870108 has a certain degree of hepatotoxicity. Serum biochemistry and hematology can be used as a surrogate approach to monitor hepatic lesion formation [34]. Hypoalbuminemia is a disease‐related consequence of impaired hepatic function [35], with circulating albumin concentrations and functionality being reduced in patients experiencing liver failure [36]. Hepatic dysfunction is known to impact ALB synthesis and reduce the A/G ratio. Here, hematological analyses revealed that LPM4870108‐treated rats exhibited reductions in the absolute numbers and NEU% and MONO%, along with mild anemia. This anemia is consistent with what has been reported in Larotrectinib‐treated rats and is likely attributable to the splenic and hepatic extramedullary hematopoietic functionality (FDA) (https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=211710). The increased NEU and MONO counts may be associated with skin injury and infection in these rats. No other serum biochemistry or hematology findings were considered to be clinically significant, given that they were not dose‐dependent and were within the historical normal ranges of our laboratory. The skin was a major target organ of LPM4870108 in rats. Trk‐deficient mice exhibit nociceptive impairment [37]. Thus, Trk inhibition may result in a treatment‐related decline in sensitivity to bodily injury, which may account for the skin damage observed in these experimental rats.

Substantial variations in measured toxicokinetic parameters were observed in this study. Dose‐dependent increases in plasma LPM4870108 levels were observed on treatment Days 1 and 28, with higher exposure levels at the end of the study in all animals likely accounting for the abnormal body weight, food intake, and neurofunctional phenotypes exhibited by these treated rats. Female rats exhibited higher plasma LPM4870108 exposure as compared to male rats. Tissue distribution analyses indicated that this drug was eliminated more rapidly in male rats as compared to females (data not shown), possibly owing to the higher cytochrome P450 expression levels in males. However, additional chronic toxicity testing will be essential to fully elucidate the toxicokinetic profiles of LPM4870108.

Common approaches for testing genotoxicity potentials include the Ames test, bone marrow micronucleus test, and analyses of chromosomal aberrations using CHL cells [19]. LPM4870108 did not show any evidence of genotoxicity or mutagenicity in any of these three studies. In the Ames test, LPM4870108 treatment did not result in a positive response for any of the five tested histidine‐auxotrophic S. typhimurium strains with or without metabolic activation. Moreover, cultured cells exhibited no evidence of chromosomal aberrations when treated with LPM4870108 concentrations of up to 300 μg/mL. Under the test conditions, LPM4870108 treatment with doses as high as 200 mg/kg failed to induce any increase in micronucleated PCE frequencies in NIH mice.

In the safety pharmacology study, oral gavage with a single dose of LPM4870108 resulted in transient reductions in motor activity (100 mg/kg) and rectal temperature (10, 30, and 100 mg/kg), both of which recovered to normal levels at 24 h post‐treatment. In rats, a single oral dose of LPM4870108 (10, 30, or 100 mg/kg) had no impact on respiratory function. In vitro, the IC₅₀ for LPM4870108‐mediated inhibition of hERG current activity was 18.2 μM, which was over 3000 times higher than its corresponding inhibitory activity at its target receptor [12]. In rhesus monkeys, a single oral dose of LPM4870108 (5, 10, or 20 mg/kg) did not affect blood pressure. Sinus arrhythmia observed in response to LPM4870108 treatment can be attributed to the effects of this compound on the autonomic nervous system, and as such, it is considered a non‐adverse reaction.

In conclusion, given that LPM4870108 exhibits promising antitumor activity and the present study demonstrated relatively low levels of toxicity, it represents a promising candidate Trk inhibitor worthy of future clinical development as a strategy to treat NTRK gene fusion‐positive tumors. The MTD of LPM4870108 determined in the single‐dose acute toxicity study in rats was 300 mg/kg, whereas its primary toxicological effects in the 4‐week subacute dosing study were primarily attributable to Trk receptor inhibition and hepatotoxicity as a result of the accumulation of LPM4870108. Regarding the observed toxicological effects, the LPM4870108 STD₁₀ was determined as 10 mg/kg/day in rats. Importantly, LPM4870108 did not exhibit any genotoxic activity, nor did it adversely affect the respiratory, neurobehavioral, and cardiovascular activities in rats or monkeys up to a dose of 100 mg/kg. Together, these results will inform the future clinical development of safety and efficacy studies with the goal of ultimately enabling the application of LPM4870108 in the clinic.

Author Contributions

Xiaochen Zhang: methodology, software, validation, formal analysis, investigation, data curation, writing – review and editing, visualization, writing – original draft. Baiyang Yuan: conceptualization, methodology, formal analysis, investigation, writing – review and editing, supervision. Chunmei Li: writing – original draft. Hongbo Wang: methodology, data curation, writing – original draft. Shujuan Wei: formal analysis, resources, supervision. Jingwei Tian: conceptualization, resources, writing – original draft, writing – review and editing, project administration. Sijin Duan: conceptualization, resources, writing – original draft, writing – review and editing, project administration, funding acquisition.

Ethics Statement

Experiments were performed at WestChina‐Frontier Pharma Tech Co. Ltd. (Chengdu, Sichuan, China). Good Laboratory Practice (GLP) principles were strictly followed throughout this study, including those from the CFDA (now renamed as National Medical Products Administration, NMPA) (September 18, 2017), those from the OECD (ENV/MC/CHEM(98)17), and the FDA GLP principles for Nonclinical Laboratory Studies (21CFR58). All animal protocols described here received Institutional Animal Care and Use Committee (IACUC) approval.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1.

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Table S10.

Zhang X., Yuan B., Li C., et al., “Preclinical Evaluation of the Oral Toxicity, Genotoxicity, and Safety Pharmacology of LPM4870108, a Novel Potent Tropomyosin Receptor Kinase Inhibitor,” Pharmacology Research & Perspectives 13, no. 4 (2025): e70153, 10.1002/prp2.70153.

Data Availability Statement

The original contributions presented in this study are included in the article/Supporting Information. Further inquiries can be directed to the corresponding author(s).