Preclinical and Toxicology Studies of LPM7100328, a Novel Oral GnRH Antagonist.

Fangxia Zou: data curation, methodology. Pengfei Yu: data curation, methodology. Chunmei Li: conceptualization, funding acquisition, data curation, project administration, investigation, writing – review and editing, supervision. Yao Wang: data curation, formal analysis, funding acquisition, project administration, writing – original draft. Xiaolin Sun: conceptualization, funding acquisition, project administration. Liang Ye: data curation, methodology. Jingwei Tian: conceptualization, investigation, funding acquisition, project administration, writing – review and editing, supervision. Junxian Zhao: data curation, funding acquisition, project administration, writing – original draft, formal analysis. Jianzhao Zhang: data curation, methodology. Wanglin Jiang: data curation, methodology.

This work was supported by Major Basic Research Special Projects of Shandong Provincial, China, ZR2024ZD21 and Natural Science Foundation of Shandong Province, China, ZR2025MS1445, ZR2025QC881.

The results of whole body plethysmography function observation combination test showed no treatment‐related abnormalities on respiratory system and central nervous system. At post‐dosing, a minor reduction in tidal volume in the 30 mg/kg group of rats, and slight alterations in respiratory frequency or respiratory minute volume were noted (Table  1 ), which were not considered to be toxicologically significant due to the small magnitude, similar alterations were also observed in the control group, or lack of dose‐dependent effects. Selected parameters of the respiratory function in rats (mean ± SD). p  ≤ 0.05 versus pre‐dose. The function observation combination test results showed that at post‐dose, LPM7660141 caused reduced rearing frequency in all groups; increased fecal pellet count at 30, 300 mg/kg; increased grip strength or decreased body temperature at 100 mg/kg (Figure  2 ; Table  2 ). Those findings were considered non‐adverse; most showed only a small magnitude in change or similar alterations compared to control group or lack of dose relationship. Selected Parameters of Open Field Test Parameters in SD Rats. Parameters include fecal pellet count (A) and rearing frequency (B). Groups were analyzed using repeated measures ANOVA with the Geisser–Greenhouse correction; multiple comparisons were performed using Tukey's post hoc test. The statistical significance of the results is represented as * p  ≤ 0.05, and the results are presented as mean ± SD ( n  = 10). Effects of LPM7660141 on grip strength and decreased body temperature in SD rats (mean ± SD). p  ≤ 0.05 versus pre‐dose. The IC 50 of LPM7660141 blocked hERG current is 9.01 μM. Statistically significant increases in Corrected QT interval were observed in dogs received 50 mg/kg LPM7660141 at 1–1.5 h, 1.5–2 h, 2–3 h, 3–4 h, 4–5 h post‐dose (Figure  3 ). And no LPM7660141‐related statistical changes in heart rate, and blood pressure parameters were noted. The results showed that LPM7660141 had no effect on the cardiovascular function in beagle dogs received oral administration of 15 mg/kg. Effects of LPM7660141 on Corrected QT interval in Conscious Beagle Dogs. Groups were analyzed using LEVENE's test, multiple comparisons were performed using Dunnett's t ‐test or Mann–Whitney U test. The statistical significance of the results is represented as * p  ≤ 0.05 versus control group, and the results are presented as mean ± SD ( n  = 6).

Here, we report the safety pharmacology and toxicology profile of the novel GnRH antagonist LPM7100328. LPM7100328 demonstrated an acceptable nonclinical toxicity in preclinical studies. LPM7660141 showed no effects on CNS or respiratory system at doses ranging from 30 to 300 mg/kg. Oral administration of LPM7660141 up to 15 mg/kg did not cause any significant cardiovascular alterations in the conscious dogs. And in 28 days chronic dog toxicity study at dose up to 50 mg/kg or in phase I/II clinical studies (data not published), no abnormal cardiovascular parameters were observed. In acute toxicity assessments, the administration of LPM7660141 was found to be well tolerated in both rats and dogs at oral dosages of 2000 mg/kg and 1000 mg/kg, respectively. The clinical manifestations observed at elevated doses in both species were nonspecific. In 28 days chronic rat toxicity study, no significant abnormal change was observed in any of the rats orally administered LPM7660141 up to 100 mg/kg. Uterine fibroids are benign solid tumors of female reproductive system; by inducing GnRH agonist, it could reduce uterine cavity and fibroids size to facilitate the surgical intervention [ 9 , 10 ]. Alarelin dose‐dependently reduces secretion of FSH and LH and uterine weight in mice [ 11 ]. According to the mechanism of GnRH antagonist, LPM7660141 does not cause enlargement of uterine cavity in female rats. In 28 days chronic dog toxicity study, no indications of toxicity or differences in clinical symptoms or pathology findings associated with daily oral gavage doses of up to 50 mg/kg were observed. Upon allometric scaling by a conventional factor of 6 or 20, the NOAEL dose 100 mg/kg (rats) or 50 mg/kg (dogs) corresponded to a human dose equivalent of 967, 1620 mg, which is 24, 40 times the intended therapeutic dose at 40 mg, respectively. The safety and effectiveness of LPM7100328 has also been demonstrated in multiple human clinical in china, include CTR20232263, CTR20241485, CTR20234209 and CTR20243419 (data not published). Findings from mutagenicity assessments of LPM7660141 suggest that the compound is unlikely to pose a genotoxic risk to humans. In the Ames test, LPM7660141 demonstrated no mutagenic effects at concentrations of ≤ 5000 μg/plate, both in the presence and absence of metabolic activation. And LPM7660141 did not induce chromosome structural aberration in the in vitro mouse lymphoma assay in the presence or absence of metabolic activation. Beside, LPM7660141 was not clastogenic in the mice bone marrow micronucleus assay. In nonclinical reproductive toxicity studies conducted in rats and rabbits, LPM7660141/LPM7100328 was found to be generally safe with no fertility and embryo toxicity or teratogenic effects in the rats and rabbits. In the other nonclinical studies, phototoxic potential was evaluated in guinea pigs by administering oral doses of LPM7100328. LPM7100328 was negative for phototoxicity. Bioequivalence analysis showed that LPM7100328 (dissolved in 1% CMC‐Na or in capsule) was complete pharmacokinetic bioequivalence to LPM7660141 (dissolved in 1% CMC‐Na solution containing 6 mg/mL citric acid) (data not shown) for rats and dogs. LPM7100328 exhibited higher solubility and selected for use in clinical trials. LPM7100328 is an antagonist (IC 50  = 8.676 nM) of the gonadotropin‐releasing hormone receptor and is presently undergoing evaluation in Phase I/II clinical trials. The findings from the preclinical studies conducted with LPM7100328, as discussed herein, indicate that it is a safe agent for the suppression of premature luteinizing hormone (LH) surges. Given its favorable safety profile demonstrated in these preclinical investigations, along with its good oral bioavailability and lower toxicity, LPM7100328 holds promise as a potential therapeutic option for the inhibition of premature LH surges in women undergoing controlled ovarian stimulation.

In the single dose toxicology studies, some rats in 1000 and 2000 mg/kg groups showed mild transient increased salivation on day 1. Nor any changes in body weight, food consumption, hematology, serum chemistry, and gross necropsy. All dogs in 1000 mg/kg group showed small amount of white liquid/foamy vomit, and some of them showed yellowish liquid stool and mild sialorrhea. On Day 14, WBC, NEU and MONO in 1000 mg/kg group significantly increased. No deaths or change in body weights, food consumption, clinical chemistry and gross lesions were observed. In the chronic toxicity studies, no apparent changes were observed in general clinical observations, body weight, food consumption, ophthalmoscopy and gross pathology in both male and female rats on days 29 and 57. There were some slight changes in hematology (BASO, BASO%, PLT), serum chemistry (TP, ALT), urine analysis (glucose, specific gravity of urine, urine pH), organ weights (thymus) and organ weight coefficient (organ to body/brain weight ratios of thymus) of male rats on days 29 or 57 (Table  3 ), which were not considered to be toxicologically significant due to the small magnitude of change from controls, lack of dose‐relationship, within the normal range, or lack of corresponding microscopic findings. After 4 weeks recovery periods, the weight and organ to body/brain weight ratios of uterus and cervix uteri were statistically increased in 100 mg/kg (Table  4 ). Histopathological examination showed enlargement of the uterine cavity in 100 mg/kg group on day 57 (Table  5 ), which were physiological changes of the rat uterus during proestrus and estrus. The NOAEL was 100 mg/kg for 28‐day consecutive i.g. dosing in SD rats. Selected hematology, serum chemistry and organ parameters in male rats (mean ± SD). p  ≤ 0.05 versus control group. Selected organ parameters in female rats (mean ± SD). p  ≤ 0.05 versus control group. Incidence and grading of histopathological lesions in SD rats at the end of the recovery phase following a 4‐week repeated dose toxicity study. No significant changes in body weight, ophthalmoscopic examination, blood pressure, hormone, examination of marrow smear, gross pathology and histopathology were observed during the study in all dogs. There were some slight changes in general clinical observations (loose or liquid stool, vomiting food or foam), food consumption, body temperature, QT interval, hematology (PT, MCV), serum chemistry (GLU), urine analysis (urine pH) and organ weight coefficient (organ to body ratio of heart) during dosing period or at the end of administration (Tables  6 , 7 , 8 ), which were not considered to be toxicologically significant owing to the small extent of change compared to control, no significant change compared with adaptation period, lack of dose/time‐relationship or corresponding microscopic findings. The NOAEL was 50 mg/kg for a 28‐day oral dose administration in dogs. Selected clinical signs in beagle dogs. Food consumption, body temperature, QT interval, hematology and serum chemistry changes in dogs (mean ± SD). p  ≤ 0.05 versus control group. Urine pH changes in female dogs. p  ≤ 0.05 versus control group. After repeated oral administration to rats, female rats in the 10 and 30 mg/kg groups exhibited higher mean plasma AUC 0‐24h of LPM7660141 compared to males, while no significant gender differences were noted in the 100 mg/kg group. The proportional increase in plasma AUC 0‐24h exceeded the dose escalation magnitude in both genders. The accumulation indexes of LPM7660141 in the rat plasma were 1.6, 2.6, 1.7 (female) and 2.5, 3.8, 1.7 (male) in the 10, 30, and 100 mg/kg/dose group, respectively. Indicating accumulation in both female and male animals (Table  9 ). Summary of LPM7660141 toxicokinetic parameters in rats. After the 1st and 28th dose in dogs, the increase in the AUC of LPM7660141 was slightly exceeded the proportional to the increase in dose from 5 to 50 mg/kg/dose, and there was no significant gender difference. The accumulation indexes of LPM7660141 in the dog plasma were 1.5, 1.2, 1.5 (female) and 1.9, 1.3, 1.9 (male) in the 5, 10, and 50 mg/kg/dose group, respectively. Indicating no accumulation in both female and male animals (Table  10 ). Summary of LPM7660141 toxicokinetic parameters in dogs. The LPM7660141 was evaluated up to a concentration of 5000 μg/plate across all the five strains both with and without the presence of the S9 metabolic activation system. No significant cytotoxicity was observed in all dose groups. There were significant increases or decreases in the number of revertant colonies at a certain concentration tested with or without S9 compared to the negative group (Table  11 ). But these were considered within normal background level or lack of dose‐dependent effects and therefore not related to treatment. Furthermore, the aberration test demonstrated that there was no statistically significant increase in the percentage of chromosome structural aberration in all groups with and without S9 metabolic activation system compared with the vehicle group, indicating that LPM7660141 did not induce chromosomal aberration (Table  12 ). Effect of LPM7660141 on the revertant colony counts in mutant strains of Salmonella typhimurium (mean ± SD). p  ≤ 0.05 versus vehicle control group. Effect of LPM7660141 on the chromosomal aberrations of CHL cells. p  ≤ 0.05 versus vehicle control group. In the micronucleus assay, there was no significant increase in the frequency of micronuclei in 500, 1000, 2000 mg/kg groups (Table  13 ). And there was statistically significant decrease in the ratio of PCE/(PCE + NCE) at 2000 mg/kg in female mice, which were not considered to be toxicologically significant due to within the range of historical data or lack of dose‐dependent effects. All the above results suggested that LPM7660141 did not exhibit any genotoxicity. Micronucleus test of LMP7660141 orally administered to NIH mice (mean ± SD). p  ≤ 0.05 versus vehicle control group. In the rat fertility and early embryonic developmental toxicity study, maternal administration of LPM7660141 showed no adverse clinical observations, no effects on maternal body weight, body weight gain, food consumption, fertility, reproductive function of male or female rats and early embryogenesis. From gestation day (GD) 2 to GD 4 and GD 13 to GD 15, the body weight gain increased in pregnant rats treated at 30 mg/kg and 100 mg/kg (Figure  4 ). In the 200 mg/kg group, a decrease in the percentage of medium‐speed sperm motility was observed. All changes noted above were statistically significant but lacked a dose–response relationship or no significant abnormal changes were observed in the other relevant parameters and were considered not toxicologically relevant. In conclusion, the NOAEL for the parental male and female rats was 200 mg/kg; the NOAEL for the reproductive toxicity in females and for embryo‐fetal development toxicity was 200 mg/kg. Effect of LPM660141 on the body weight gain of pregnant rats. Groups were analyzed using LEVENE's test, multiple comparisons were performed using Dunnett's t ‐test or Mann–Whitney U test. The statistical significance of the results is represented as * p  ≤ 0.05 versus control group, and the results are presented as mean ± SD ( n  = 24). To evaluate the administration of GnRH antagonists in clinical assisted reproductive indications, LPM7100328 was orally administered once daily to rabbits at dose of 0, 5, 15, 50 mg/kg for 14 days before mating. There was a statistically significant increase in the mating day compared to vehicle control group at 50 mg/kg (Table  14 ), which was considered to be related to pharmacological effect of LPM7100328 [ 8 ]. And administration of LPM7100328 caused reduced female rabbits weight gain at 25 mg/kg/day on day 1–2; brain weight of pregnant rabbits at 25, 50 mg/kg, which were not deemed toxicologically significant due to the minimal magnitude. No mortality was observed at all dose groups. No treatment‐related clinical observations were noted for the treated females. There were no effects on body weights, weight gain, and food consumption. No gross pathological findings were treatment‐related. No treatment‐related findings suggesting embryo‐fetal deaths, teratogenicity or depression of fetal growth were observed in any group. The NOAEL for maternal toxicity and embryo‐fetal development was 50 mg/kg/day. Effect of LPM7100328 on the mating day and mating rate of female rabbits. p  ≤ 0.05 versus vehicle control group. Following oral administration of LPM7100328 in guinea pigs, no apparent changes in locomotor activity, body weight, mental state or secretion. No treatment‐related skin erythema or oedema was observed at doses up to 100 mg/kg (data not shown). However, in the 8‐Methoxypsoralen group, mild to moderate erythema and oedema were noted in all animals.

Gonadotropin‐Releasing Hormone (GnRH) is a crucial part of the sexual maturation process and reproductive function of human cells [ 1 ]. GnRH is a peptide hormone produced and secreted in a pulsatile manner by hypothalamic neurons. GnRH plays a pivotal role in promoting the synthesis and release of the gonadotropins, specifically follicle‐stimulating hormone (FSH) and luteinizing hormone (LH), from the pituitary gland [ 2 ]. Therefore, it plays a crucial role in reproductive functions. GnRH receptor is the target of GnRH agonist (receptor desensitization) and GnRH antagonist (receptor inhibition) for treatment of female infertility, gynecological disorders and hormone‐dependent tumors [ 2 , 3 ]. GnRH agonists bind to pituitary GnRH receptors, induce the release of FSH and LH (flare‐up effect), and upregulate the GnRH receptor. Subsequently, agonists lead to desensitization of the GnRH receptors and a decrease in the number of GnRH receptors [ 4 ]. GnRH antagonists bind competitively to pituitary GnRH receptors thus inhibiting secretion of gonadotrophin [ 3 , 5 ]. This suppression of secretion occurs within hours of initiating antagonist treatment, and notably, no flare‐up effect is observed [ 4 ]. Upon cessation of GnRH antagonist therapy, there is an immediate restoration of the pituitary‐gonadal axis [ 3 ]. Due to these beneficial characteristics, GnRH antagonist protocols have been extensively employed in the context of in vitro fertilization (IVF) [ 6 ]. The only GnRH antagonists approved for pituitary suppression in controlled ovarian stimulation during IVF treatment are ganirelix and cetrorelix, which are the injectable types. In these protocols, the patients are required to have multiple injections, both increasing the financial and physical burdens. A local skin reaction with redness, swelling and pain can be observed in patients using the drugs. Some oral GnRH antagonists are already commercially available (elagolix, relugolix, linzagolix) for cancer, uterine fibroids or endometriosis. Relugolix is a newly developed orally administered GnRH antagonist which was approved by the FDA, PMDA or EMA for the management of advanced prostate cancer in adult patients, as well as for alleviating symptoms associated with uterine fibroids. However, relugolix has low bioavailability and high within‐subject pharmacokinetic variability (NDA 214, 621). Maybe the low logD value of relugolix leads to its poor membrane permeability and bioavailability [ 7 ]. We designed and developed a highly potent and orally active GnRH antagonist LPM7100328 as a clinical candidate [ 7 ]. In this study, we present the findings from the preclinical safety pharmacology and toxicology assessments of LPM7660141/LPM7100328. The results of these studies are instrumental in facilitating the approval for clinical trials.

The authors declare no conflicts of interest.

The preclinical and toxicology studies were approved by the respective Institutional Animal Care and Use Committees (IACUC) and performed in compliance with the pertinent sections of Good Laboratory Practice for Non‐clinical Studies of Drugs (2017) as established by the China Food and Drug Administration, as well as Good Laboratory Practice for Non‐clinical Laboratory Studies in 21 CFR Part 58 by the Food and Drug Administration, and OECD Principles of Good Laboratory Practice (as revised in 1997), ENV/MC/CHEM (98)17. LPM7660141/LPM7100328 (Figure  1 ) was synthesized at Shandong Luye Pharmaceutical Co. Ltd. The purity of the compound used in these studies was > 94%. LPM7100328 is fumaric acid salt form of LPM766014. All doses in this study are expressed as LPM7660141. For safety pharmacology, acute and chronic toxicology studies, reproductive and developmental toxicology studies, and phototoxicity study, LPM7660141 was administered in 1% (w/v) CMC‐Na solution containing 6 mg/mL citric acid, and LPM7100328 was administered in 1% (w/v) CMC‐Na solution. Chemical structure of LPM7100328. In the context of mutagenicity studies, LPM7660141 was formulated as a solution in dimethyl sulfoxide (DMSO) for the Ames assay and mouse lymphoma assay, while a 1% (w/v) sodium carboxymethyl cellulose (CMC‐Na) solution containing 6 mg/mL citric acid was utilized for the micronucleus assay. NIH mice were obtained from SPF (Beijing) Biotechnology Co. Ltd. Sprague–Dawley rats and guinea pigs were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. Beagle dogs were obtained from Yizheng Anlimao Biotechnology Co. Ltd. NZW rabbits were obtained from Pizhou Dongfang breeding Co. Ltd. The potential of LPM7660141 to inhibit hERG channel current was evaluated in a stably transfected CHO cell line at concentrations of 1, 3, 10, 30 and 45 μM. Channel currents were measured using the whole‐cell patch clamp method. The effect of LPM7660141 on the respiratory system was studied in SD rats by using whole body plethysmography. Forty SD rats (5 animals/sex/group) were given a single of LPM7660141 orally at 30, 100, 300 mg/kg or vehicle. The respiratory rate, tidal volume, and respiratory minute volume in rats were measured pre‐dose and about 4 h and 24 h post‐dose. The effect of LPM7660141 on the central nervous system in SD rats was evaluated by the function observation combination test. Forty SD rats (5/sex/group) received a single oral administration of LPM7660141 at 30, 100, 300 mg/kg or vehicle. The neurobehavioral assessments of rats were observed pre‐dose and at about 0.25 h, 1 h, 24 h post‐dose, including home cage observation (asleep, walking or climbing, piloerection, self‐harm or attacking other animals, grooming, convulsions, catalepsy, death), hand‐held observation (aggressiveness, bare‐hands restraint and grasping, salivation, lacrimation, secretion, skin color, piloerection, breathing), open field observation (locomotor, posture, gait, tail status when moving forward, clonic involuntary movement, defecation, urination, rearing times), stimulus–response observation (alertness, abdominal tone, pinna reflex, tail pinch response, righting reflex, pupil reflex), grip strength and body temperature. The effect of LPM7660141 on the cardiovascular system was evaluated in conscious beagle dogs using the telemetry system. Six beagle dogs (3/sex/group) were fasted overnight and then received a single oral administration of LPM7660141 at 5, 15, 50 mg/kg or vehicle. On dosing day, the electrocardiogram (ECG), heart rate, and blood pressure of all tested animals were continuously recorded from at least 1.5 h pre‐dose to about 24 h post‐dose.