Zebra fish drug developmental toxicity optimization evaluation method conforming to ICH S5 (R3) guidance principle substitution test requirements and application of zebra fish drug developmental toxicity optimization evaluation method

Abstract

According to the zebra fish drug developmental toxicity evaluation method meeting the requirements of an ICHS5 (R3) guidance principle substitution method and the application, a classical scheme is adopted, in 31 ICH positive compounds, 23 compounds have teratogenicity in zebra fish, 5 compounds are false negative, and 3 compounds cannot judge teratogenicity or non-teratogenicity; in 14 ICH negative compounds, 12 compounds have no teratogenicity in zebra fish, and 2 compounds have false positive results. After an additional TI value (2dpf) is added on the basis of the original 5dpf, a new category is proposed as an unclassified compound, the TI value of the unclassified compound is smaller than 2dpf and 5dpf, but the unclassified compound induces toxic phenotypes, the test concentration range is refined, the optimized TI cutoff value is larger than or equal to 10 and larger than or equal to 3, and the optimized zebra fish development test reaches 90.3% of sensitivity and 88.9% of overall predictability.

Description

Technical Field

[0001] The present invention belongs to the technical field of toxicity evaluation, and specifically relates to a zebrafish drug developmental toxicity optimization evaluation method and application that complies with the requirements of the ICH S5 (R3) guideline for alternative testing. Background Art

[0002] Developmental toxicity and teratogenicity are serious safety issues, with teratogens causing congenital anomalies in approximately 5-10% of human newborns. After the developmental effects of thalidomide were confirmed in 1966, the U.S. Food and Drug Administration (FDA) established a program to evaluate the effects of drugs on reproduction and development before human use. In addition, there are currently more than 143,835 pre-registered chemicals that may contaminate food and the environment, but approximately 86% of these chemicals and other existing chemicals have no safety testing data.

[0003] Due to concerns about the safety of chemicals, REACH plans to establish protocols for collecting such data for all chemicals produced or sold in quantities exceeding 1 tonne per year. It is estimated that the need to evaluate the developmental toxicity of one sample would require 5,816 traditional test animals and a cost of £1,883,200, according to the guidelines of the Organization for Economic Co-operation and Development (OECD). This situation has prompted the study of alternative methods for developmental toxicity testing, namely the whole embryo culture (WEC) test, the embryonic cell micromass test (MM), and the embryonic stem cell test. The first two models still use intact mammals as test systems; the latter has been an area of ​​controversy. However, the above tests do not cover the entire stage of embryonic development.

[0004] As a non-mammalian vertebrate, zebrafish has several advantages in alternative toxicity testing, such as economical breeding requirements, embryo transparency, high fecundity, and high-throughput screening in 6-384-well plates. In addition, zebrafish also seems to be a suitable model for developmental toxicity testing. In the past 10 years, the developmental toxicity of drugs and compounds has actually been evaluated in zebrafish, as shown in Table 1. In a blinded study conducted by 4 laboratories, 20 non-patented compounds were tested in zebrafish for developmental toxicity; each testing laboratory achieved an overall agreement similar to the mammalian data. Based on the optimization of experimental parameters and consideration of zebrafish embryo absorption, 38 proprietary drug compounds were evaluated in two laboratories; the overall agreement reached 62-82%. In other studies, at the Flanders Institute of Technology and Phylonix Pharmaceuticals, the overall predictive value of zebrafish developmental toxicity analysis was 50-60% and 92%, respectively. It is clear that these developmental toxicity reports from zebrafish experiments are not uniform: the definition of teratogenicity, the predictability of the results, multiple details related to the experimental conditions and data analysis, and the rationality of the concentration settings have not been fully optimized and validated.

[0005] Table 1. Reported evaluation schemes for developmental toxicity and teratogenicity in zebrafish

[0006]

[0007] The release and implementation of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Harmonized Guideline S5(R3) (2020) is driving the progress of alternative methods for developmental toxicity testing in China. The China National Medical Products Administration participated in the revision of ICHS5(R3), in which the introduction of alternative test systems was a major update, and it was also the first time that a large number of alternative test methods were added to the ICH safety series of guidelines.

[0008] ICH guidelines recommend the use of alternative in vitro and non-mammalian in vivo reproduction tests for embryo-fetal developmental toxicity (EFD) risk assessment. The final version of ICH S5(R3) (2020) explicitly states that data generated from qualified alternative assays conducted alone or in combination with one or more in vivo studies can be used to support hazard identification and risk assessment in limited circumstances. However, data generated from zebrafish developmental toxicity studies have been used for investigational new drug (IND) applications both domestically and internationally. Summary of the invention

[0009] In view of the problems arising from the above background technology, the purpose of the present invention is to provide a zebrafish drug developmental toxicity evaluation method and application that meets the requirements of the ICHS5 guidelines, which is specifically achieved through the following technical solutions:

[0010] The zebrafish drug developmental toxicity evaluation method guided by ICHS5(R3) includes the following steps:

[0011] 1) Zebrafish rearing and egg collection: Zebrafish were reared in 28°C fish water (water quality: 200 mg instant sea salt was added to each 1L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3); zebrafish embryos produced by natural mating were collected at 4-6 h post fertilization (4-6 hpf);

[0012] 2) Selection of validation compounds: Select at least 45 compounds from the list of positive and negative reference compounds in the ICHS5 guidelines (covering all categories in the reference compound list); the ratio of compounds used for positive detection to compounds used for negative detection in the compounds to be validated is approximately 2:1;

[0013] 3) Compound exposure method: 6hpf zebrafish embryos were transferred to 24-well plates containing negative compounds, positive compounds, solvent controls (0.5% DMSO solution) or normal controls (water for fish farming) for exposure, 1mL / well, 1 tail / well, and the samples were continuously treated with zebrafish for 5 days until 5dpf. The development of the embryos was observed every day during this period and dead zebrafish were removed in time. The morphological characteristics of each individual zebrafish were evaluated under a stereomicroscope at 48hpf (2 days after fertilization, dpf) and 120hpf (5dpf); false positive, false negative and unclassified compounds were repeated at least once;

[0014] 4) 25% lethal concentration (LC 25 ) and no observed adverse effect level (NOAEL): According to the cumulative mortality at 2dpf and 5dpf, the 25% lethal concentration LC was calculated respectively. 25 If the lethality at the highest detected concentration is <25%, the LC 25 The value is higher than the highest detected concentration; the determination of NOAEL involves evaluating the concentration-response relationship of abnormal effects. The abnormal phenotype caused by NOAEL should be equal to or lower than the phenotype observed in the solvent and normal control. Two NOAEL values ​​were observed at 2 dpf and 5 dpf respectively;

[0015] 5) Developmental toxicity morphological assessment: The developmental toxicity of zebrafish was assessed at 2 dpf and 5 dpf. After the sample exposure treatment, the zebrafish were fixed with 3% methylcellulose, and morphological abnormalities were photographed using a stereo microscope camera or image acquisition system for developmental toxicity morphological assessment;

[0016] 6) Calculation of teratogenicity and teratogenicity index (TI), the specific steps are:

[0017] Step A: Classical TI calculation

[0018] Based on NOAEL and LC at 5 dpf 25 value, calculate TI, that is, LC 25 / NOAEL ratio. In the classical scheme, compounds are classified into nonteratogens and teratogens according to their TI values ​​at 5 dpf;

[0019] Step B: Optimize the TI calculation (only for uncertain suspected test substances)

[0020] The optimization of the classic scheme includes calculating additional TI values ​​at 2 dpf, adding a new category of unclassified compounds, narrowing the test concentration range of certain compounds determined in the initial test, and proposing new teratogenicity judgment criteria. Furthermore, the developmental toxicity assessment includes the evaluation of tissues and organs, including heart, circulation, hemorrhage / thrombosis, head, pharyngeal arch / jaw, eyes, sac / otoliths, liver, kidney, swim bladder, intestine, notochord / somite / tail, trunk muscle, pectoral fins and body pigmentation.

[0021] Furthermore, in step A), a TI value of ≥10 at 5 dpf indicates that the compound is predicted to induce teratogens; a TI value of <10 indicates that the compound is predicted to be non-teratogens; and an LC value cannot be determined because the concentration is >1000 μM. 25 Compounds with a low / NOOAEL ratio were predicted to be non-teratogens.

[0022] Furthermore, the optimized teratogenicity judgment criteria in step B) are:

[0023] (1) Calculation method based on the classical scheme

[0024] At 2 dpf or 5 dpf, TI values ​​≥ 10 represented teratogenic compounds; to reduce possible false-negative results, compounds that showed obvious morphological abnormalities in zebrafish but had TI values ​​< 10 were classified as unclassified compounds;

[0025] (2) Unclassified and false positive compounds identified in the classical protocol were further retested using an optimized smaller test concentration range to obtain more accurate LC 25 and NOAEL; OriginPro 8.0 software was used to calculate the concentration-mortality curve and obtain the LC at 2dpf and 5dpf 25 and NOAEL to obtain more precise TI values; if the TI at 2 dpf or 5 dpf was ≥ 3, these compounds were retested at the precise concentration and judged to be teratogens, otherwise they were judged to be non-teratogens.

[0026] The ICHS5(R3)-guided zebrafish drug developmental toxicity evaluation method was used as an alternative in vivo method to screen and evaluate the teratogenicity of candidate drugs.

[0027] After adding an additional 2dpf TI value to the classic 5dpf TI, the compounds that showed obvious morphological abnormalities in zebrafish but had a TI value of <10 were classified into a new category - "unclassified compounds"; for unclassified or suspected positive compounds, the test concentration range was refined, and the TI judgment value was optimized from ≥10 to ≥3. The sensitivity of the optimized zebrafish development test was increased from 74.2% (23 / 31, positive compounds were teratogenic in zebrafish) to 90.3% (28 / 31), and the overall predictability was increased from 77.8% (35 / 45) to 88.9% (40 / 45). BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a typical picture of abnormal developmental phenotype in zebrafish;

[0029] a. Typical image of dead embryos 24 hours after fertilization: no heartbeat, egg condensation (a left), and unformed body segments (a right); bf. Abnormal phenotypes 120 hours after fertilization: abnormal liver (b right, yellow L is the liver); abnormal heart (red dashed line / H is the zebrafish heart); muscle degeneration (d lower left and right, red arrow / M), intestinal development abnormalities (d lower left and right, red dashed line / In:); shortened body length (e lower); renal edema (f lower middle, red arrow / K), eye edema (d lower middle, blue arrow / E);

[0030] Figure 2 These are pictures showing developmental abnormalities in zebrafish at 5 dpf after treatment with triptolide, aconitine, and chlorogenic acid; A. Untreated; B. 0.5% DMSO solvent; C. Exposure to 0.1 μM triptolide causes renal edema, pericardial edema, and cardiovascular toxicity in zebrafish; D. Exposure to 100 μM aconitine causes renal edema and pericardial edema in zebrafish; E. Treatment with 100 μM chlorogenic acid causes swim bladder loss and delayed yolk sac absorption in zebrafish. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0032] Example: Validation of the developmental toxicity evaluation method for zebrafish using the classical and optimized protocols for 45 known compounds

[0033] Zebrafish husbandry and egg collection: Adult AB strain zebrafish were obtained from the Chinese Zebrafish Resource Center (Shanghai, China) and maintained in a light- and temperature-controlled aquaculture facility with a standard 14:10-h light / dark cycle and fed brine shrimp twice and dried flakes once a day. Four to five pairs of zebrafish were naturally mated each time. An average of 200-300 embryos were produced. Embryos were maintained at 28°C in fish water (0.2% instant sea salt added to deionized water, pH

[0034] 6.9-7.2, conductivity 480-510mS.cm-1, hardness 53.7-71.6mg / L CaCO3). Embryos were collected 4-6 hours after fertilization. At the end of the experiment, all zebrafish were euthanized by anesthesia with 0.25g / L tricaine methanesulfonate.

[0035] Selection of test compounds: The 45 positive (teratogenic) and negative (non-teratogenic) compounds used for the validation of the zebrafish developmental toxicity test method were selected according to the requirements of the ICH S5 (R3) step 2 draft (2017). The draft requires that: in order to be applicable for regulation, the alternative test method should be tested using compounds from the ICH reference compound list; at least 45 compounds in total should be tested; the reference compounds are divided into 10 categories according to the mechanism of action, and all categories should be covered when selecting compounds (at least two to three compounds in each category); the ratio of positive and negative compounds tested is approximately 2:1. . All of these compounds are ICH-approved drugs for clinical use in patients and have demonstrated developmental toxicity and teratogenicity in humans or mammals. The compounds used in this method were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Aladdin) or Wuhan Dahua Pharmaceutical Co., Ltd. (WD Pharma), China, and dimethyl sulfoxide (DMSO) was purchased from Sigma. The compounds were dissolved in 100% DMSO, and different volumes of the master solution were added directly to the test fish water at the specified test concentration, with a final DMSO concentration of 0.5% (v / v). All main solutions were prepared before each experiment and no manual adjustment was made to the pH value.

[0036] Compound exposure: The evaluation of malformation and mortality in zebrafish after exposure to compounds was conducted based on previous reports from multiple inter-laboratory studies. At 4-6 hpf, zebrafish were manually transferred to 24-well plates containing negative compounds, positive compounds, or solvent (0.5% DMSO), 1 tail / well, and 1 mL / well fish water was added, with 12 wells per group. The water-soluble exposure of zebrafish was selected to start at 4-6 hours because most unfertilized zebrafish embryos can be easily identified and removed at 4 hpf, and most zebrafish laboratories in China and Europe and the United States perform zebrafish egg washing and grading at 4-6 hpf. As reported in our previous studies and by others, the use of high-quality fertilized eggs at 4-6 hpf for developmental toxicity testing will produce more reliable results. Untreated (fish water) zebrafish were also tested. The test concentrations were set to 0.1, 1, 10, 100, and 1000 μM to evaluate the teratogenicity of the test compounds. The exposure was continuous and static, without feeding; in daily observation, the dead zebrafish were recorded and removed from the solution daily, and the morphological characteristics of each individual zebrafish were evaluated under a stereomicroscope (Nikon, SMZ645, Tokyo, Japan) at 48 hpf (2 days post fertilization, dpf) and 120 hpf (5 dpf). False positive, false negative and unclassified compounds were repeated at least once.

[0037] LC 25 Determination of NOAEL: According to the cumulative mortality at 2dpf and 5dpf, the 25% lethal concentration (LC 25 If the lethality at the highest concentration tested is <25%, the LC 25 The value is higher than the highest concentration tested. The determination of no observed adverse effect level (NOAEL) involves evaluating the concentration-response relationship for abnormal effects. The abnormal phenotypes induced at the NOAEL dose should be equal to or lower than those observed in the solvent and negative control. Two NOAEL values ​​were observed at 2 dpf and 5 dpf, respectively.

[0038] Morphological evaluation of developmental toxicity: In the original classical protocol, the developmental toxicity of zebrafish was only evaluated at 5 dpf, which resulted in a high false negative result. In the present invention, it was found that evaluating the developmental toxicity of zebrafish at 2 dpf and 5 dpf can better predict positive teratogenic compounds. After sample exposure treatment, zebrafish were fixed with 3% methylcellulose, and morphological abnormalities were photographed using a 1× DF PLAPO objective (Olympus, Japan) and a VertA1 camera (Sony Exmor CCD Sensor). The structural / organ developmental malformations observed morphologically in zebrafish are detailed in Table 2. The tissues and organs evaluated included heart, circulation, hemorrhage / thrombosis, head, pharyngeal arch / jaw, eyes, sac / otoliths, liver, kidney, swim bladder, intestine, notochord / somite / tail, trunk muscles, pectoral fins, and body pigmentation. In previous work, it was found that swim bladder loss or delayed yolk sac absorption occurred quite frequently in zebrafish embryos exposed to various drugs and environmental factors, and was not a specific developmental toxicity. Therefore, swim bladder loss and yolk sac resorption were recorded in Table 2 but were not used in the developmental toxicity assessment and TI calculation.

[0039] Table 2. Observation indexes of developmental toxicity and teratogenicity in zebrafish

[0040]

[0041] Teratogenicity and teratogenicity index calculation: Step 1 (validation of the classic scheme): based on the NOAEL and LC at 5 dpf 25 value, calculate the teratogenicity index (TI), i.e. LC 25 / NOAEL ratio. In the classic approach, compounds are classified as non-teratogens and teratogens based on their TI values ​​at 5 dpf. A TI value ≥ 10 at 5 dpf indicates that the compound is predicted to be a teratogen; if the TI value is < 10, the compound is predicted to be a non-teratogen; the LC cannot be determined because the concentration is > 1000 μM 25 Compounds with a low / NOOAEL ratio were predicted to be non-teratogens.

[0042] Step 2 (validation of the optimized protocol): The optimization of the classical protocol included calculating additional TI values ​​at 2 dpf, adding a new category of unclassified compounds, narrowing the test concentration range for some compounds identified in the classical protocol testing, and proposing new teratogenicity judgment criteria.

[0043] (1) Calculation method based on the classical scheme: At 2dpf or 5dpf, TI values ​​≥ 10 represent teratogenic compounds. To reduce possible false negative results, compounds that show obvious morphological abnormalities in zebrafish but TI values ​​< 10 are classified as unclassified compounds, whose toxicity may mask the teratogenic potential and require further confirmation of their teratogenicity.

[0044] (2) Unclassified or false positive compounds identified in the classical protocol were further retested using an optimized smaller test concentration range to obtain more accurate LC 25 and NOAEL. Ceritinib and warfarin were retested at concentrations of 5, 10, 15, 20, 25 μM and 10, 25, 50, 75, 100 μM, respectively. Cyproheptadine hydrochloride was retested at 5, 10, 25, 50, 100 μM and cyclobenzaprine hydrochloride was retested at 62.5, 125, 250, 500, 1000 μM, respectively. In addition, since the positive teratogenic compound aspirin showed cardiovascular toxicity at 2 dpf, but this toxicity was completely restored at 5 dpf, its concentration was set at 10, 25, 50, 75, and 100 μM. The concentration-mortality curve was calculated using OriginPro 8.0 software to obtain the LC at 5 dpf. 25 If the TI at 2dpf or 5dpf was ≥ 3, these compounds were retested at the exact concentration and judged to be teratogens, otherwise they were judged to be non-teratogens.

[0045] Comparison with human data: The ICH reference compound list is based on data available in 2017 and is derived from human drug reproductive toxicity test data. Further literature on the developmental toxicity of compounds was retrieved from databases such as PubMed (Medline) and the Reproductive Developmental Toxicology Database and the Hazardous Substances Database. The results of the zebrafish developmental toxicity test were compared with the human teratogenicity data in the ICH Harmonized Guideline S5 (R3) to obtain the overall predictability of zebrafish developmental toxicity and teratogenicity evaluation. In traditional mammalian studies (rats, mice, rabbits, etc.), a compound is considered a developmental toxicant (teratogen) if it exhibits one of the following four manifestations: functional defects, growth changes, structural abnormalities, or death. The literature shows that for most compounds, teratogenicity data for laboratory animal development from different strains of a species or multiple species are available. The overall predictability of the zebrafish developmental toxicity evaluation results was obtained by calculating the sensitivity (teratogens) and specificity (non-teratogens) between human / mammal and zebrafish data.

[0046] Data analysis: The mortality data were imported into Origin (OriginPro, version 8.0, 2007), curve fitting was performed, concentration-effect curves were generated, and NOAEL and LC were calculated. 25values ​​and TI values. Overall agreement was used to evaluate the performance of the analytical method. True positive (Y) and true negative (N) compounds are compounds whose zebrafish teratogenicity classification is consistent with the mammalian data. False positive (FP) and false negative (FN) are compounds whose zebrafish teratogenicity classification is inconsistent with the mammalian results. Unclassified (U) refers to compounds that induce visible malformations(s) in zebrafish, but have a TI value of <10. The analysis included the determination of the following endpoints: Teratogen detection sensitivity = Y / (Y+FN+U)×100%; non-teratogen detection specificity = N / (N+FP)×100%; overall agreement = (Y+N) / 45×100%.

[0047] result:

[0048] Validation and evaluation of developmental toxicity and teratogenicity of ICH reference compounds using the classic zebrafish model

[0049] The results in Tables 3, 4 and 5 show that, according to the results of the evaluation using the "before optimization" classical scheme, among the 31 ICH-positive compounds, 23 compounds (74.2%) were teratogenic in zebrafish, and 8 compounds were false negative results; among the 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish, and 2 compounds had false positive results, with an overall consistency of 77.8% (35 / 45).

[0050] Optimization of the zebrafish model to validate and evaluate the developmental toxicity and teratogenicity of ICH reference compounds

[0051] In general, the results in Tables 3, 4 and 5 show that, among the 31 ICH-positive compounds evaluated using the "optimized" scheme, 28 compounds (90.3%) were teratogenic in zebrafish, and 3 compounds were false-negative results; among the 14 ICH-negative compounds, 12 compounds (85.7%) were non-teratogenic in zebrafish, and 2 compounds had false-positive results, with an overall consistency of 88.9% (40 / 45).

[0052] Table 3. Developmental toxicity and teratogenicity results of positive (teratogenic) compounds in zebrafish

[0053]

[0054] Y = false positive, FN = false positive, and U = unclassified

[0055] √: correct prediction; ×: wrong prediction.

[0056] (U): Unclassified compounds were judged based on the TI values ​​of optimization step 2 only.

[0057] *: False negative compounds in the original TI (5 dpf ) and add 2 dpf The TI was later corrected as a teratogenic compound.

[0058] #: False negative or unclassified compounds were finally determined as teratogenic compounds after optimizing concentration and TI parameters.

[0059] Table 4. Developmental toxicity and teratogenicity results of negative (non-teratogenic) compounds in zebrafish

[0060]

[0061] N = non-teratogenic. FP = false positive.

[0062] √: correct prediction; ×: wrong prediction.

[0063] Table 5. Comparison of the prediction accuracy of developmental toxicity and teratogenicity before and after optimization

[0064]

[0065] Optimize detection time and define unclassified compounds

[0066] The present invention found that although the TI value of acitretin was 1 at 5 dpf, the TI value at 2 dpf was >1000, which had obvious teratogenicity and was ultimately determined to be a teratogen; the TI values ​​of aspirin, acitretin and isotretinoin at 2 dpf were respectively greater than 10, 1000 and 100, therefore, they were all reclassified as teratogens, while the TI values ​​of cytarabine and ribavirin at 2 dpf or 5 dpf were both false negatives.

[0067] The toxicity of ceritinib was manifested by swim bladder loss at 10 μM, 100% mortality at 100 and 1000 μM, and TI less than 10; warfarin caused 75% swim bladder loss and 100% delayed yolk sac absorption at 10 μM, 100% mortality at 100 and 1000 μM, and TI less than 10; hydroxyurea caused 100% swim bladder loss at 1000 μM, but the TI was also less than 10. Therefore, based on the fact that the TI values ​​of hydroxyurea, warfarin and ceritinib at 2 dpf and 5 dpf were all less than 10, but all induced morphological abnormalities, they were all classified as unclassified compounds (Table 3).

[0068] Test concentration refinement and TI value optimization

[0069] The optimal concentration and TI values ​​of 1 positive compound, 2 unclassified compounds and 2 false positive compounds were further determined. In the refined concentration test, the TI value of the positive compound aspirin was optimized to 3.9. At 25μM, 1 zebrafish showed pericardial edema, delayed yolk sac absorption, and swim bladder loss; 5 zebrafish showed delayed yolk sac absorption, swim bladder loss, cardiovascular toxicity and renal edema; at 75μM, 12 zebrafish died. After the concentration was refined and the curve was calculated, the TI values ​​of the unclassified compounds ceritinib and warfarin were 3.5 and 23.9, respectively. The developmental toxicity phenotype of ceritinib showed that 3 zebrafish had delayed yolk sac absorption and swim bladder loss at 15μM, and 12 zebrafish died at 20mM. Warfarin produced cardiovascular toxicity at 10 μM; 4 zebrafish died at 50 mM; at 5 dpf, the TI value of zebrafish treated with ceritinib was less than 10 but greater than 3, and that of warfarin was greater than 10. If the TI cutoff value is lowered from 10 to 3 based on the new results of the refined concentration experiment, ceritinib and warfarin are redefined as teratogens from unclassified compounds.

[0070] At the refined test concentration, the TI values ​​of the two false positive compounds, cyproheptadine hydrochloride and cyclobenzaprine hydrochloride, were 13.8 and 11.7, respectively. Obviously, even in the optimized concentration test, the TI values ​​of cyproheptadine hydrochloride and cyclobenzaprine hydrochloride were still ≥10, so they were both true positives in the zebrafish developmental toxicity test.

[0071] in conclusion:

[0072] After using the optimized method of the present invention, the sensitivity of the zebrafish test for detecting teratogens was significantly improved from 74.2% (23 / 31) before optimization to 90.3% (28 / 31), and the overall predictability of all positive and negative compounds was improved from 77.8% (35 / 45) before optimization to 88.9% (40 / 45).

[0073] Application example: Evaluation of developmental toxicity and teratogenicity of three unknown compounds in zebrafish

[0074] The compounds selected for the application test were triptolide (Batch No.: Y29S9H71524; Shanghai Yuanye Biotechnology Co., Ltd.), aconitine (Batch No. 00001340-718; Chromadex, USA), and chlorogenic acid (Batch No. ZZS19041904; Shanghai Pearl Biotechnology Co., Ltd., China). These three compounds are not included in the ICH reference compound list, and their developmental toxicity and teratogenicity in zebrafish are still unclear.

[0075] The optimized scheme and standards developed in the present invention were used to test the developmental toxicity and teratogenicity of the above three unknown compounds in zebrafish. Zebrafish were treated with 0.1, 1, 10, 100 and 1000 μM doses of triptolide, aconitine and chlorogenic acid, respectively, and their LC values ​​were calculated according to their respective lethality curves. 25 And adverse effect level (NOAEL) concentration. After obtaining the initial TI value, further refine the test concentration to obtain the optimal TI value.

[0076] result:

[0077] As shown in Table 6, Figure 2 As shown, at 2 dpf, zebrafish treated with 0.1 μM triptolide showed no compound-related toxic phenotypes and death, 12 zebrafish showed pericardial edema at 1 μM, and 12 zebrafish died at 10 μM. At 5 dpf, 0.1 μM triptolide treatment caused renal edema, pericardial edema, and cardiovascular toxicity in 12 zebrafish, 3 zebrafish died, and 9 zebrafish showed deformities at 1 μM. Therefore, LC 25 The concentration of 1 μM and NOAEL was < 0.1 μM.

[0078] In 2dpf zebrafish, aconitine induced pericardial edema and cardiovascular toxicity in 2 zebrafish, no mortality at 100μM, and no compound-related dysplasia at 10μM; no mortality at 1000μM, and pericardial edema and cardiovascular toxicity in 7 zebrafish. 25 The NOAEL was >1000μM and 10μM. At 5dpf, 10μM aconitine treatment caused renal edema and delayed yolk sac absorption in 12 zebrafish; at 100μM, 2 zebrafish died, and 10 zebrafish had renal edema and pericardial edema. LC 25 The concentration of 5-nitro-1-nitro-2 ...

[0079] In 2dpf zebrafish, at 10μM, no compound-related toxicity or death was found in chlorogenic acid treatment; at 100μM, 2 zebrafish died, and no other abnormal development was observed. 25 The NOAEL was 10 μM. At 5 dpf, no toxicity was detected at 10 μM. At 100 μM, 2 zebrafish died, 6 zebrafish had swim bladder loss, 5 zebrafish had delayed yolk sac absorption, and LC 25 The concentration of 5-nitro-2-nitro-1-oxo-2-nitro ... Figure 2 These results were further confirmed by detailed concentration experiments shown in Table 6.

[0080] Table 6. Evaluation of developmental toxicity and teratogenicity of chlorogenic acid, triptolide, and aconitine using the optimized zebrafish protocol

[0081]

[0082] in conclusion:

[0083] The TI values ​​of triptolide, aconitine, and chlorogenic acid were >10, >100, and >10, respectively, and all three compounds were classified as zebrafish teratogens.

[0084] discuss

[0085] Zebrafish, with similar physiology, morphology, and functions to mammals, have been recognized as a valuable in vivo model for toxicity and safety assessment of candidate drugs and chemicals. In recent years, zebrafish embryonic developmental toxicity testing has been reported by several laboratories in the United States and Europe, but their experimental methods, parameters, and sensitivity are not consistent (Table 1). Most zebrafish embryonic developmental toxicity testing requires 5dpf viability and morphological assessment. According to the EU Directive on the Protection of Laboratory Animals (2010), independent feeding is the stage at which zebrafish embryos are subject to animal experimentation regulations. Traditionally, hatched embryos >5dpf are considered protected because the swim bladder is inflated, free to swim, and autonomous to feed. Therefore, the design of zebrafish whole organism experiments within 5dpf meets the requirements of the relevant definition of using non-protected species.

[0086] With reference to the internationally reported zebrafish developmental toxicity and teratogenicity evaluation methods, this patent hopes to establish an optimized, rapid, and reliable alternative method that is expected to be used for new drug registration applications in China and the world. Step 1 in this patent mainly refers to the method reported by AstraZeneca et al. in 2012 and 2014 (Gustafson et al., 2012; Ball et al., 2014). This method has high throughput and saves time and cost in new drug development; however, in the validation study, the classic protocol had 8 false negatives and the correct predictability was only 74.2%. It was found that: (1) Using only the TI value at 5 dpf in the classic protocol will lead to false negative results. For example, isotretinoin, as a relatively strong teratogenic drug, causes a high proportion of zebrafish deaths at 5 dpf, thus affecting the calculation of the TI value (lower TI values ​​are judged as false negatives). In addition, it was found that the cardiovascular system of zebrafish was more sensitive to toxic substances at 2 dpf. For example, aspirin caused pericardial edema at 2 dpf, but this cardiovascular toxicity was restored at 5 dpf. (2) The classic scheme is tested within a 10-fold concentration design range. A large number of compounds can be quickly screened in one test, but certain categories of compounds with a narrow safety window are easily lost, resulting in false negative results. For example, ceritinib induces zebrafish deformities, but LC 25The TI value is quite close to the adverse event level (NOAEL) value, so it is judged as a false negative; (3) The classic scheme proposes TI ≥ 10 as the teratogenicity judgment value, which may misclassify some teratogenic positive compounds as teratogenic negative compounds.

[0087] Based on the experience in zebrafish toxicity and safety analysis and initial validation data, the present invention adds a new TI value at 2dpf. If any of the compounds has one or both TI values ​​greater than the judgment value in the evaluation at 2dpf and 5dpf, the compound is considered to induce teratogenesis. The newly added 2dpf TI helps reduce false negatives.

[0088] In the classical scheme, there are only two categories of teratogenic and non-teratogenic compounds. Some compounds with TI values ​​less than 10 at 2dpf and 5dpf but inducing toxic phenotypes do not belong to teratogenic and non-teratogenic compounds. Therefore, this patent defines these as "unclassified compounds". In further optimization studies, the multiples between each test concentration group were reduced to obtain more accurate TI values. After optimizing the TI judgment value from 10 to 3, the unclassified compounds with TI ≥ 3 were reclassified as positive (teratogenic) compounds.

[0089] After optimization, the sensitivity of the zebrafish developmental toxicity test (positive compound detection rate) increased from 74.2% (23 / 31) to 90.3% (28 / 31), and the overall consistency increased from 77.8% (35 / 45) to 88.9% (40 / 45). The predictive value of a new test method was evaluated according to the ECVAM (European Center for Validation of Alternative Methods) criteria, and the test method ranked excellent (>85%) in identifying developmental toxic and teratogenic compounds.

[0090] In addition to obtaining TI values, morphological observations of developmental toxicity and teratogenicity also play an important role in judging drug toxicity, especially drug-related morphological abnormalities. Figure 1 Common and rarely observed developmental toxic and teratogenic morphological abnormalities in zebrafish are summarized in .

[0091] In the application example, three compounds extracted from traditional Chinese medicine that were not approved by ICH (triptolide, chlorogenic acid, and aconitine) were selected to evaluate the applicability of the optimization scheme for developmental toxicity and teratogenicity in zebrafish. Triptolide has an effect on the reproductive system of humans and male mice; aconitine induces embryo toxicity in vitro in rats, and the developmental toxicity of chlorogenic acid is still unclear. Experimental data show that these three compounds all induce malformations in zebrafish. The results of triptolide and aconitine in zebrafish are highly consistent with the literature.

Claims

1. An optimized evaluation method for the developmental toxicity of zebrafish drugs that complies with the ICHS5 (R3) guidelines for alternative testing, characterized in that: The following steps are involved: 1) Zebrafish rearing and egg collection: Zebrafish were reared in fish culture water at 28°C; Zebrafish embryos produced by natural mating were collected at 4-6 h after fertilization, i.e., 4-6 hpf; 2) Selection of validation compounds: Select at least 45 compounds from the list of positive and negative reference compounds in the ICH S5 guidelines; the ratio of compounds used for positive detection to compounds used for negative detection in the validation compounds is 2:1; 3) Compound exposure method: 6hpf zebrafish embryos were transferred to 24-well plates containing negative compounds, positive compounds, solvent controls or normal controls for exposure, 1mL / well, 1 tail / well, and the samples were continuously treated with zebrafish for 5 days to 5dpf. During this period, the development of the embryos was observed every day and the dead zebrafish were removed in time. The morphological characteristics of each zebrafish individual were evaluated under a stereomicroscope at 48hpf (2 days after fertilization), 2dpf and 120hpf (5dpf); false positive, false negative and unclassified compounds were repeated at least once; the solvent control was 0.5% DMSO solution, and the normal control was water for fish farming; 4) 25% lethal concentration LC 25 Determination of no-observed-adverse-effect level (NOAEL): Calculate the 25% lethal concentration (LC) based on the cumulative mortality at 2 dpf and 5 dpf, respectively. 25 If the lethality at the highest detected concentration is <25%, the LC 25 The value is higher than the highest detected concentration; the determination of NOAEL involves evaluating the concentration-response relationship of abnormal effects. The abnormal phenotype caused by NOAEL should be equal to or lower than the phenotype observed in the solvent and normal control. Two NOAEL values ​​were observed at 2 dpf and 5 dpf respectively; 5) Developmental toxicity morphological assessment: The developmental toxicity of zebrafish was assessed at 2 dpf and 5 dpf. After the sample exposure treatment, the zebrafish were fixed with 3% methylcellulose, and morphological abnormalities were photographed using a stereo microscope camera or image acquisition system for developmental toxicity morphological assessment; 6) Calculation of teratogenicity and teratogenicity index (TI), the specific steps are: Step A: Classical TI calculation Based on NOAEL and LC at 5 dpf 25 value, calculate TI, that is, LC 25 / NOAEL ratio. In the classical scheme, compounds are classified into nonteratogens and teratogens according to their TI values ​​at 5 dpf; Step B: Optimize the TI calculation (only for uncertain suspected test substances) The optimization of the classical protocol included calculating additional TI values ​​at 2 dpf, adding a new category of unclassified compounds, narrowing the test concentration range for some compounds identified in the classical protocol testing, and proposing new criteria for teratogenicity judgment.

2. The method for optimizing the evaluation of zebrafish drug developmental toxicity according to the ICHS5 (R3) guidelines as claimed in claim 1, characterized in that The tissues and organs evaluated in the developmental toxicity morphological assessment included heart, circulation, hemorrhage / thrombosis, head, pharyngeal arches / jaw, eyes, otoliths, olfactory region, liver, kidney, swim bladder, intestine, notochord / somite / tail, trunk muscles, fins, body pigmentation, and yolk sac.

3. The method for optimizing the evaluation of zebrafish drug developmental toxicity according to the ICHS5 (R3) guidelines as claimed in claim 1, characterized in that In step 1), the water quality for fish farming is: 200 mg of instant sea salt is added to every 1 L of reverse osmosis water, the conductivity is 450-550 μS / cm; the pH is 6.5-8.5; and the hardness is 50-100 mg / L CaCO3.

4. The method for optimizing the evaluation of zebrafish drug developmental toxicity according to the ICHS5 (R3) guidelines as claimed in claim 1, characterized in that In the calculation step A) of teratogenicity and teratogenicity index, a TI value ≥ 10 at 5 dpf indicates that the compound is predicted to be a teratogen; a TI value < 10 indicates that the compound is predicted to be a non-teratogen; and the LC value cannot be determined because the concentration is > 1000 μM. 25 Compounds with a low / NOOAEL ratio were predicted to be non-teratogens.

5. The method for optimizing the evaluation of zebrafish drug developmental toxicity according to the ICHS5 (R3) guidelines as claimed in claim 1, characterized in that The optimized teratogenicity judgment criteria in the teratogenicity and teratogenicity index calculation step B) are: (1) Calculation method based on the classical scheme At 2 dpf or 5 dpf, TI values ​​≥ 10 represented teratogenic compounds; to reduce possible false-negative results, compounds that showed obvious morphological abnormalities in zebrafish but had TI values ​​< 10 were classified as unclassified compounds; (2) Unclassified or false positive compounds identified in the classical protocol were further retested using an optimized smaller test concentration range to obtain more accurate LC 25 and NOAEL; OriginPro 8.0 software was used to calculate the concentration-mortality curve and obtain the LC at 2dpf and 5dpf 25 and NOAEL to obtain more accurate TI values; If the TI at 2 dpf or 5 dpf was ≥ 3, these compounds were retested at the exact concentration and judged as teratogens, otherwise they were judged as non-teratogens.

6. The application of the zebrafish drug developmental toxicity optimization evaluation method as described in any one of claims 1-5 that complies with the ICHS5 (R3) guidelines for alternative testing requirements in the zebrafish model as an alternative method to the traditional mammalian in vivo embryo-fetal developmental toxicity test (EFD) to screen and evaluate the developmental toxicity and teratogenicity of candidate drugs.

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