Preclinical efficacy against acute myeloid leukaemia of SH1573, a novel mutant IDH2 inhibitor approved for clinical trials in China

Abstract

Acute myeloid leukaemia (AML) is the most common form of acute leukaemia in adults, with increasing incidence with age and a generally poor prognosis. Almost 20% of AML patients express mutant isocitrate dehydrogenase 2 (mIDH2), which leads to the accumulation of the carcinogenic metabolite 2-hydroxyglutarate (2-HG), resulting in poor prognosis. Thus, global institutions have been working to develop mIDH2 inhibitors. SH1573 is a novel mIDH2 inhibitor that we independently designed and synthesised. We have conducted a comprehensive study on its pharmacodynamics, pharmacokinetics and safety. First, SH1573 exhibited a strong selective inhibition of mIDH2 R140Q protein, which could effectively reduce the production of 2-HG in cell lines, serum and tumors of an animal model. It could also promote the differentiation of mutant AML cell lines and granulocytes in PDX models. Then, it was confirmed that SH1573 possessed characteristics of high bioavailability, good metabolic stability and wide tissue distribution. Finally, toxicological data showed that SH1573 had no effects on the respiratory system, cardiovascular system and nervous system, and was genetically safe. This research successfully promoted the approval of SH1573 for clinical trials (CTR20200247). All experiments demonstrated that, as a potential drug against mIDH2 R140Q acute myeloid leukaemia, SH1573 was effective and safe.

Keywords: 2-HG, 2-hydroxyglutaric acid; 2-Hydroxyglutarate; ADME, absorption, distribution, metabolism and excretion; AG-221, enasidenib; AML, acute myeloid leukemia; AUC, area under the cure; Acute myeloid leukaemia; BCRP, breast cancer resistance protein; CDX, cell-line-derived xenograft; CYP, cytochrome P450; Differentiation; EPO, erythropoietin; IC50, half maximal inhibitory concentration; LC–MS/MS, liquid chromatography–tandem mass spectrometry; MDR1, multidrug resistance protein 1; Mutant isocitrate dehydrogenase 2 (mIDH2); OAT, organic anion transporter; OATP, organic anion transporting polypeptide; OCT, organ cation transporter; PD, pharamacodynamics; PDX, patient-derived tumor xenograft; PK, pharmacokinetics; Papp, apparent permeability coefficient; Preclinical efficacy; SH1573; Tumor metabolism; mIDH2 inhibitor; mIDH2, mutant isocitrate dehydrogenase; α-KG, α-ketoglutaric acid.

Graphical abstract

Figure 1

The enzyme activity assay of SH1573. (A) Chemical structure of SH1573. (B) and (C) The molecular dynamics were evaluated using GROMACS and the ligand with lowest energy and most favorable orientation was selected. (D)–(H) The directly inhibitory effects of SH1573 and AG-221 on mIDH2 R140Q/R172K, wild-type IDH 1/2 and 23 types enzymes (AKT1, ABL1, AKT2, ALK, Aurora B, CDK 2/cycin A, CHK1, c-Kit, c-Src, EGFR, FGR, FLT3, GSK3b, JAK2, LCK, JNK2, PDGFRa, LYN, PKCa, PDGFRb, SYK, and ROCK2). All data are expressed as mean ± SD (n = 3).

Figure 2

The effects of SH1573 in vitro. (A) and (B) SH1573 reduced the production of 2-HG in TF-1 (mIDH2 R140Q), U87 (mIDH2 R140Q) and SW1353. (C) and (D) SH1573 had moderate proliferation inhibition on TF-1 (wild-type) and TF-1 (mIDH2 R140Q). (E) and (F) SH1573 reversed the cell differentiation by promote the mRNA and protein expression of homoglobin. All data are expressed as mean ± SD (n = 3); ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns: no significant difference (E).

Figure 3

The PK–PD analysis of SH1573 in subcutaneous xenograft model. (A) and (B) Mice were inoculated TF-1 (mIDH2 R140Q) cells subcutaneously and treated for once with vehicle (n = 6), 45 mg/kg SH1573 i.g. (n = 18) or 45 mg/kg AG-221 i.g. (n = 18), and LC–MS/MS was used to analysis the concentration of 2-HG, SH1573 and AG-221 in plasma or tumor. SH1573 and AG-221 reduced the 2-HG level in plasma and tumor after 6, 24 and 50 h. (C)–(E) The PK–PD analysis of SH1573 or AG-221 in plasma, tumor and in both of them (n = 6). All data are expressed as mean ± SD; ∗P < 0.05; ∗∗P < 0.01, ns: no significant difference (A and B).

Figure 4

The anti-tumor effects of SH1573 in PDX model. (A) and (B) Mice were injected primary AML cell obtained from a patient via tail vein and treated with vehicle QD p.o., 45 mg/kg AG-221 QD p.o., 5 mg/kg SH1573 QD p.o., 15 mg/kg SH1573 QD p.o. or 5 mg/kg SH1573 QD p.o. Six mice in each group was for PD analysis and nine mice was for survival analysis. The SH1573 could maintain the body weight (n = 6) and prolong survival time of mice (n = 9). (C) In peripheral blood and marrow bone, SH1573 did not significantly reduce tumor burden (n = 6). (D) SH1573 promote cell differentiation (n = 6). (E) SH1573 reduced 2-HG level (n = 6). Data are expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 versus Vehicle QD group (B–E).

Figure 5

PK process of SH1573. (A) The P_app, recovery rate and efflux rate of SH1573 detected by Caco-2 model (n = 3). (B) The binding rate of SH1573 to plasma protein of mouse, rat, monkey and human (n = 3). (C) Four metabolites of SH1573 in liver microsomes detected by LC–MS/MS. (D) SD rats were treated for once with 1 mg/kg SH1573 i.v., 5 mg/kg SH1573 i.g., 10 mg/kg SH1573 i.g. or 20 mg/kg SH1573 i.g., and plasma drug concentration was detected by LC–MS/MS (n = 6). (E) Monkeys were treated for once with 1 mg/kg SH1573 i.v., 1 mg/kg SH1573 i.g., 3 mg/kg SH1573 i.g. or 10 mg/kg SH1573 i.g., and plasma drug concentration was detected by LC–MS/MS (n = 6). (F) Rats and monkey were treated for 14 days with 5 or 1 mg/kg SH1573 i.g., respectively. Plasma drug concentration on Day 1 and Day 14 was detected by LC–MS/MS (n = 6). (G) The drug distribution in rats treated with 10 mg/kg SH1573 i.g. was detected by LC–MS/MS (n = 18). (H) The [¹⁴C]SH1573 in urine, fieces and bile of rats was traced by HPLC radio chromatography (n = 18). (I) Five metabolites were detected in plasma of monkey using LC–MS/MS (n = 6). Date are expressed as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ns: no significant difference (A).

Figure 6

Drug interaction of SH1573. (A) The IC₅₀ of SH1573 to CYPs in human liver microsomes (n = 2). (B) and (C) The inducing effect of SH1573 on CYP1A2, CYP2B6 or CYP3A4 in ZVZ, BXW and HNN cell; NC: negative control; PC: positive control (n = 3). (D) The residual quantity of SH1573 after incubating with recombinant CYP enzymes for 60 min (n = 3). (E) The inhibitory effect of SH1573 to transport activity of BCRP and MDR1 (n = 3). (F) Inhibition effect of SH1573 on transport activity of OATPs, OATs and OCT2 (n = 3). (G) Uptake ratio of BCRP and MDR1 to SH1573; LFY: Lucifer yellow carbohydrazide; NMQ: N-methyl quinidine (n = 3). Data are expressed as mean (A) and mean ± SD (B–G); ∗∗∗P < 0.001, ns (no significant difference) versus NC group (B, C); ∗∗∗P < 0.001, versus ATP group (G), ns: no significant difference.

Figure 7

Toxicity evaluation of SH1573. (A) Rats were treated for once with vehicle i.g., 12.5 mg/kg SH1573 i.g., 25 mg/kg SH1573 i.g. or 50 mg/kg SH1573 and they minute ventilation was recorded for 96 h (n = 16). (B) The potassium channel inhibition rate (n = 3) of SH1573 on HEK293 cell expressed hERG. (C) and (D) Monkeys were treated for once with vehicle i.g., 12.5 mg/kg SH1573 i.g., 25 mg/kg SH1573 i.g. or 50 mg/kg SH1573. They heart rate and mean blood pressure (MBP) were record for 480 h (n = 8). (E) Rats were treated for once with vehicle i.g., 50 mg/kg SH1573 i.g., 100 mg/kg SH1573 i.g. or 200 mg/kg SH1573 and they body weight was recorded for 14 days (n = 10). (F) and (G) The body weight and organ coefficient of monkeys (n = 4) treated for once with vehicle i.g., 50 mg/kg SH1573 i.g., 100 mg/kg SH1573 i.g. or 200 mg/kg SH1573. The organ coefficient of monkeys (n = 10) treated for 28 days with vehicle i.g., 1.5 mg/kg SH1573 i.g., 3 mg/kg SH1573 i.g. or 6 mg/kg SH1573. i.g. (H) The amount of reverse mutaion in ames test (n = 3). (I) The micronucleus rate of rats in repeated administration toxicity test (n = 3). All data are expressed as mean ± SD; ∗∗∗P < 0.001 versus Vehicle QD group (H); ns: no significant difference versus Vehicle group (I).