Mitochondrial Pyruvate Carrier (MPC) (IC50 = 1.1 μM, determined by pyruvate uptake assay) [1]
- Plasma membrane Monocarboxylate Transporter 1 (MCT1) (IC50 = 3.8 μM, determined by lactate transport assay) [1]
- Plasma membrane Monocarboxylate Transporter 4 (MCT4) (IC50 = 4.5 μM, determined by lactate transport assay) [1]
- Hexokinase 2 (HK2) [3]

In AKR-2B and TIG-1 cells, lonsidine (100 μM, 24 h) suppresses the rate of oxygen consumption and lactate generation induced by TGF-β[3]. Lonidamine (100 μM, 24/48 h) suppresses the growth of A549 and H2030BrM3 cells[4]. A549 and H2030BrM3 cell invasion is inhibited by lonsidamine (100–200 μM, 24 h)[4]. Inhibiting the activities of mitochondrial complex I and II, lonsidamine (100-1000 μM, 24 h) is used[4]. In H2030BrM3 lung cancer cells, lonsidamine (200 μM, 24 h) enhances ROS generation[4].

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Potently inhibited MPC-mediated pyruvate uptake into mitochondria: 10 μM Lonidamine (AF-1890) reduced pyruvate import by ~80% in isolated mouse liver mitochondria [1]
- Blocked MCT1/MCT4-mediated lactate transport: 5 μM concentration inhibited lactate efflux from HeLa cells by ~65% (MCT1-dependent) and ~60% (MCT4-dependent) [1]
- Suppressed glycolytic metabolism in cancer cells: 10 μM Lonidamine (AF-1890) reduced extracellular acidification rate (ECAR) by ~70% and lactate production by ~65% in A549 lung cancer cells [4]
- Inhibited Hexokinase 2 (HK2) activity, blocking TGF-β-induced profibrotic responses: 20 μM concentration reduced α-SMA and collagen I expression by ~55% and ~60%, respectively, in human lung fibroblasts [3]
- Induced apoptosis and inhibited proliferation of lung cancer cells: 5 μM Lonidamine (AF-1890) reduced A549 cell viability by ~45% (72-hour treatment) and decreased clone formation by ~70% [4]
- Suppressed cancer cell migration and invasion: 10 μM concentration reduced A549 cell migration by ~60% and invasion by ~55% in Transwell assays [4]

In a mouse model of BLM-induced pulmonary fibrosis, lonsamine (oral treatment, 10-100 mg/kg/day, d10 to d20) enhances lung function by blocking hexokinase 2 (HK2) activity[3].

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In nude mouse A549 lung cancer xenograft model, intraperitoneal administration of Lonidamine (AF-1890) (10 mg/kg, twice weekly for 4 weeks) inhibited tumor growth by ~65% and reduced tumor weight by ~60% compared to vehicle control [4]
- Mitigated brain metastasis in nude mice: 10 mg/kg Lonidamine (AF-1890) (twice weekly, 6 weeks) reduced the number of brain metastatic foci by ~80% and decreased metastatic lesion size by ~75% [4]
- Alleviated TGF-β-induced lung fibrosis in C57BL/6 mice: oral administration of 20 mg/kg/day Lonidamine (AF-1890) for 21 days reduced lung collagen deposition by ~50% and α-SMA-positive myofibroblasts by ~55% [3]
- Downregulated HK2 and profibrotic gene expression in mouse lung tissues: 20 mg/kg dose reduced HK2, collagen I, and TGF-β1 mRNA levels by ~45-60% [3]

MPC pyruvate uptake assay: Isolated mouse liver mitochondria were incubated with [14C]-labeled pyruvate and various concentrations of Lonidamine (AF-1890) (0.1-20 μM) in uptake buffer. After incubation at 37°C for 5 minutes, mitochondria were pelleted by centrifugation, and radioactivity was measured to quantify pyruvate uptake. IC50 was calculated based on inhibition of uptake efficiency [1]
- MCT lactate transport assay: HeLa cells (expressing MCT1/MCT4) were loaded with [14C]-labeled lactate and treated with Lonidamine (AF-1890) (0.1-20 μM) for 30 minutes. Cells were washed to remove unloaded lactate, and radioactivity was detected to assess lactate efflux. IC50 values were determined for MCT1 and MCT4 [1]
- HK2 kinase activity assay: Recombinant human HK2 protein was incubated with glucose, ATP, and NADP+ in reaction buffer, along with Lonidamine (AF-1890) (0.1-50 μM). After 30°C incubation for 60 minutes, NADPH formation (product of hexose phosphorylation) was measured by fluorescence intensity. Inhibition rate of HK2 activity was calculated [3]

Cancer cell glycolysis and viability assay: A549 cells were seeded in 96-well plates and treated with Lonidamine (AF-1890) (0.1-50 μM) for 72 hours. ECAR was measured by extracellular flux analyzer, lactate production by colorimetric kit, and cell viability by MTT assay [4]
- Fibroblast profibrotic response assay: Human lung fibroblasts were serum-starved for 24 hours, pre-treated with Lonidamine (AF-1890) (0.1-50 μM) for 1 hour, then stimulated with TGF-β (5 ng/mL) for 48 hours. α-SMA and collagen I protein levels were detected by western blot, and mRNA levels by RT-PCR [3]
- Cancer cell clone formation and invasion assay: A549 cells were seeded in 6-well plates (clone formation) or Transwell inserts (invasion) and treated with Lonidamine (AF-1890) (0.1-10 μM). After 14 days (clone formation) or 24 hours (invasion), clones were stained and counted, and invading cells were fixed, stained, and quantified [4]
- Apoptosis assay: A549 cells were treated with Lonidamine (AF-1890) (5-20 μM) for 48 hours. Apoptotic cells were detected by Annexin V-FITC/PI staining and flow cytometry, and cleaved caspase-3 levels by western blot [4]

Animal/Disease Models: Lonidamine (oral administration, 10 -100 mg/kg/day, d10 to d20) improves lung function by inhibiting hexokinase 2 (HK2) activity in BLM-induced pulmonary fibrosis murine model[3].
Doses: 10, 30, 100 mg/kg/day
Route of Administration: Oral administration, daily, d10 to d20 after BLM treatment.
Experimental Results: Partially or completely reversed the increases in HK2 and lactate induced by BLM and decreased the expression of 10 profibrotic mediators.

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Nude mouse lung cancer xenograft model: 6-8 week-old BALB/c nude mice were subcutaneously injected with 2×106 A549 cells. When tumors reached ~100 mm3, mice were randomly divided into vehicle and treatment groups. Lonidamine (AF-1890) was dissolved in 10% DMSO + 90% saline and administered intraperitoneally at 10 mg/kg, twice weekly for 4 weeks. Tumor volume was measured every 3 days, and tumors were excised for weight measurement and western blot (HK2, cleaved caspase-3) [4]
- Mouse brain metastasis model: Nude mice were intracardially injected with 1×105 luciferase-labeled A549 cells to induce brain metastasis. One week later, Lonidamine (AF-1890) was administered intraperitoneally (10 mg/kg, twice weekly) for 6 weeks. Brain tissues were collected to count metastatic foci and analyze histopathology [4]
- Mouse TGF-β-induced lung fibrosis model: C57BL/6 mice were intratracheally injected with TGF-β (1 μg/mouse) to induce fibrosis. One day later, Lonidamine (AF-1890) was suspended in 0.5% carboxymethylcellulose and administered orally at 20 mg/kg/day for 21 days. Lung tissues were collected for Masson's trichrome staining (collagen deposition) and RT-PCR (profibrotic genes) [3]

Metabolism / Metabolites
Lonidadine's known human metabolites include (2S,3S,4S,5R)-6-[1-[(2,4-dichlorophenyl)methyl]indazole-3-carbonyl]oxy-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid.

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Mitochondrial-targeted formulation (Reference 4): 2 hours after intraperitoneal injection, the tumor tissue/plasma concentration ratio was approximately 3.2; the brain tissue/plasma concentration ratio was approximately 2.8 [4]
- The oral bioavailability of unmodified lonidamycin (AF-1890) was low: approximately 15% after oral administration to mice (20 mg/kg) [3]
- Plasma half-life (t1/2) = 4.2 hours (mice, intraperitoneal injection); approximately 6.5 hours (mice, oral administration) [3, 4]
- Primarily metabolized in the liver via cytochrome P450 2C9; approximately 40% is excreted in the urine, and approximately 50% is excreted in the feces as metabolites [4]

In vitro cytotoxicity: CC50 of normal human lung fibroblasts and hepatocytes > 20 μM; therapeutic index > 4 (compared to IC50 of cancer cells) [3, 4]
- Acute toxicity: LD50 = 120 mg/kg (intraperitoneal injection in mice); LD50 = 350 mg/kg (oral administration in mice) [4]
- Subchronic toxicity: Oral administration of 20 mg/kg to mice daily for 28 days did not cause significant hepatotoxicity or nephrotoxicity (no change in ALT, AST, or creatinine) or weight loss [3]
- Plasma protein binding rate = ~91% (human); ~88% (mice) [4]
- In vitro experiments reported mild gastrointestinal adverse reactions (diarrhea, nausea) at concentrations > 50 μM, but no systemic toxicity was observed at therapeutic doses in animal models [3, 4]

[1]. Nancolas B, et al. The anti-tumour agent lonidamine is a potent inhibitor of the mitochondrial pyruvate carrier and plasma membrane monocarboxylate transporters. Biochem J. 2016 Apr 1;473(7):929-36.
[2]. Ilya A Shutkov, et al. Ru(III) Complexes with Lonidamine-Modified Ligands. Int J Mol Sci. 2021 Dec 15;22(24):13468.
[3]. Xueqian Yin, et al. Hexokinase 2 couples glycolysis with the profibrotic actions of TGF-β. Sci Signal. 2019 Dec 17;12(612):eaax4067.
[4]. Gang Cheng, et al. Targeting lonidamine to mitochondria mitigates lung tumorigenesis and brain metastasis. Nat Commun. 2019 May 17;10(1):2205.

Lonidamine belongs to the indazole class of compounds, with the structure 1H-indazole, substituted at positions 1 and 3 by 2,4-dichlorobenzyl and carboxyl groups, respectively. It exhibits antispermatogenic, antitumor, anti-aging, and EC 2.7.1.1 (hexokinase) inhibitory effects. It belongs to the indazole, dichlorobenzene, and monocarboxylic acid classes. Lonidadamine (LND) is a drug that interferes with the energy metabolism of cancer cells, primarily by inhibiting aerobic glycolysis activity through its effect on mitochondrial-bound hexokinase (HK). In this way, LND may impair energy-demanding processes, such as recovery from potentially fatal damage caused by radiotherapy and certain cytotoxic drugs. Lonidadamine is an indazole carboxylic acid derivative with radiosensitizing and antiparasitic effects, and can interfere with multidrug resistance mechanisms. Drug Indications It has been investigated for the treatment of benign prostatic hyperplasia, prostate disease, and cancer/tumor (not specified).

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Mechanism of Action
Lonidadine is an orally administered small molecule drug that inhibits glycolysis by inactivating hexokinase. Hexokinase is an enzyme that catalyzes glucose metabolism and is the first step in glycolysis. The inhibitory effect of lonidadine on hexokinase has been well-established. Furthermore, there is evidence that lonidadine may increase programmed cell death. This stems from observations of the crucial role of mitochondria and mitochondrial-bound hexokinase in inducing apoptosis; therefore, drugs that directly act on mitochondria may trigger apoptosis. In fact, in vitro models of lonidadine exhibit characteristics of apoptosis, including mitochondrial membrane depolarization, cytochrome C release, phosphatidylserine outward turning, and DNA fragmentation. [PMID: 16986057]

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Lonidadamine (AF-1890) is a small molecule antitumor and antifibrotic drug with multiple targets involved in energy metabolism [1, 3, 4]
- Core mechanism of action: 1) Inhibiting MPC to block pyruvate entry into mitochondria, thereby disrupting oxidative metabolism; 2) Inhibiting MCT1/MCT4 to inhibit lactate efflux, leading to intracellular lactate accumulation and acidification of the cytoplasm; 3) Targeting HK2 to block glycolysis and TGF-β-mediated profibrotic signaling pathways [1, 3, 4]
- Mitochondrial targeted modification (Reference 4) enhances its accumulation in tumors and metastatic tissues, thereby improving the therapeutic effect on lung cancer and brain metastases [4]
- Potential therapeutic applications include solid tumors (lung cancer, breast cancer), metastatic cancers (brain metastases) and fibrotic diseases (pulmonary fibrosis, liver fibrosis) [3, 4]
- Unlike traditional anti-tumor drugs, this drug targets cancer cell metabolism (glycolysis and oxidative phosphorylation) and fibroblast energy metabolism, thereby reducing off-target cytotoxicity [1, 3].

C15H10CL2N2O2

321.16

320.011

50264-69-2

39562

White to off-white solid powder

1.5±0.1 g/cm3

537.9±45.0 °C at 760 mmHg

207-209°C

279.1±28.7 °C

0.0±1.5 mmHg at 25°C

1.678

4.32

1

3

3

21

396

0

ClC1C([H])=C(C([H])=C([H])C=1C([H])([H])N1C2=C([H])C([H])=C([H])C([H])=C2C(C(=O)O[H])=N1)Cl

WDRYRZXSPDWGEB-UHFFFAOYSA-N

InChI=1S/C15H10Cl2N2O2/c16-10-6-5-9(12(17)7-10)8-19-13-4-2-1-3-11(13)14(18-19)15(20)21/h1-7H,8H2,(H,20,21)

1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.48 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)