By occupying the proline and tRNA binding sites of prolyl-tRNA synthetase, halofuginone competitively inhibits prolyl-tRNA synthetase [1]. In KYSE70 and A549 cells, halofuginone (1, 10, 100, 1000, and 10,000 nM; 48 hours) had an IC50 of 114.6 and 58.9 nM, respectively. In KYSE70 and A549 cells, the IC50 of halofuginone (1, 10, 100, and 1000 nM; 24 hours) against NRF2 protein was 22.3 and 37.2 nM, respectively. In KYSE70 and A549 cells, halofuginone's IC50 values for total protein synthesis were 22.6 and 45.7 nM, respectively [1]. Halofuginone raises K+ currents via KCNA5 channels in HEK cells transfected with the KCNA5 gene and voltage-gated K+ (Kv) currents in pulmonary artery smooth muscle cells (PASMC). In HEK cells transfected with calcium-sensing receptor genes, halofuginone (0.03-1μM) suppresses receptor-operated Ca2+ entry (ROCE), and in PASMC, it attenuates store-operated Ca2+ entry (SOCE) [5].

In mice with anterior cruciate ligament transection (ACLT), halofuginone (0.2, 0.5, 1, or 2.5 mg/kg; i.p. every other day for one month) attenuates the progression of osteoarthritis (OA). Lower concentrations (0.2 or 0.5 mg/kg) have minimal effects on subchondral bone, and higher concentrations (2.5 mg/kg) cause loss of proteoglycans in articular cartilage [3]. Halofuginone (0.25 mg/kg; i.p.; daily; 16 days) reduces NRF2 protein levels in tumors; however, there were no significant differences in tumor volume between treatments with vehicle, halogenone (0.25 mg/kg, daily intraperitoneal injection), or cisplatin alone. Combination treatment with halopentone and cisplatin significantly suppressed tumor volume compared with halopentone or cisplatin alone [1]. Halopentide (0.3 mg/kg) was administered intraperitoneally every two weeks.

Cell viability assay [1]
Cell Types: from human esophageal cancer KYSE70 cells carrying NRF2 gene mutations and A549 cells carrying KEAP1 gene mutations
Tested Concentrations: 1, 10, 100, 1000, 10000 nM
Incubation Duration: 48 hrs (hours)
Experimental Results: IC50 KYSE70 and The concentrations in A549 cells were 114.6 and 58.9 nM, respectively.

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Western Blot Analysis [1]
Cell Types: KYSE70 cells derived from human esophageal cancer contain NRF2 gene mutations; A549 cells contain KEAP1 gene mutations.
Tested Concentrations: 1, 10, 100, 1000 nM
Incubation Duration: 24 hrs (hours)
Experimental Results: The IC50 of NRF2 protein in KYSE70 and A549 cells were 22.3 and 37.2 nM respectively.

Animal/Disease Models: Male nude mice (BALB/C nu/nu (nude) mice) (6-8 weeks) [1]
Doses: 0.25 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily; 16 days
Experimental Results: Combined treatment with cisplatin demonstrated significant Dramatically inhibited tumor volume. NRF2 protein levels were indeed diminished in the tumors.

Absorption, Distribution and Excretion
Readily bioavailable and rapidly absorbed following oral administration.

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Biological Half-Life
23.8 to 72.1 hours

[1]. Tsuchida K, et al. Halofuginone enhances the chemo-sensitivity of cancer cells by suppressing NRF2 accumulation. Free Radic Biol Med. 2017 Feb;103:236-247.
[2]. Keller TL, et al. Halofuginone and other Febrifugine derivatives inhibit prolyl-tRNA synthetase. Nat Chem Biol. 2012 Feb 12;8(3):311-7.
[3]. Cui Z, et al. Halofuginone attenuates osteoarthritis by inhibition of TGF-β activity and H-type vessel formation in subchondral bone. Ann Rheum Dis. 2016 Sep;75(9):1714-21.
[4]. Tracy L McGaha, et al. Halofuginone, an inhibitor of type-I collagen synthesis and skin sclerosis, blocks transforming-growth-factor-beta-mediated Smad3 activation in fibroblasts. J Invest Dermatol. 2002 Mar;118(3):461-70.
[5]. Pritesh P Jain, et al. Halofuginone, a Promising Drug for Treatment of Pulmonary Hypertension. Br J Pharmacol. 2021 Mar 10.

Halofuginone is a low molecular weight quinazolinone alkaloid, and a potent inhibitor of collagen alpha1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also effectively suppresses tumor progression and metastasis in mice. Collgard Biopharmaceuticals is developing halofuginone for the treatment of scleroderma and received orphan drug designation from the U.S. Food and Drug Administration in March, 2000.
Halofuginone has been reported in Hydrangea febrifuga with data available.
Halofuginone is an orally-active quinazolinone alkaloid with potential antineoplastic activity. Halofuginone interferes with the signaling pathway of transforming growth factor beta (TGF beta) and inhibits expression of matrix metalloproteinase 2, thereby inhibiting collagen type I synthesis and inducing extracellular matrix degradation, resulting in inhibition of angiogenesis, tumor growth, or metastasis.

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Drug Indication
For the treatment of scleroderma, cancer, and restenosis.
In new born calves: - Prevention of diarrhoea due to diagnosed Cryptosporidium parvum, in farms with history of cryptosporidiosis,Administration should start in the first 24 to 48 hours of age- Reduction of diarrhoea due to diagnosed Cryptosporidium parvum. Administration should start within 24 hours after the onset of diarrhoea. In both cases, the reduction of oocysts excretion has been demonstrated.
In newborn calves: Prevention of diarrhoea due to diagnosed Cryptosporidium parvum infection, in farms with history of cryptosporidiosis. Administration should start in the first 24 to 48 hours of age. Reduction of diarrhoea due to diagnosed Cryptosporidium parvum infection. Administration should start within 24 hours after the onset of diarrhoea. In both cases, the reduction of oocysts excretion has been demonstrated.
In newborn calvesPrevention of diarrhoea due to diagnosed Cryptosporidium parvum in farms with history of cryptosporidiosis. Administration should start in the first 24 to 48 hours of age. Reduction of diarrhoea due to diagnosed Cryptosporidium parvum. Administration should start within 24 hours after the onset of diarrhoea. In both cases, the reduction of oocyst excretion has been demonstrated.

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Mechanism of Action
Halofuginone is a potent inhibitor of collagen a1(I) and matrix metalloproteinase 2 (MMP-2) gene expression. Halofuginone also suppresses extracellular matrix deposition and cell proliferation. The profound antitumoral effect of halofuginone is attributed to its combined inhibition of the tumor stromal support, vascularization, invasiveness, and cell proliferation.

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Pharmacodynamics
Halofuginone, a fully synthetic small molecule, is a potent and selective regulator of stromal cell activation, cell migration and Collagen type I synthesis, a process that has been identified as a 'master switch' in the body's tissue repair process.

413.014

55837-20-2

Halofuginone hydrobromide;64924-67-0

456390

White to off-white solid powder

1.7±0.1 g/cm3

595.8±60.0 °C at 760 mmHg

>150ºC dec.

314.1±32.9 °C

0.0±1.8 mmHg at 25°C

1.712

1.24

2

5

4

24

533

2

C1C[C@@H]([C@H](NC1)CC(=O)CN2C=NC3=CC(=C(C=C3C2=O)Cl)Br)O

LVASCWIMLIKXLA-CABCVRRESA-N

InChI=1S/C16H17BrClN3O3/c17-11-6-13-10(5-12(11)18)16(24)21(8-20-13)7-9(22)4-14-15(23)2-1-3-19-14/h5-6,8,14-15,19,23H,1-4,7H2/t14-,15+/m1/s1

7-bromo-6-chloro-3-[3-[(2R,3S)-3-hydroxypiperidin-2-yl]-2-oxopropyl]quinazolin-4-one

RU 19110 Halofuginone TempostatinRU19110 RU-19110

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)

DMSO : ~20 mg/mL (~48.23 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.82 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 20.0 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 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 0.67 mg/mL (1.62 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 6.7 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.)