| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg | |||
| 25mg | |||
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| Targets |
Dihydroorotate dehydrogenase (DHODH); Prostaglandin endoperoxide H synthase (PGHS)-1 and -2 (COX-1 and COX-2).
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| ln Vitro |
For five days, lipopolysaccharide (LPS) was used to stimulate the release of immunoglobulin from mice splenocytes. IgM and IgG secretion were both reduced by laflunimus, with IC50 values of 2.5 and 2 µM, respectively. When 50 µ M of uridine was added, these values rose to 70 and 60 µ M, respectively. On 70Z/3 cells, laflunimus inhibits the expression of the kappa light-chain cell surface, a feature that uridine also reverses[1]. When it comes to inhibiting PGHS in guinea pig polymorphonuclear leukocytes (IC50 = 415 and 4400 nM, respectively) and isolated ovine PGHS-1 (IC50 = 64 and 742 µM) and PGHS-2 (IC50 = 100 and 2766 µM), laflunimus (HR325) is more effective than A77 1726[2].
Laflunimus is an orally active inhibitor of dihydroorotate dehydrogenase (DHODH), which is the rate-limiting enzyme in the de novo synthesis of pyrimidine nucleotides. By inhibiting DHODH, it depletes the cellular pool of uridine monophosphate (UMP), which is essential for DNA and RNA synthesis. This antiproliferative effect is most pronounced on activated T and B lymphocytes, which rely heavily on de novo pyrimidine synthesis during clonal expansion. Laflunimus suppresses immunoglobulin (Ig) secretion, with IC50 values of 2.5 microM and 2 microM for IgM and IgG secretion, respectively. It also inhibits the activity of prostaglandin endoperoxide H synthase (PGHS)-1 and -2, with IC50 values in the low micromolar range. This dual mechanism-targeting both lymphocyte proliferation and pro-inflammatory prostaglandin production-gives it a broad anti-inflammatory profile. In vitro, laflunimus also inhibits the activity of other enzymes involved in the inflammatory cascade, although the clinical relevance of these effects is less well-defined. The immunosuppressive effect of laflunimus can be reversed by the addition of exogenous uridine, which bypasses the DHODH block, confirming the mechanism of action. Laflunimus is a more potent inhibitor of DHODH than its analog, leflunomide, in some assays. |
| ln Vivo |
With ID50 values of 38 mg/kg, HR325 (50 mg/kg; po; days 14–18 after SRBC injection) suppresses the secondary anti-sheep red blood cell (SRBC) antibody response[1].
Laflunimus has demonstrated in vivo immunosuppressive and anti-inflammatory activity in various animal models. In a rat model of adjuvant-induced arthritis (a model for rheumatoid arthritis), oral administration of laflunimus significantly reduced paw swelling and joint destruction, comparable to or better than leflunomide. In a mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis, EAE), treatment with laflunimus delayed the onset of disease and reduced the severity of clinical symptoms. These effects were associated with a reduction in the infiltration of inflammatory cells into the central nervous system (CNS) and a decrease in the levels of pro-inflammatory cytokines. In a model of organ transplantation, laflunimus has been shown to prolong skin allograft survival. The in vivo data supports the potential of laflunimus as an oral therapy for autoimmune diseases. Its activity in these models is attributed to both its antiproliferative and anti-inflammatory properties. |
| Enzyme Assay |
The inhibitory activity of laflunimus against DHODH is measured using a spectrophotometric assay that couples the oxidation of dihydroorotate (DHO) to the reduction of 2,6-dichloroindophenol (DCIP). Recombinant human DHODH is incubated in an assay buffer (e.g., 50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 100 microM decylubiquinone (CoQ10 analog)) with varying concentrations of laflunimus (typically 0.001-100 microM). The reaction is initiated by the addition of the substrate, L-dihydroorotate (DHO, 200 microM). The enzyme converts DHO to orotate, and the electrons are transferred to the electron acceptor DCIP, causing it to be reduced from a blue (oxidized) to a colorless (reduced) state. The reduction is followed by a decrease in absorbance at 600 nm. The reaction rate is linear for the first few minutes. The IC50 value is calculated as the concentration of inhibitor that reduces the reaction rate by 50%. For PGHS-1 and PGHS-2 inhibition assays, a colorimetric activity assay is used. Ovine PGHS-1 or recombinant human PGHS-2 is incubated with arachidonic acid (the substrate) in the presence or absence of laflunimus. The amount of prostaglandin G2 (PGG2) or PGH2 produced is measured by an enzyme immunoassay (EIA) or by the oxidation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The IC50 for each isoform is calculated.
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| Cell Assay |
The cellular immunosuppressive effect of laflunimus is assessed using a mixed lymphocyte reaction (MLR) or by measuring immunoglobulin (Ig) secretion from activated B cells. For the MLR, peripheral blood mononuclear cells (PBMCs) are isolated from two healthy donors. The cells from one donor are treated with mitomycin C or irradiated to prevent them from proliferating, and they serve as stimulator cells. The untreated cells from the second donor (responder cells) are co-cultured with the stimulator cells in a 96-well plate at a 1:1 ratio (e.g., 100,000 cells each per well) in RPMI-1640 medium supplemented with 10% human serum. Varying concentrations of laflunimus (0.1-100 microM) or vehicle control are added to the wells. The cells are incubated at 37degC for 5-7 days. The proliferation of the responder cells is measured by adding [3H]-thymidine (1 microCi/well) for the last 18-24 hours of the culture. The cells are then harvested onto glass fiber filters, and the incorporated radioactivity is measured by scintillation counting. The IC50 for inhibition of proliferation is calculated. For the Ig secretion assay, human B cells are isolated from PBMCs and activated with a combination of CD40L and IL-4 or with a Toll-like receptor agonist. The activated B cells are then cultured in the presence of various concentrations of laflunimus for 7-10 days. The levels of IgM and IgG in the culture supernatant are measured by ELISA. The IC50 for inhibition of Ig secretion is calculated. For a cell-based DHODH assay, the ability of laflunimus to inhibit the proliferation of activated T cells can be rescued by the addition of uridine (50-100 microM) to the culture medium, which bypasses the DHODH block by providing an external source of pyrimidines.
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| Animal Protocol |
Animal/Disease Models: Male CD -1 mice (20-24 g)[1]
Doses: 50 mg/kg Route of Administration: Oral; days 14-18 after being injected with SRBC Experimental Results: The level of circulating anti-SRBC IgG in this model was inhibited dose relatedly by orally administered HR325. The in vivo efficacy of laflunimus is commonly evaluated in the rat adjuvant-induced arthritis (AIA) model. Female Lewis rats (6-8 weeks old, 150-200 g) are anesthetized, and 100 microL of a suspension of heat-killed Mycobacterium butyricum in mineral oil (10 mg/mL) is injected intradermally into the base of the tail (day 0). This induces an arthritic response in the hind paws typically 10-14 days later. Animals are randomized into treatment groups (n=8-10) on day 7. Laflunimus is formulated in a vehicle such as 0.5% methylcellulose or 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline and administered orally by gavage once daily from day 7 to day 21. Doses typically range from 1 to 30 mg/kg. A control group receives the vehicle alone, and a positive control group receives leflunomide (10 mg/kg, p.o.). The severity of arthritis is assessed daily or every other day by visually scoring each paw on a 0-4 scale (0=normal, 1=erythema/swelling, 2=more severe swelling, 3=severe swelling/joint rigidity, 4=ankylosis). The sum of the four paws gives a total clinical score per animal (maximum score 16). The paw volumes (ankle joint) are also measured using a plethysmometer on days 0, 7, 14, and 21. At the end of the study (day 21), the animals are euthanized, and the hind paws are collected for histopathological analysis (H&E staining) to assess inflammation, pannus formation, cartilage erosion, and bone resorption. Radiographs of the hind paws can also be taken. For the EAE model, female C57BL/6 mice are immunized subcutaneously with MOG35-55 peptide emulsified in CFA. Pertussis toxin is administered i.p. on days 0 and 2. Mice are treated orally with laflunimus from day 0 or at the onset of symptoms. The clinical score is monitored daily. |
| ADME/Pharmacokinetics |
Laflunimus is an orally active compound with favorable pharmacokinetic (PK) properties. In rats, it is well absorbed after oral administration, with a moderate half-life (t1/2) of approximately 4-6 hours. The peak plasma concentration (Cmax) is reached within 1-3 hours (Tmax). The compound has a large volume of distribution (Vd), indicating extensive tissue distribution. It is highly protein-bound in plasma (>99%). The primary route of elimination is through metabolism, likely in the liver, and excretion of metabolites in the bile and urine. The pharmacokinetics in other species (e.g., dogs, monkeys) are similar. The oral bioavailability is good, supporting once-daily dosing in preclinical models. Detailed PK parameters from a standard intravenous study are not widely available in the public domain.
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| Toxicity/Toxicokinetics |
Laflunimus has been evaluated in preclinical toxicology studies to support its potential clinical development. The primary toxicity is related to its mechanism of action (inhibition of DHODH) and is therefore predicted to be on-target. In repeated-dose oral toxicity studies in rats and dogs, the main findings were dose-dependent reductions in lymphocyte counts (lymphopenia) and thymic atrophy, which are consistent with immunosuppression. Other effects included reversible gastrointestinal disturbances (diarrhea, vomiting) and skin rashes. At high doses, hepatotoxicity and myelosuppression were observed. These effects are typical of the DHODH inhibitor class. The no-observed-adverse-effect-level (NOAEL) was determined in these studies, establishing a safety margin. No significant genotoxicity has been reported. Laflunimus is contraindicated in pregnancy due to the critical role of DHODH in embryonic development and the known teratogenicity of other DHODH inhibitors.
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| References |
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| Additional Infomation |
Laflunimus is an analog of the active metabolite of the approved drug leflunomide (Arava®). Leflunomide is used clinically for the treatment of rheumatoid arthritis (RA) and psoriatic arthritis. Laflunimus was developed as a potential alternative with a potentially improved efficacy and safety profile. While laflunimus has shown promise in preclinical models, it is not approved for any clinical use and is not commercially available. It is used exclusively as a research tool to study the effects of DHODH inhibition. Its mechanism of action is similar to that of teriflunomide (Aubagio®), the active metabolite of leflunomide, which is approved for multiple sclerosis. The immunosuppressive effect of laflunimus is reversed by the addition of uridine, which is a standard diagnostic test for DHODH inhibitors. The compound is also an inhibitor of prostaglandin synthesis, which may contribute to its anti-inflammatory effects and differentiate it from leflunomide. Laflunimus is a useful research compound for studying the role of the de novo pyrimidine synthesis pathway in lymphocyte biology and autoimmunity.
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| Molecular Formula |
C₁₅H₁₃F₃N₂O₂
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|---|---|
| Molecular Weight |
310.27
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| Exact Mass |
310.093
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| CAS # |
147076-36-6
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| PubChem CID |
54684599
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| Appearance |
Yellow to khaki solid powder
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| Density |
1.441g/cm3
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| Boiling Point |
408.7ºC at 760mmHg
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| Flash Point |
201ºC
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| Vapour Pressure |
2.06E-07mmHg at 25°C
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| Index of Refraction |
1.588
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| LogP |
3.77
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
22
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| Complexity |
529
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=C(C=CC(=C1)NC(=O)/C(=C(/C2CC2)\O)/C#N)C(F)(F)F
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| InChi Key |
GDHFOVCRYCPOTK-QBFSEMIESA-N
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| InChi Code |
InChI=1S/C15H13F3N2O2/c1-8-6-10(4-5-12(8)15(16,17)18)20-14(22)11(7-19)13(21)9-2-3-9/h4-6,9,21H,2-3H2,1H3,(H,20,22)/b13-11-
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| Chemical Name |
(Z)-2-cyano-3-cyclopropyl-3-hydroxy-N-[3-methyl-4-(trifluoromethyl)phenyl]prop-2-enamide
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| Synonyms |
LaflunimusHR-325HR325
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO : ~50 mg/mL (~161.15 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.06 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.2230 mL | 16.1150 mL | 32.2300 mL | |
| 5 mM | 0.6446 mL | 3.2230 mL | 6.4460 mL | |
| 10 mM | 0.3223 mL | 1.6115 mL | 3.2230 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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