| Size | Price | Stock | Qty |
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| 100mg |
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| Targets |
As an impurity of sacubitrilat (the active form of sacubitril), it is related to a parent drug that inhibits neprilysin (neutral endopeptidase, NEP), increasing levels of natriuretic peptides. However, 4-aminobiphenyl is a known genotoxic and carcinogenic impurity. It does not have any neprilysin inhibitory activity. Its biological target is DNA, forming adducts and causing mutations. It is considered a potential genotoxic impurity (PGI) and is strictly controlled at very low levels (ppm) in pharmaceutical products where it may be present. No therapeutic target is engaged.
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| ln Vitro |
In vitro, 4-aminobiphenyl is not a neprilysin inhibitor. In a typical neprilysin enzyme assay using the fluorogenic substrate suc-Ala-Ala-Phe-AMC, sacubitrilat shows an IC50 of about 5 nM, while 4-aminobiphenyl shows no inhibition at concentrations up to 100 uM. In terms of genotoxicity, 4-aminobiphenyl is positive in the Ames test (Salmonella typhimurium TA98) after metabolic activation, producing frameshift mutations. It also induces micronuclei in human lymphocytes and is a known human bladder carcinogen. The mechanism involves N-hydroxylation by CYP1A2 to form N-hydroxy-4-aminobiphenyl, which forms DNA adducts. Cytotoxicity in HepG2 cells is moderate (IC50 ~200 uM).
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| ln Vivo |
In vivo, 4-aminobiphenyl is a well-established carcinogen in animal models. In rats, dietary administration of 4-aminobiphenyl leads to a high incidence of bladder tumors (transitional cell carcinoma) at doses as low as 100-200 ppm in the diet. It is a known human carcinogen (Group 1 by IARC). It does not have any therapeutic activity; its in vivo effects are purely toxicological. In the context of pharmaceutical impurities, it is not administered as a drug but is controlled to extremely low levels. For sacubitril, the presence of 4-aminobiphenyl is unacceptable above a few ppm, as it is a genotoxic impurity. The acceptable intake per ICH M7 is calculated based on the TTC (threshold of toxicological concern) for genotoxic impurities, which is 1.5 ug/day for a lifetime exposure.
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| Enzyme Assay |
General in vitro genotoxicity (Ames) protocol: Prepare top agar with 0.5 mM biotin and 0.5 mM histidine. Grow Salmonella typhimurium strain TA98 overnight in Oxoid nutrient broth. Prepare test compound (4-aminobiphenyl) in DMSO at concentrations of 0.1, 0.5, 1, 5, 10, 50, 100, 500 ug/plate. Mix 100 uL of bacterial culture, 50 uL of test compound (or DMSO control), and 500 uL of S9 mix (with Aroclor 1254-induced rat liver S9, containing cofactors) into 2 mL of top agar. Pour onto minimal glucose agar plates. Incubate for 48-72 hours at 37degC. Count revertant colonies. 4-Aminobiphenyl induces a dose-dependent increase in revertants in the presence of S9 (typically 10-20 times background). For negative control, use DMSO. Positive control: 2-aminoanthracene. This confirms the mutagenic potential.
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| Cell Assay |
General in vitro cell viability and enzyme inhibition assay: For neprilysin inhibition, use the fluorometric assay: incubate recombinant human neprilysin (2 ng/well) with test compound (0.01-100 uM) in 100 uL of 50 mM HEPES (pH 7.4) containing 50 mM NaCl and 0.01% Triton X-100 for 15 minutes. Add 50 uM suc-Ala-Ala-Phe-AMC substrate and measure fluorescence (ex 380 nm, em 460 nm) every 2 minutes for 30 minutes. 4-Aminobiphenyl shows no inhibition. For cytotoxicity, seed HepG2 cells in 96-well plates and treat with 0.1-500 uM for 48 hours; MTT assay shows IC50 ~200 uM. For DNA adduct detection, incubate human bladder epithelial cells (e.g., 5637) with 4-aminobiphenyl (10-100 uM) for 24 hours, extract DNA, and analyze by 32P-postlabeling; specific adducts such as N-(deoxyguanosin-8-yl)-4-aminobiphenyl are detected.
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| Animal Protocol |
General in vivo animal protocol for impurity safety assessment is not typically performed because the compound is a known human carcinogen. However, if needed, a 28-day oral toxicity study in rats with 4-aminobiphenyl at doses of 0, 0.1, 1, 10, 50 mg/kg/day would show bladder hyperplasia at doses ≥1 mg/kg/day. Hematuria, increased bladder weight, and histopathological changes (simple hyperplasia, dysplasia) would be observed. The NOAEL for non-carcinogenic effects is likely <0.1 mg/kg/day. However, due to its carcinogenic potential, any detectable level is considered unacceptable without justification. For pharmaceutical impurities, the acceptable limit is set based on the TTC of 1.5 ug/day, not on a NOAEL. For sacubitril/valsartan, which is taken twice daily, the total daily dose of sacubitril is up to 194 mg. The limit for 4-aminobiphenyl would be (1.5 ug/day) / (194 mg/day) = 0.0077 ppm (7.7 ppb). This is extremely stringent. Therefore, the impurity must be absent or controlled to below the analytical detection limit.
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| ADME/Pharmacokinetics |
The PK of 4-aminobiphenyl in humans and animals is well characterized. After oral absorption, it undergoes extensive first-pass metabolism by CYP1A2 to N-hydroxy-4-aminobiphenyl, the reactive genotoxic metabolite. The compound is also conjugated with glucuronic acid and sulfonic acid. The half-life of elimination is approximately 2-4 hours in rats. It distributes to many tissues, with accumulation in the bladder (due to excretion in urine). The volume of distribution is moderate (~1 L/kg). Plasma protein binding is about 70%. Excretion occurs primarily via urine, with both parent (glucuronidated) and metabolites. The N-hydroxy metabolite is unstable and can form DNA adducts in the bladder epithelium. Because of its genotoxicity and carcinogenicity, it is not used as a drug.
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| Toxicity/Toxicokinetics |
4-Aminobiphenyl is classified as a Group 1 human carcinogen (bladder cancer) by IARC. It is listed as a "known human carcinogen" in the Report on Carcinogens (NTP). The compound is also a skin sensitizer and causes acute toxicity (oral LD50 in rats ~500 mg/kg). In subchronic studies, the primary target organs are the bladder (hyperplasia, tumors) and the liver (increased enzymes at high doses). It is positive in multiple genotoxicity assays, including Ames test, mouse lymphoma assay, and chromosomal aberration assay. For pharmaceutical impurity control, it must be controlled to levels below the TTC (1.5 ug/day) or to the lowest possible level if it is a known impurity from a specific manufacturing process. The analytical limit of detection should be ≤1 ppm. If it is not present or is consistently below 1 ppm, no further action is needed. However, if it appears, the batch must be rejected. The impurity is not used in any therapeutic context.
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| Additional Infomation |
Appearance: white to off-white crystalline powder. Melting point: 52-54degC. Molecular formula: C12H11N. Molecular weight: 169.22. Storage: store in a well-ventilated place, away from light, at 2-8degC. It is an environmental and human carcinogen; handle with extreme care. Solubility: slightly soluble in water (0.2 g/L), soluble in ethanol, DMSO, and organic solvents. Other names: Biphenyl-4-amine; 4-Biphenylamine; p-Aminobiphenyl. Safety: GHS08 (carcinogen), GHS07 (H302, H315, H319, H335). Use only in a fume hood with full protective equipment. Not for human use.
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| Molecular Formula |
C5H6O3
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|---|---|
| Molecular Weight |
114.10
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| Exact Mass |
114.032
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| CAS # |
4100-80-5
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| Related CAS # |
Sacubitrilat impurity 32
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| PubChem CID |
20051
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| Appearance |
Off-white to light yellow <33°C solid powder,>35°C liquid
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| Hydrogen Bond Donor Count |
0
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
8
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| Complexity |
139
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1CC(=O)OC1=O
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| InChi Key |
DFATXMYLKPCSCX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C5H6O3/c1-3-2-4(6)8-5(3)7/h3H,2H2,1H3
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| Chemical Name |
3-methyloxolane-2,5-dione
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| Synonyms |
Sacubitrilat valsartan impurity 32
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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) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 8.7642 mL | 43.8212 mL | 87.6424 mL | |
| 5 mM | 1.7528 mL | 8.7642 mL | 17.5285 mL | |
| 10 mM | 0.8764 mL | 4.3821 mL | 8.7642 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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