| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| 25mg |
|
||
| 50mg |
|
||
| 100mg |
|
||
| Other Sizes |
| Targets |
Anhydrotetracycline has multiple distinct targets. First, it acts as a competitive inhibitor of tetracycline destructase enzymes (Tet(50), Tet(X), Tet(X)_3), which are bacterial enzymes that degrade tetracycline antibiotics . Second, it serves as an effector for tetracycline repressor (TetR) and reverse tetracycline repressor (revTetR) transcriptional repressors in eukaryotic cells, binding tightly to these regulatory proteins and modulating gene expression in Tet-controlled systems . Importantly, anhydrotetracycline binds poorly to the bacterial 30S ribosomal subunit, which explains its lack of direct antibacterial activity . Unlike class 1 tetracyclines (e.g., tetracycline, doxycycline) that target the ribosome, anhydrotetracycline belongs to class 2 tetracyclines, whose primary site of action appears to be the cytoplasmic membrane rather than the ribosome .
|
|---|---|
| ln Vitro |
Anhydrotetracycline (0.1 μM–10 mM) exhibits a potent and dose-dependent inhibitory impact on enzymes that break down tetracycline, according to in vitro tests [1].
In vitro studies have demonstrated that anhydrotetracycline is a potent inhibitor of tetracycline destructase enzymes. It inhibits Tet(50)-mediated tetracycline degradation with an IC₅₀ of 210 μM, Tet(X)-mediated degradation with an IC₅₀ of 41 μM, and Tet(X)_3-mediated degradation with an IC₅₀ of 3 μM . For chlortetracycline degradation, anhydrotetracycline shows IC₅₀ values of 210 μM (Tet(50)), 75 μM (Tet(X)), and 26 μM (Tet(X)_3) . The compound exhibits dose-dependent inhibition in vitro across concentrations ranging from 0.1 μM to 10 mM . Unlike class 1 tetracyclines, anhydrotetracycline fails to inhibit cell-free translation directed by E. coli or B. subtilis extracts and is a very poor inhibitor of protein synthesis . Instead, it rapidly inhibits the in vivo incorporation of precursors into DNA and RNA, suggesting a mode of action distinct from ribosomal inhibition . |
| ln Vivo |
In vivo studies have shown that anhydrotetracycline exhibits significant biological activity in animal models. In E. coli expressing tetracycline destructase enzymes, anhydrotetracycline acts synergistically with tetracycline to inhibit bacterial growth, functionally rescuing tetracycline antibiotic activity . The fractional inhibitory concentration index (FICI) for the combination is 0.1875, indicating strong synergy . In eukaryotic systems, treatment of mice bearing NIH3T3-HER2 cell-based tumor xenografts with anhydrotetracycline hydrochloride (10 mg/kg, subcutaneous) resulted in tumor regression of more than 95% within 7 days . The compound effectively downregulates HER2/ERBB2 gene expression in vivo, leading to rapid reduction in tumor volume . In E. coli, low concentrations of anhydrotetracycline (20 μg/mL and below) completely inhibit RNA accumulation, as well as protein, DNA, and guanosine 5'-diphosphate 3'-diphosphate (ppGpp) synthesis .
|
| Enzyme Assay |
Methodology for Tetracycline Destructase Inhibition Assay: The inhibitory activity of anhydrotetracycline against tetracycline destructase enzymes (Tet(50), Tet(X), Tet(X)_3) is determined using a spectrophotometric assay. The enzyme is incubated with varying concentrations of anhydrotetracycline (typically ranging from 0.1 μM to 10 mM) in reaction buffer containing the tetracycline substrate (e.g., tetracycline, chlortetracycline, or demeclocycline). The reaction is initiated by the addition of the tetracycline substrate, and the consumption of tetracycline is monitored by measuring the change in absorbance at 400 nm over time . Reaction velocity is calculated from the linear portion of the absorbance decay curve. IC₅₀ values are determined by fitting the dose-response data using non-linear regression analysis. For each enzyme-inhibitor pair, reactions are performed in triplicate, and results are expressed as mean ± standard deviation .
|
| Cell Assay |
Methodology for Gene Expression Modulation in Mammalian Cells: The ability of anhydrotetracycline to modulate gene expression in Tet-controlled systems is assessed using established mammalian cell lines (e.g., NIH3T3 or N1 cells) expressing a tetracycline-regulated reporter gene. Cells are cultured in appropriate medium (e.g., DMEM with 10% FBS) at 37°C with 5% CO₂. Anhydrotetracycline hydrochloride is added to the culture medium at concentrations ranging from 10 ng/mL to 200 nM for specified durations (e.g., 3 days for HER2 downregulation studies; 7 days for GUS gene expression studies) . Following treatment, cells are harvested for analysis. Gene expression levels are quantified by RT-qPCR (mRNA) or Western blotting (protein). For reporter gene assays, enzymatic activity (e.g., GUS) is measured using fluorogenic substrates. Treatment with 10 ng/mL anhydrotetracycline for 3 days has been shown to reduce HER2 gene expression to below the detection limit in NIH3T3-HER2 cells .
|
| Animal Protocol |
Methodology for Tumor Xenograft Regression Study in Mice: The in vivo efficacy of anhydrotetracycline is evaluated using a mouse xenograft model. Female immunocompromised mice (e.g., nude mice) are subcutaneously implanted with NIH3T3-HER2 cells (or other tetracycline-responsive cell lines). Once tumors reach a measurable size (e.g., approximately 100-200 mm³), animals are randomly assigned to treatment groups (n≥5 per group). Anhydrotetracycline hydrochloride is administered at a dose of 10 mg/kg body weight via subcutaneous injection or intraperitoneal injection, typically once daily . Control animals receive vehicle alone (e.g., saline or appropriate buffer). Tumor volumes are measured every 2-3 days using calipers, and body weights are monitored to assess toxicity. After the treatment period (typically 7-14 days), animals are euthanized, and tumors are excised for further analysis (e.g., histology, gene expression analysis). Anhydrotetracycline treatment (10 mg/kg, s.c.) has been shown to induce more than 95% tumor regression within 7 days, with associated downregulation of ERBB2 mRNA and protein .
|
| ADME/Pharmacokinetics |
The compound is known to be active in vivo following both subcutaneous and intraperitoneal administration, as demonstrated by tumor regression studies in mice at a dose of 10 mg/kg . The compound's ability to modulate gene expression in vivo suggests adequate bioavailability and tissue distribution. Anhydrotetracycline can enter the cytoplasm of bacterial cells, as evidenced by its ability to inhibit macromolecular synthesis in vivo . For research purposes, the compound is supplied as a solid and should be stored at 2°C to 8°C to maintain stability . The hydrochloride salt form (anhydrotetracycline hydrochloride, CAS: 13803-65-1) is also available and offers improved aqueous solubility compared to the free base .
|
| Toxicity/Toxicokinetics |
Anhydrotetracycline exhibits significant toxicity, distinguishing it from the parent antibiotic tetracycline. Toxicity studies in mice have determined the intravenous LD₅₀ of anhydrotetracycline to be 134.2 mg/kg, compared to 160.7 mg/kg for tetracycline . The compound is less toxic than 4-epitetracycline (LD₅₀ 85.8 mg/kg) but more toxic than anhydro-4-epi-tetracycline (LD₅₀ 193 mg/kg) . In sub-acute toxicity studies, anhydro-4-epi-tetracycline (the 4-epimer of anhydrotetracycline) exhibited the most severe effects, causing diabetic urine, proteinuria, and serious pathological changes in renal convoluted tubules at doses as low as 1 mg/day intravenously or 12 mg/day orally . Research indicates that anhydro-4-epi-tetracycline may be the primary compound responsible for Fanconi syndrome, a form of kidney dysfunction associated with outdated tetracycline preparations . These findings highlight the importance of preventing tetracycline degradation to anhydro forms during storage and formulation.
|
| References |
[1]. Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes. ACS Infect Dis. 2019 Apr 12;5(4):618-633.
|
| Additional Infomation |
Anhydrotetracycline is a tertiary α-hydroxy ketone, belonging to the tetracycline class of compounds. It is the tautomer of the Anhydrotetracycline zwitterion.
|
| Molecular Formula |
C22-H22-N2-O7
|
|---|---|
| Molecular Weight |
426.42
|
| Exact Mass |
426.143
|
| CAS # |
1665-56-1
|
| Related CAS # |
Anhydrotetracycline hydrochloride;13803-65-1
|
| PubChem CID |
54675758
|
| Appearance |
Typically exists as solid at room temperature
|
| Density |
1.6 g/cm3
|
| Boiling Point |
618.6ºC at 760 mmHg
|
| Flash Point |
327.9ºC
|
| Vapour Pressure |
3.66E-16mmHg at 25°C
|
| Index of Refraction |
1.76
|
| LogP |
1.156
|
| Hydrogen Bond Donor Count |
5
|
| Hydrogen Bond Acceptor Count |
8
|
| Rotatable Bond Count |
2
|
| Heavy Atom Count |
31
|
| Complexity |
855
|
| Defined Atom Stereocenter Count |
3
|
| SMILES |
N/C(=C1/C(=O)[C@@H](N(C)C)[C@@H]2CC3=C(C)C4C=CC=C(O)C=4C(O)=C3C(=O)[C@]2(O)C/1=O)/O
|
| InChi Key |
KTTKGQINVKPHLY-DOCRCCHOSA-N
|
| InChi Code |
InChI=1S/C22H22N2O7/c1-8-9-5-4-6-12(25)13(9)17(26)14-10(8)7-11-16(24(2)3)18(27)15(21(23)30)20(29)22(11,31)19(14)28/h4-6,11,16,25-26,29,31H,7H2,1-3H3,(H2,23,30)/t11-,16-,22-/m0/s1
|
| Chemical Name |
(4S,4aS,12aR)-4-(dimethylamino)-1,10,11,12a-tetrahydroxy-6-methyl-3,12-dioxo-4a,5-dihydro-4H-tetracene-2-carboxamide
|
| Synonyms |
Anhydrotetracycline; 1665-56-1; 5a,6-Anhydrotetracycline; (4S)-Anhydrotetracycline; (4S,4aS,12aR)-4-(dimethylamino)-1,10,11,12a-tetrahydroxy-6-methyl-3,12-dioxo-4a,5-dihydro-4H-tetracene-2-carboxamide;
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 2.3451 mL | 11.7255 mL | 23.4511 mL | |
| 5 mM | 0.4690 mL | 2.3451 mL | 4.6902 mL | |
| 10 mM | 0.2345 mL | 1.1726 mL | 2.3451 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
