| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
Milbemycin A4 oxime targets the glutamate-gated chloride channels (GluCls) present in the nerve and muscle cells of nematodes (roundworms) and arthropods. These ion channels are unique to invertebrates; mammals do not have GluCls. The compound binds to the GluCl with high affinity (Kd in the low nanomolar range), acting as an allosteric activator that keeps the channel open, causing a continuous influx of chloride ions. This hyperpolarizes the membrane, making the neuron or muscle cell unresponsive to normal stimuli, resulting in flaccid paralysis and death of the parasite. At higher concentrations, milbemycin A4 oxime also potentiates GABA-gated chloride channels, enhancing its paralytic effect. The selectivity for invertebrate over mammalian receptors is the basis for its safety margin in pets.
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| ln Vitro |
In vitro, milbemycin A4 oxime has been tested in cell-free systems and on isolated parasites. In Caenorhabditis elegans, the EC₅0 for pharyngeal pumping inhibition (a measure of paralysis) is 0.5-1 nM. Against the canine heartworm Dirofilaria immitis microfilariae, the IC₅0 for motility inhibition is about 0.1 nM after 24 h of exposure. In cell-based assays using HEK293 cells expressing C. elegans GluClalpha, milbemycin A4 oxime (0.1-100 nM) induces chloride ion influx with an EC₅0 of 2 nM. The compound also has activity against adult heartworms, but the IC₅0 is higher (approximately 10 nM). Against the intestinal worm Toxocara canis, the EC₅0 for larval motility inhibition is 0.3 nM. Milbemycin A4 oxime has no activity against mammalian cell lines up to 10 uM, confirming selective toxicity.
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| ln Vivo |
In vivo, milbemycin A4 oxime is highly effective as a heartworm preventive. In dogs, a single oral dose of 0.05 mg/kg (as milbemycin oxime) eliminates all Dirofilaria immitis microfilariae within 7-14 days and prevents the development of immature larvae (L3 to L4 stage) for one month. It also effectively treats intestinal nematodes: a single dose of 0.5-1.0 mg/kg in dogs results in >99% reduction in Toxocara canis, Ancylostoma caninum, and Trichuris vulpis egg counts. In cats, a dose of 2 mg/kg is used. For sarcoptic mange, 2 mg/kg weekly for 3 weeks is effective. The compound has a rapid onset of action: parasites are killed within 24-48 h. The efficacy is dose-dependent and persists for about 30 days due to the long half-life.
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| Enzyme Assay |
For radioligand binding assays, cell membranes expressing C. elegans GluClalpha are prepared from HEK293 cells. The membranes (50 ug protein) are incubated with [3H]-milbemycin A4 oxime (0.01-10 nM) in 50 mM Tris-HCl (pH 7.4) containing 1 mM EDTA and 0.1% BSA for 1 h at 25degC. Non-specific binding is determined in the presence of 10 uM unlabeled ivermectin. Bound radioactivity is separated by rapid filtration through GF/B glass fiber filters, washed three times with cold buffer, and counted by liquid scintillation. Specific binding is calculated as total minus non-specific. Kd and Bmax are determined by Scatchard analysis. For GluCl activation, a fluorescence-based membrane potential assay uses a dye that redistributes upon depolarization.
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| Cell Assay |
For functional cell-based assays, HEK293 cells stably expressing C. elegans GluClalpha are seeded in 96-well black plates at 2 × 10⁴ cells/well. After 24 h, the medium is replaced with assay buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4) containing the membrane potential dye (e.g., FLIPR Membrane Potential Assay Kit). The plate is incubated for 30 min at 37degC. Then, milbemycin A4 oxime (0.01-1000 nM, diluted in assay buffer) is added using a plate reader equipped with an injector. Fluorescence (ex 530 nm, em 565 nm) is recorded for 5 min. The change in fluorescence (deltaF) is normalized to baseline, and EC₅0 is calculated. For cytotoxicity, the same cells are treated with 0.1-10 uM milbemycin A4 oxime for 72 h and viability is measured by MTT.
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| Animal Protocol |
In vivo efficacy studies are performed in dogs (beagles) naturally or experimentally infected with heartworms or intestinal parasites. For heartworm prevention, dogs (n=8 per group) are inoculated subcutaneously with 50-100 Dirofilaria immitis L3 larvae on day 0. Treatment (milbemycin A4 oxime at 0.05 mg/kg, oral) is given on day 30, day 60, and day 90 post-infection. At day 120, the dogs are euthanized, and the adult heartworms are recovered from the heart and pulmonary arteries. Efficacy is calculated as (mean worm count in control group - mean worm count in treated group) / mean worm count in control group × 100%. For intestinal worms, fecal egg counts are performed before and 7 days after treatment. The reduction in eggs per gram (EPG) is calculated. For safety studies, dogs receive up to 5 mg/kg (100× therapeutic dose) and are observed for clinical signs (vomiting, ataxia, salivation).
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| ADME/Pharmacokinetics |
Pharmacokinetics of milbemycin A4 oxime have been studied in dogs. After a single oral dose of 0.05 mg/kg (tablet formulation), the mean Cmax is 75 ng/mL reached at a Tmax of 3 h. The terminal half-life (t1/2) is approximately 2 days (48 h). The oral bioavailability is about 30-40% due to first-pass hepatic metabolism. The compound is highly bound to plasma proteins (>90%). The volume of distribution (Vd) is large (>10 L/kg), indicating extensive tissue distribution, especially to adipose tissue and skin. Metabolism is primarily via CYP3A4, with major metabolites being hydroxylated and demethylated derivatives. Excretion is mainly fecal (80-90%) via bile, with less than 5% excreted in urine. In cats, the half-life is similar (about 2 days). The long half-life allows once-monthly dosing. Food intake does not significantly affect absorption.
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| Toxicity/Toxicokinetics |
Milbemycin A4 oxime has a wide safety margin in most dog breeds. The acute oral LD₅0 in dogs is >100 mg/kg. At the therapeutic dose of 0.05 mg/kg, no adverse effects are observed. At 1-2 mg/kg (20-40× therapeutic), mild salivation and mydriasis may occur. At 5-10 mg/kg, transient ataxia, tremors, and depression are seen. Certain dog breeds with the MDR1 (multidrug resistance protein 1) gene mutation (e.g., Collies, Australian Shepherds, Shetland Sheepdogs) are highly sensitive: doses as low as 0.1 mg/kg can cause neurotoxicity (ataxia, muscle tremors, drooling, stupor) due to drug accumulation in the brain. Therefore, milbemycin oxime is contraindicated in MDR1-deficient dogs unless specifically tested. In cats, the safety margin is narrower: doses above 2 mg/kg can cause ataxia and mydriasis; the lethal dose is about 10 mg/kg. No teratogenicity or genotoxicity has been reported.
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| References | |
| Additional Infomation |
Milbemycin A4 oxime is a component of milbemycin oxime, which is an FDA-approved veterinary drug (brand names: Interceptor, MilbeGuard, Sentinel). It is available by prescription for dogs and cats. Milbemycin oxime is often formulated in combination with praziquantel (tapeworm control) or afoxolaner (flea/tick control). The compound is not approved for use in food-producing animals. It is on the World Health Organization's list of essential veterinary medicines. Clinical trials have demonstrated >99% efficacy against heartworm larvae and intestinal worms. No generic version is currently available? Several generics exist. The compound has been on the market since 1997. Milbemycin A4 oxime is not used in humans. It is classified as a macrocyclic lactone endectocide. The mechanism of action is similar to that of ivermectin, but milbemycins are generally more potent against some nematodes. No resistance has been reported in heartworm populations, but resistance in intestinal nematodes of livestock is known for related compounds.
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| Molecular Formula |
C32H45NO7
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|---|---|
| Molecular Weight |
555.70
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| Exact Mass |
555.32
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| CAS # |
93074-04-5
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| PubChem CID |
91617829
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| Appearance |
Typically exists as solids at room temperature
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| LogP |
5.394
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
40
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| Complexity |
1140
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| Defined Atom Stereocenter Count |
9
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| SMILES |
O1[C@]([H])(C([H])([H])C([H])([H])[H])[C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[C@]1([H])C([H])([H])[C@@]([H])(C([H])([H])C([H])=C(C([H])([H])[H])C([H])([H])[C@@]([H])(C([H])([H])[H])C([H])=C([H])C([H])=C3C([H])([H])O[C@]4([H])C(C(C([H])([H])[H])=C([H])[C@@]([H])(C(=O)O1)[C@@]43O[H])=NO[H])O2 |c:38,54,58|
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| InChi Key |
YCAZFHUABUMOIM-OWOPNLEVSA-N
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| InChi Code |
InChI=1S/C32H45NO7/c1-6-27-21(4)12-13-31(40-27)17-25-16-24(39-31)11-10-20(3)14-19(2)8-7-9-23-18-37-29-28(33-36)22(5)15-26(30(34)38-25)32(23,29)35/h7-10,15,19,21,24-27,29,35-36H,6,11-14,16-18H2,1-5H3/b8-7+,20-10+,23-9+,33-28?/t19-,21-,24+,25-,26-,27+,29+,31+,32+/m0/s1
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| Chemical Name |
(1R,4S,5'S,6R,6'R,8R,10E,13R,14E,16E,20R,24S)-6'-ethyl-24-hydroxy-21-hydroxyimino-5',11,13,22-tetramethylspiro[3,7,19-trioxatetracyclo[15.6.1.14,8.020,24]pentacosa-10,14,16,22-tetraene-6,2'-oxane]-2-one
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| Synonyms |
5-Ketomilbemycin A4 oxime; 5-Oxomilbemycin A4 5-oxime
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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 | 1.7995 mL | 8.9977 mL | 17.9953 mL | |
| 5 mM | 0.3599 mL | 1.7995 mL | 3.5991 mL | |
| 10 mM | 0.1800 mL | 0.8998 mL | 1.7995 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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