Cell viability is lowered by emmemectin benzoate (MK-244; 2.5–40 μM; 12 and 24 hours) in a dose- and time-dependent way [1]. 16HBE cells are exposed to 2.5–20 μM of emmemactin benzoate for 24 hours, which causes DNA damage and ROS generation [1]. Emamectin Benzoate (2.5 – 20 μM; 12 hours) raises the levels of cleaved-PARP, Bax/Bcl-2, caspase-3, caspase-9, and cytochrome c [1]. With an IC50 of 3.72 μM, emamectin benzoate (2.5, 5, 10, 15 μM; 72 hours) decreases the viability of Trichopodia exigua Tn5B1-4 cells. Condensates and cytochromes undergo nuclear staining when exposed to emamectin benzoate [2]. Examine[1]

In liver tissue, emamectin benzoate (MK-244; sidewall; 25–100 mg/kg/day; for 14 days) dramatically increases oxidative damage [3].

Cell Viability Assay[1]
Cell Types: Human normal bronchial epithelial cell line 16HBE
Tested Concentrations: 2.5, 5, 7.5, 10, 15, 20, 40 μM
Incubation Duration: 12 and 24 hrs (hours)
Experimental Results: Cell viability diminished in a time- and dose-dependent manner at 12 hrs (hours) The IC50 values within 24 hrs (hours) were 11.88 μM and 9.67 μM, respectively.

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Apoptosis analysis [1]
Cell Types: Human normal bronchial epithelial cell line 16HBE
Tested Concentrations: 2.5, 5, 10, 20 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Induced cell apoptosis and caused chromatin shrinkage and nuclear fragmentation.

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Western Blot Analysis [1]
Cell Types: Human normal bronchial epithelial cell line 16HBE
Tested Concentrations: 2.5, 5, 10, 20 μM
Incubation Duration: 12 hrs (hours)
Experimental Results: Cytochrome-c, caspase-3, and cas-pase-9 levels increased, Cleavage-PARP, Bax/Bcl-2.

Animal/Disease Models: 10weeks old Swiss albino male mice (25-30 g) [3]
Doses: 25, 50, 100 mg/kg
Route of Administration: oral; daily; lasting for 14 days
Experimental Results: Lead to significant induction of oxidation in liver tissue injury, as evidenced by increased TBARS levels and diminished GSH levels.

Absorption, Distribution and Excretion
This study was conducted in two groups, each consisting of two male beagle dogs. Group 1 received 0.5 mg/kg of (3)H-MK-0243 benzoate (dissolved in 5% ethanol, dose 1 mL/kg, radioactivity 0.239 mCi/mg, radiochemical purity 98.8%) on day 1 and 0.5 mg/kg of (3)H-MK-0243 hydrochloride (dissolved in deionized water, dose 1 mL/kg, radioactivity 0.229 mCi/mg, radiochemical purity 98.7%) on day 15. Group 2 received the reverse dosing regimen. Body weight was measured before each administration. Following each administration, 2 mL of blood was drawn at 0.5, 1, 2, 4, 6, 8, 24, 48, 96, and 168 hours for drug concentration determination. Urine and fecal samples were collected at 0–24 hours and 72–96 hours post-administration for drug concentration analysis. No drug effect was observed. The mean plasma half-lives of benzoate and hydrochloride were 35.7 ± 3.4 hours and 35.5 ± 4.4 hours, respectively. The mean plasma area under the curve (AUC) of benzoate and hydrochloride were 4479 ± 1476 and 4574 ± 1514 ng/g plasma/7 days, respectively. The mean peak plasma MAB1a (the major component of MK-0243, accounting for 90% to 95% of its content) of both salts was approximately 100 ng equivalents/g plasma, reaching peak concentration approximately 6 hours post-administration. Drug recoveries in feces and urine on days 1 and 4 were approximately 40% and 0.01% of the administered dose, respectively. The study concludes that these two salts are bioequivalent in male beagle dogs.
This study investigated the skin absorption of the experimental avermectin insecticide methylaminoavermectin benzoate in rhesus monkeys. Skin absorption was calculated by comparing fecal radioactivity levels after skin administration with those after intravenous administration of the same dose. After intravenous injection of 300 μg of (3)H-MAB1a (prepared with propylene glycol: saline 1:1) into three monkeys, plasma drug concentrations showed a biphasic decline, with a rapid decrease in radioactivity in the first 15 minutes, followed by a slow decrease to background levels. Approximately 90% and 5% of the administered radioactivity were recovered in feces and urine, respectively, 7 days after administration. After a period of washout, 300 μg of [(3)H]MAB1a (dissolved in emulsion) was topically applied to the shaved forearms of the same group of monkeys. After 10 hours of exposure, the exposed forearms were washed with soap and water, and approximately 90% of the radioactive material was recovered. Although plasma radioactivity levels were generally below background levels, approximately 1.5% of the administered dose was recovered in excrement. The skin absorption rate of [(3)H]emamectin benzoate was calculated to be 1.6%. The low skin penetration of emamectin benzoate suggests that the likelihood of actual exposure to this compound by agricultural workers is extremely low. This study aimed to investigate the levels of emamectin in blood, mucus, and muscle after field administration of the recommended dose, and its correlation with sea lice infection in the same group of fish (elimination study). Whole-body autoradiography and scintillation counting (distribution study) were used to investigate the tissue distribution of tritium-labeled emamycin benzoate in Atlantic salmon after a single oral dose. In the elimination study, peak concentrations of emamycin benzoate in blood, mucus, and muscle were reached on the last day of administration (day 7), at 128, 105, and 68 ng/g (ppb), respectively. From day 7 onwards, the concentration in blood gradually decreased until it fell below the detection limit on day 77. Except for days 7 and 21, the concentration in mucus was higher than that in plasma (P < 0.05). From the end of treatment (day 7) to day 70, the concentration of emamycin benzoate gradually decreased, with half-lives of 9.2 days, 10.0 days, and 11.3 days in muscle, plasma, and mucus, respectively. Distribution studies showed high levels of radioactive material in mucous membranes (gastrointestinal tract, gills) throughout the observation period (56 days). High levels of radioactive material were also observed in the condyles, pituitary gland, and olfactory rosette during the study period. The highest radioactive material concentration was found in bile, indicating that bile is an important excretion route. Distribution studies confirmed the results of elimination studies, namely, the concentrations of radioactive material in blood, skin mucous membranes, and muscle. Atlantic salmon (approximately 1.3 kg) were housed in seawater tanks at 5±1°C, and 3H-emamycin B1 benzoate was added to their diet for 7 consecutive days at a nominal dose of 50 micrograms of emamycin benzoate/kg/day. Tissue, blood, and bile samples were collected from 10 fish at 3 hours, 12 hours, and 1, 3, 7, 15, 30, 45, 60, and 90 days after the last administration. Fecal samples were collected daily from the fish tanks starting before administration and continuing for 90 days after the last administration. The highest total residual radioactivity (TRR) in the daily fecal samples during the administration period was 0.25 ppm, and over 97% of the TRR in all fecal samples during the administration period was emamycin B1a. The fecal TRR then decreased rapidly, reaching approximately 0.05 ppm one day after the last administration. The mean TRR ranges for each tissue over 90 days after administration were as follows: kidney, 1.4–3 ppm; liver, 1.0–2.3 ppm; skin, 0.04–0.09 ppm; muscle residue, 0.02–0.06 ppm; and bone residue, less than 0.01 ppm. Residual components in liver, kidney, muscle, and skin samples, summarized by dosing interval, were epimedin B1a (total residual rate 81-100%) and demethylemamedin B1a (total residual rate 0-17%). Trace amounts (<2%) of N-formylemamedin B1a were detected in some muscle samples. Epimedin B1a was selected as a marker residue for epimedin residue monitoring. Epimedin B1a levels in individual skin and muscle samples were quantified using high-performance liquid chromatography-fluorescence assay (HPLC-fluorescence). Epimedin B1a levels in all analyzed samples (3 hours to 30 days post-dose) were below 85 ppb. For more complete data on the absorption, distribution, and excretion of epimedins (a total of 8), please visit the HSDB record page.

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Metabolism/Metabolites
Atlantic salmon (approximately 1.3 kg) were kept in seawater tanks at 5±1°C and fed with [(3)H]emamycin B1 benzoate for 7 consecutive days at a nominal dose of 50 μg emamycin benzoate/kg/day. Tissue, blood, and bile samples were collected from 10 fish at 3 hours, 12 hours, and 1, 3, 7, 15, 30, 45, 60, and 90 days after the last administration. Fecal samples were collected daily from the tanks from before administration until 90 days after the last administration. The highest total radioactive residue (TRR) in the daily fecal samples during the administration period was 0.25 ppm, and more than 97% of the TRR in all fecal samples during the administration period was emamycin B1a. The fecal TRR then decreased rapidly, reaching approximately 0.05 ppm 1 day after the last administration. The mean TRR ranges for various tissues within 90 days post-administration were as follows: kidney, 1.4–3 ppm; liver, 1.0–2.3 ppm; skin, 0.04–0.09 ppm; muscle, 0.02–0.06 ppm; and bone, less than 0.01 ppm. Residual components in liver, kidney, muscle, and skin samples, aggregated at post-administration intervals, were epimedin B1a (total residual rate 81–100%) and demethyleimamedin B1a (total residual rate 0–17%). Trace amounts (<2%) of N-formyleimamedin B1a were detected in some muscle samples. Emamedin B1a was selected as the marker residue for regulatory monitoring of epimedin residues. Emamedin B1a levels in skin and muscle samples were quantified using high-performance liquid chromatography-fluorescence assay (HPLC-FISA). Levels in all analyzed samples (3 hours to 30 days post-administration) were less than 85 ppb. …A mammalian metabolite has been identified. The metabolite was identified as an N-demethylation byproduct of emamectin. This study investigated the metabolism of (3)H/(14)C-labeled 4"-deoxy-4"-epimycin B1a (MAB1a) benzoate (a major homologue of the emamectin insecticide emamectin benzoate, with a content ≥90%) in laying hens. Ten Gallus domesticus chickens were orally administered once daily for 7 consecutive days (1 mg/kg body weight/day). Eggs and feces were collected daily, and eggs were separated into albumen and yolk. Chickens were euthanized within 20 hours of the last administration, and liver, kidneys, heart, muscle, fat, ovaries, gizzard, gastrointestinal tract and its contents, and carcass were collected. Approximately 70% and 6% of the total administered dose were recovered from feces, gastrointestinal tract and its contents, and tissues and eggs, respectively. Two novel metabolites were identified: a 24-hydroxymethyl derivative of the parent compound (24-hydroxymethyl-4"-deoxy-4"-epimycin B1a) and an N-demethylated derivative of 24-hydroxymethyl-4"-deoxy-4"-epimycin B1a (24-hydroxymethyl-4"-deoxy-4"-epimycin B1a). Furthermore, eight fatty acid conjugates of these two metabolites were isolated and identified, accounting for 8-75% of the total radioactive residues in tissues and eggs. Although this is one of the most extensive cases of in vivo fatty acid binding to exogenous substances reported to date, the likelihood of human exposure to MAB1a residues through chicken consumption is extremely low, as the dose levels in this study were approximately 1000 times higher than those in crops, and most of the administered dose was recovered from feces. Based on these findings, the biotransformation of MAB1a in poultry differs significantly from that in mammals.
Although emamectin benzoate is not extensively metabolized in mammals, limited information about its metabolites suggests that metabolism does not detoxify it. One plant metabolite of emamectin benzoate is slightly more toxic than the emamectin benzoate itself.
Biological Half-Life

Emamectin benzoate is rapidly cleared from rat plasma after oral or intravenous administration, with a half-life of approximately 15 to 28 hours.
Emamectin benzoate is a relatively large molecule (actually a mixture of four closely related molecules), poorly absorbed orally, poorly absorbed through the skin, and rapidly excreted in feces, with a systemic half-life of approximately 1.5 days.
This study also investigated the tissue distribution of tritium-labeled emamectin benzoate in Atlantic salmon after a single oral administration using whole-body autoradiography and scintillation counting (distribution study). From the end of treatment (day 7) to day 70, the concentration of emamycin benzoate gradually decreased, with half-lives of 9.2 days, 10.0 days, and 11.3 days in muscle, plasma, and mucus, respectively.

Toxicity Summary
This substance has low water solubility and broad non-specific binding capacity. It can open GABA-insensitive chloride ion channels, reduce membrane resistance, and increase inward conductivity. (T10)

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Interactions The housefly (Musca domestica L.) is a major pest in the global dairy industry, exhibiting extremely strong adaptability. Currently, various insecticides are used to control houseflies, but the development of resistance is a serious problem. Insecticide mixtures can enhance the toxicity of resistant pests to insecticides, thus potentially becoming a tool for resistance management. This study evaluated the toxicity of bifenthrin, cypermethrin, deltamethrin, chlorpyrifos, profenofos, emamectin benzoate, and fipronil to houseflies, as well as their toxicity when used in combination. Compared to laboratory-sensitive strains, field-collected housefly populations showed significant resistance to all the insecticides studied. Most insecticide mixtures, such as a mixture of pyrethroids with other compounds, significantly increased the toxicity of pyrethroids to field populations under both conditions (1:1 – “A” and LC50:LC50 – “B”). Under both conditions, the combination index of pyrethroids with other compounds was significantly lower than 1 in most cases, indicating a synergistic effect. When enzyme inhibitors PBO and DEF were used in combination with insecticides to combat resistant populations, the toxicity of bifenthrin, cypermethrin, deltamethrin, and emamectin was significantly increased, indicating the existence of resistance mechanisms based on esterases and monooxygenases. The combined use of bifenthrin, cypermethrin, and deltamethrin with chlorpyrifos, profenofos, emamectin, and fipronil enhanced their toxicity to resistant housefly populations. The results of this study may have practical implications for the management of insecticide resistance in houseflies.

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Non-human toxicity values
LD50 Mice (female) Oral (gavage) 165 mg/kg / imamectin benzoate-methyl tert-butyl ether (MBTE) solvate, purity 96.4%/
LD50 Mice (female) Oral (gavage) 141 mg/kg / imamectin benzoate monohydrate solvate, purity 99.1%/
LD50 Rats (female) Oral (gavage) 53 mg/kg / imamectin benzoate-methyl tert-butyl ether (MBTE) solvate, purity 96.4%/
LD50 Rats (female) Oral (gavage) 58 mg/kg / imamectin benzoate monohydrate solvate, purity 99.1% pure product/
LD50 Mice (gavage) Oral (gavage) 107 mg/kg

[1]. Chenguang Niu, et al. Toxic effects of the Emamectin Benzoate exposure on cultured human bronchial epithelial (16HBE) cells. Environ Pollut. 2020 Feb;257:113618.
[2]. Shaorong Luan, et al. Emamectin benzoate induces ROS-mediated DNA damage and apoptosis in Trichoplusia Tn5B1-4 cells. Chem Biol Interact. 2017 Aug 1;273:90-98.
[3]. Özge Temiz, et al. Biopesticide emamectin benzoate in the liver of male mice: evaluation of oxidative toxicity with stress protein, DNA oxidation, and apoptosis biomarkers. Environ Sci Pollut Res Int. 2020 Jun;27(18):23199-23205.

Emamycin B1a is a type of emamycin drug. Avermectin is a series of macrolide derivatives with potent anthelmintic activity. In recent years, oral or injectable avermectin has become a common treatment method. Avermectin is a macrolide produced by the soil microorganism Streptomyces avermitilis through fermentation in carefully prepared laboratory culture media. They are active against a variety of livestock nematodes and arthropod parasites at doses of 300 μg/kg or lower. Unlike macrolide or polyene antibiotics, they lack significant antibacterial or antifungal activity. (L829) Mechanism of Action: Emamectin benzoate is the 4'-deoxy-4'-epio-methyl-aminobenzoate of avermectin B1 (avermectin), and its structure is similar to the natural fermentation product of Streptomyces avermitilis. Emamectin benzoate is being developed as a novel broad-spectrum insecticide for vegetables, requiring extremely low application rates. Its mechanism of action involves stimulating high-affinity GABA receptors, thereby increasing chloride ion permeability of cell membranes. Overexpression of P-glycoproteins (Pgps) is considered a major mechanism of macrolide resistance in nematodes and arthropods. This study used quantitative RT-PCR (Q-RT-PCR) to detect changes in the transcriptional levels of two putative P-glycoprotein genes (named SL0525 and SL-Pgp1, respectively) in sea lice (Lepeophtheirus salmonis) after exposure to emamectin benzoate (EMB). Larval L. salmonis were cultured in an EMB bioassay for 24 hours, and gene expression in sea lice surviving at EMB concentrations of 0, 10, and 30 ppb was analyzed. Gene expression levels were determined using Q-RT-PCR, with elongation factor 1 (eEF1α) as an internal control gene. The results showed that exposure to 10 ppb EMB significantly increased the expression levels of both target genes, SL0525 and SL-Pgp1 (p=0.11 and p=0.17, respectively), while in the group exposed to 30 ppb EMB, only SL-Pgp1 expression was nearly significant (p=0.053). At a concentration of 10 ppb EMB, the gene expression of both SL0525 and SL-Pgp1 increased more than fivefold. Therefore, when lice are exposed to EMB, the upregulation of these target genes may provide protection by increasing Pgp expression. Optimized quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) can be used to determine whether the overexpression of these genes is the basis for drug resistance in sea lice, thereby evaluating appropriate alternative chemotherapeutic treatments. Macrolides, including avermectin and milbemycin, are novel parasite and insecticides produced by fermentation by soil microorganisms. Although different macrolides may vary in efficacy and safety, they are believed to share a common pharmacological/toxicological mechanism: paralysis and death of parasites and other target organisms by activating glutamate-gated chloride channels in invertebrate nerve and muscle cells and/or acting on γ-aminobutyric acid (GABA) receptors. Ivermectin was the first macrolide approved for use in animals and humans, demonstrating excellent efficacy and good tolerability in treating parasitic infections. Other macrolides, such as avermectin, emamectin methylaminoavermectin, and moxicillin, have subsequently been commercialized and used as insecticides and acaricides for crop protection, or as parasite control agents for animal health.

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Therapeutic Use
Therapeutic Category (Veterinary): Antiparasitic Drugs

C56H81NO15

1008.2401

885.523

155569-91-8

11549937

White to off-white powder
Off-white crystalline powder

141-146ºC

4.628

3

14

9

63

1750

20

O1C([H])([H])C2=C([H])C([H])=C([H])[C@]([H])(C([H])([H])[H])[C@@]([H])(C(C([H])([H])[H])=C([H])C([H])([H])[C@]3([H])C([H])([H])[C@@]([H])(C([H])([H])[C@]4(C([H])=C([H])[C@]([H])(C([H])([H])[H])[C@@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])O4)O3)OC([C@]3([H])C([H])=C(C([H])([H])[H])[C@]([H])([C@]1([H])[C@]32O[H])O[H])=O)O[C@@]1([H])C([H])([H])[C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)O[C@@]1([H])C([H])([H])[C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O1)[N+]([H])([H])C([H])([H])[H])OC([H])([H])[H])OC([H])([H])[H].[O-]C(C1C([H])=C([H])C([H])=C([H])C=1[H])=O |c:8,23,t:4|

CXEGAUYXQAKHKJ-NSBHKLITSA-N

InChI=1S/C49H75NO13/c1-12-26(2)44-29(5)18-19-48(63-44)24-35-21-34(62-48)17-16-28(4)43(27(3)14-13-15-33-25-56-46-42(51)30(6)20-36(47(52)59-35)49(33,46)53)60-40-23-38(55-11)45(32(8)58-40)61-39-22-37(54-10)41(50-9)31(7)57-39/h13-16,18-20,26-27,29,31-32,34-46,50-51,53H,12,17,21-25H2,1-11H3/b14-13+,28-16+,33-15+/t26-,27-,29-,31-,32-,34+,35-,36-,37-,38-,39-,40-,41-,42+,43-,44+,45-,46+,48+,49+/m0/s1

(1'R,2R,3S,4'S,6S,8'R,10'E,12'S,13'S,14'E,16'E,20'R,21'R,24'S)-2-[(2S)-butan-2-yl]-21',24'-dihydroxy-12'-[(2R,4S,5S,6S)-4-methoxy-5-[(2S,4S,5S,6S)-4-methoxy-6-methyl-5-(methylamino)oxan-2-yl]oxy-6-methyloxan-2-yl]oxy-3,11',13',22'-tetramethylspiro[2,3-dihydropyran-6,6'-3,7,19-trioxatetracyclo[15.6.1.14,8.020,24]pentacosa-10,14,16,22-tetraene]-2'-one

Emamectin benzoate; Avermectin b1, 4''-deoxy-4''-(methylamino)-, (4''R)-, benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 31 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 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: ≥ 2.08 mg/mL (Infinity 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 20.8 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.)