Interactions
Alcalase (9014011), Savinase (9014011), Termoyl (9000902), or Lipolase (9001621) in binary or ternary mixtures at concentrations of 0 to 0.3 μg were administered weekly via intratracheal infusion for 10 weeks. Alcalase and Savinase are proteases, Termoyl is an α-amylase, and Lipolase is a lipase. The mixture always contains either Alcalase or Savinase, or Termoyl, Lipolase, or both. Some mixtures contain Alcalase inactivated by pretreatment with hexamethylene isocyanate or diisopropyl fluorophosphate. Control animals were given only the single enzyme. Animals were observed for signs of respiratory distress, such as periodic diaphragmatic spasms or retractions. Blood samples were collected from the retro-orbital venous plexus before exposure and at 4, 5, 6, 8, or 10 weeks after exposure. Serum was isolated and circulating immunoglobulin (Ig)-G1 isotropic antibodies (HARs) were analyzed by passive cutaneous anaphylaxis. The number and severity of respiratory symptoms in guinea pigs exposed to mixtures containing Alcalase or Savinase were not significantly different from those exposed to Alcalase or Savinase alone. HAR titers against Lipolase and Termonyl were significantly increased when mixed with Alcalase or Savinase. The presence of other enzymes in the mixture did not significantly increase HAR titers against Alcalase or Savinase. The enhanced response produced in mixtures containing Alcalase or Savinase disappeared when inactivated forms of the enzymes were used in the mixture. Other guinea pigs were inhaled daily for 20 minutes for 5 days with a mixture of 500 μg/m³ lipase or lipase and 2000 μg/m³ alkaline protease. Challenge tests were performed again with the same aerosol 14 or 21 days after the last exposure. Blood samples were collected, and lipase-specific IgG antibodies were analyzed by enzyme-linked immunosorbent assay (ELISA). Co-exposure to alkaline protease significantly increased the level of lipase IgG antibodies compared to exposure to lipase alone. The authors concluded that the presence of proteolytic enzymes such as alkaline protease and saverase in the mixture enhances antibody responses to other enzymes in the mixture. This should be considered when developing exposure guidelines for mixtures containing proteases.

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Non-human toxicity values
Oral LD50 in rats: 3700 mg/kg

[1]. Philipp M, et al. Kinetics of subtilisin and thiolsubtilisin. Mol Cell Biochem. 1983;51(1):5-32.
[2]. Azrin NAM, et al. Versatility of subtilisin: A review on structure, characteristics, and applications. Biotechnol Appl Biochem. 2022 Dec;69(6):2599-2616.

Reports indicate that Bacillus subtilis and Saccharomyces cerevisiae contain bacillus toxins, and relevant data is available for reference.

0

9014-01-1

3086051

Light-colored, free-flowing powders
Light-colored prills

1.3

13

15

18

70

1860

8

N#CC1=CC=C(CCl)C=C1

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)

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

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)