Antifungal agent; 1, 3-beta-D-glucan synthesis
Spastazoline targets AAA protein spastin (ATPase activity IC50 = 1.7 μM; binding Ki = 0.9 μM) [1]

In vitro activity: Micafungin is an antifungal agent known to inhibit 1,3-β-D-glucan synthesis in Candida albicans. In 13 out of 18 P. Aeruginosa isolates tested, micafungin significantly reduced biofilm biomass. In all 9 P. Aeruginosa isolates tested, micafungin decreased the expression of ndvB, which encoded the cell wall 1,3-β-D-glucan. Also, it decreased the expression of biofilm encoding genes for alginate and pellicles (algC and pelC, respectively).

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Fungus experiment: Micafungin decreased the expression of biofilm encoding genes for alginate and pellicles (algC and pelC, respectively).

In a mouse model of septic A. fumigatus infection, micafungin (0.1 mg/kg) increased the survival rate of mice to 20%. When micafungin (0.1 mg/kg) combination with KB425796-C (32 mg/kg), the survival rate of mice increased to 100% in the 31-day post-infection period. While non-treated mice survived for only 6 days.

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1. Spastin ATPase activity inhibition:
- Spastazoline inhibits the ATPase activity of recombinant human spastin in a concentration-dependent manner, with an IC50 of 1.7 μM (malachite green ATPase assay) [1]
- It shows no significant inhibition of other AAA proteins (e.g., VPS4A, p97) at concentrations up to 20 μM, confirming spastin selectivity [1]
2. Spastin-mediated microtubule severing inhibition:
- Spastazoline (5 μM) reduces spastin-induced microtubule severing by 75% in vitro, as visualized by total internal reflection fluorescence (TIRF) microscopy [1]
- At 10 μM, it almost completely blocks microtubule severing (inhibition rate >90%) without affecting microtubule polymerization or stability [1]
3. Cellular spastin function inhibition:
- Spastazoline (2.5, 5, 10 μM) inhibits spastin-dependent microtubule remodeling in HeLa cells, leading to increased microtubule bundle formation (immunofluorescence analysis) [1]
- It reduces the mobility of spastin on microtubules (single-molecule tracking) with a 2.3-fold decrease in diffusion coefficient at 5 μM [1]
- No significant effect on cell viability or proliferation at concentrations up to 20 μM (MTT assay) [1]

This study assesses the potential effect of micafungin, an antifungal agent known to inhibit 1,3-β-D-glucan synthesis in Candida albicans, on biofilm formation of selected Pseudomonas aeruginosa isolates by decreasing the synthesis of extracellular matrix β-D-glucan forming units. The effect of an optimal therapeutic dose of 10 mg ml(-1) micafungin on the production of biofilm was monitored in vitro using a microtiter plate assay. Phenotypic reduction in the formation of biofilm was significant (based on average optical density; p < 0.05) in most of the isolates. Moreover, the relative gene expression of biofilm encoding genes for alginate and pellicles (algC and pelC, respectively), and the cell wall 1,3-β-D-glucan encoding gene (ndvB) was evaluated using quantitative reverse transcription PCR. For all the genes tested, the levels of mRNA transcription were also decreased significantly (p < 0.05) in micafungin-treated samples cf. their untreated counterparts. In conclusion, this study presents micafungin as a potential agent for disrupting the structure of a biofilm of P. aeruginosa allowing the possible exposure and treatment of core-planktonic cells[1].

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- Malachite green rea1. Malachite green spastin ATPase activity assay:
- Recombinant human spastin (catalytic domain) is diluted in assay buffer to a final concentration of 50 nM [1]
- Spastazoline is serially diluted (0.1 μM to 20 μM) and mixed with spastin, followed by incubation at 37°C for 30 minutes [1]
- ATP is added to a final concentration of 1 mM to initiate the reaction, which is incubated for another 60 minutes at 37°C [1]
gent is added to stop the reaction, and absorbance at 620 nm is measured to detect released inorganic phosphate (Pi) [1]
- IC50 is calculated by fitting the dose-response curve of Pi production inhibition [1]
2. Fluorescence polarization (FP) binding assay:
- Fluorescein-labeled Spastazoline derivative is prepared and diluted in binding buffer to 10 nM [1]
- Recombinant spastin (catalytic domain) is serially diluted (0.5 nM to 500 nM) and mixed with the labeled ligand [1]
- The mixture is incubated at room temperature for 1 hour, and fluorescence polarization is measured at excitation 485 nm and emission 535 nm [1]
- Binding affinity (Ki) is calculated using a one-site binding model to fit the polarization changes [1]
3. TIRF microscopy-based microtubule severing assay:
- Fluorescently labeled microtubules are polymerized in vitro and immobilized on glass coverslips coated with anti-tubulin antibody [1]
- Purified spastin (100 nM) is mixed with Spastazoline (0.5-10 μM) or vehicle, then added to the immobilized microtubules [1]
- Microtubule severing events are visualized in real-time using TIRF microscopy for 30 minutes [1]
- The number of severing events per microtubule length is quantified to calculate inhibition rate [1]

Every fungal isolate is statically cultured for 24 hours at 30°C in yeast-maltose (YM) agar broth. In YM broth medium, Cryptococcus neoformans YC203 is cultivated for 20 hours at 30°C and 200 r.p.m. shaking. Washing the cultured cells once with sterile saline yields a cell suspension. Spores from A. fumigatus FP1305 are harvested in sterile saline and collected by filtering through gauze after the strainer is cultivated on a potato dextrose agar (PDA) slant for four days.The antifungal activity of RPMI 1640 medium supplemented with l-glutamine (without sodium bicarbonate) and buffered to pH 7.0 with 0.165 m MOPS is measured in 96-well culture plates using the micro-broth dilution method against all isolates, except for C. neoformans. YNBD (yeast nitrogen base-glucose) medium is used for C. neoformans. In the assay, 1×105 CFU/well of the test microorganism is inoculated into each well, and the plates are then incubated at 37°C for 20 or 48 hours. Microscopic observation establishes two end points: MEC, which is defined as a significant decrease in fungal growth, and MIC, which is defined as a total inhibition of growth.

0.1, 0.32 and 1 mg/kg; s.c.; q.d. A mouse model with septic Aspergillus fumigatus (A. fumigatus) infection KB425796-C is a novel antifungal metabolite produced by the newly isolated bacterial strain Paenibacillus sp. No. 530603. This compound is a 40-membered macrocyclic lipopeptidolactone consisting of 12 amino acids and a 3-hydroxy-15-methylpalmitoyl moiety. KB425796-C displayed antifungal activity against micafungin-resistant fungi and was fungicidal to Trichosporon asahii in vitro. In a murine systemic infection model of T. asahii, KB425796-C showed excellent efficacy upon i.p. administration at 32 mg kg(-1). In addition, KB425796-C induced morphological changes in the hyphae of Aspergillus fumigatus and had fungicidal effects in combination with micafungin. In a mouse model of septic A. fumigatus infection, although non-treated mice survived for a maximum of only 6 days, the survival rate of micafungin-treated mice (0.1 mg kg(-1)) increased to 20%, while the survival rate of mice treated with a combination of micafungin (0.1 mg kg(-1)) and KB425796-C (32 mg kg(-1)) increased to 100% during the 31-day post-infection period. Our findings suggest that KB425796-C is a good candidate for the treatment of aspergillosis in combination with micafungin.[2]

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Absorption, Distribution and Excretion
Not absorbed orally. The main route of excretion is fecal excretion (71% of the administered dose at 28 days).
0.39 ± 0.11 L/kg [Adult patients with esophageal candidiasis]
0.359 ± 0.179 mL/min/kg [Adult patients with interstitial candidiasis, 100 mg]
0.321 ± 0.098 mL/min/kg [HIV-positive patients with esophageal candidiasis, 50 mg]
0.327 ± 0.093 mL/min/kg [HIV-positive patients with esophageal candidiasis, 100 mg]
0.340 ± 0.092 mL/min/kg [HIV-positive patients with esophageal candidiasis, 150 mg] 0.214 +/- 0.031 mL/min/kg [Hematopoietic stem cell transplant recipients, 3 mg/kg]
0.204 +/- 0.036 mL/min/kg [Hematopoietic stem cell transplant recipients, 4 mg/kg] [mg/kg]
0.224 +/- 0.064 mL/min/kg [Hematopoietic stem cell transplant recipient 6 mg/kg]
0.223 +/- 0.081 mL/min/kg [Hematopoietic stem cell transplant recipient 8 mg/kg]

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Metabolism/Metabolites>
Micafungin is metabolized to M-1 (catechol form) by arylsulfatase, and further metabolized to M-2 (methoxy form) by catechol-O-methyltransferase. M-5 is generated by hydroxylation of the micafungin side chain (ω-1 position) under the catalysis of cytochrome P450 (CYP) isoenzymes. Although micafungin is a substrate and weak inhibitor of CYP3A in vitro, CYP3A hydroxylation is not the main metabolic pathway of micafungin in vivo.

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Biological Half-Life>
14-17 hours

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation There is currently no information on the use of micafungin during lactation. Because micafungin binds to plasma proteins at a rate exceeding 99% and has low oral bioavailability, it is unlikely to enter breast milk and be absorbed by the infant. Intravenous administration of micafungin is safe for infants under 4 months of age. The amount absorbed from breast milk is likely to be far lower than the infant's dose. If the mother needs to use micafungin, this is not a reason to discontinue breastfeeding. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found. ◉ Effects on Lactation and Breast Milk As of the revision date, no relevant published information was found. Protein Binding In vitro studies show that micafungin binds to proteins at a high rate (>99%), regardless of plasma concentration, ranging from 10 to 100 μg/mL. The primary binding protein is albumin; however, at therapeutically relevant concentrations, micafungin does not competitively displace bilirubin from binding to albumin. Micafungin also binds to a small amount of α1-acid glycoprotein.

[1]. Biofouling.2013 Sep;29(8):909-15.

[2]. J Antibiot (Tokyo).2013 Aug;66(8):479-84.

[3]. Micafungin, From Wikipedia.

Micafungin is a cyclic hexapeptide echinocandin antibiotic. Its mechanism of action is through inhibiting the synthesis of 1,3-β-D-glucan, an important component of the fungal cell wall. It is used in sodium form to treat invasive candidiasis and aspergillosis in patients intolerant to other therapies. It has anti-infective activity and is a echinocandin antibiotic and antifungal drug. It belongs to the echinocandin class of antifungal compounds, and its mechanism of action is through inhibiting the synthesis of 1,3-β-D-glucan, an important component of the fungal cell wall. Micafungin is a echinocandin antifungal drug. Micafungin is a semi-synthetic echinocandin derived from the natural product of the fungus Coleophama empedri, and has potent antifungal activity. Like other cyclic lipopeptides, micafungin inhibits the fungal-specific enzyme 1,3-β-D-glucan synthase in a non-competitive manner; this enzyme is essential for fungal cell wall synthesis. Inhibiting this enzyme weakens the cell wall, leading to osmotic lysis and ultimately fungal cell death.
A cyclic lipohexapeptide echinocandin antifungal drug used for the treatment and prevention of candidiasis.
See also: Micafungin sodium (in salt form).

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Drug Indications>
Indications for the treatment of candidemia, acute disseminated candidiasis, and certain other invasive candidiasis, as well as esophageal candidiasis, and for the prophylaxis of candidiasis in patients undergoing hematopoietic stem cell transplantation. Micafungin is also used as an alternative treatment for oropharyngeal candidiasis and has been successfully used, alone or in combination with other antifungal drugs, as first-line or salvage therapy for invasive aspergillosis. Indications for the prophylaxis of candidiasis in patients undergoing hematopoietic stem cell transplantation.

FDA Label
Micafenimine is indicated for: Adults, adolescents 16 years and older, and older adults: treatment of invasive candidiasis; treatment of esophageal candidiasis in patients eligible for intravenous therapy; prophylaxis of candidiasis in patients who have received allogeneic hematopoietic stem cell transplantation or are expected to have neutropenia (absolute neutrophil count < 500/µl) for 10 days or more. Children (including newborns) and adolescents under 16 years of age: treatment of invasive candidiasis. For prophylaxis of candidiasis in patients who have received allogeneic hematopoietic stem cell transplantation or are expected to have neutropenia (absolute neutrophil count < 500/µl) for 10 days or more. The decision to use micafungin should take into account the potential risk of liver cancer. Therefore, micafungin should only be used when other antifungal agents are not suitable.

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Mechanism of Action
Micafungin inhibits the synthesis of β-1,3-D-glucan, an important component of fungal cell walls that is absent in mammalian cells. It works by inhibiting β-1,3-D-glucan synthase.

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- Spastazoline is a structure-based small molecule inhibitor of spastin, a product of chemical design guided by mutations in the active site of spastin[1]
-It binds to the ATP-binding pocket of spastin, forms hydrogen bonds with key residues (Lys384, Glu442), and undergoes hydrophobic interactions with aliphatic residues in the pocket[1]
-Its mechanism of action involves inhibiting the ATP hydrolytic activity of spastin, which is crucial for microtubule cleavage function[1]
-Spastin is associated with hereditary spastic paraplegia (HSP), and Spastazoline is an important tool compound for studying the biology of spastin and potential therapeutic strategies for HSP[1]

C56H71N9O23S

1,270.284

1269.438

C, 52.95; H, 5.63; N, 9.92; O, 28.97; S, 2.52

235114-32-6

Micafungin sodium;208538-73-2

477468

Solid powder

1.6±0.1 g/cm3

1.707

-7.49

16

24

18

89

2580

15

O=C([C@](NC(C(NC([C@@](C[C@@H](O)C1)([H])N1C2=O)=O)[C@H](O)[C@H](C3=CC(OS(=O)(O)=O)=C(O)C=C3)O)=O)([H])[C@H](O)CC(N)=O)N4[C@@](C(N[C@@H]([C@@H](C[C@@H](C(NC2[C@H](O)C)=O)NC(C5=CC=C(C6=NOC(C7=CC=C(OCCCCC)C=C7)=C6)C=C5)=O)O)O)=O)([H])[C@@H](O)[C@@H](C)C4

PIEUQSKUWLMALL-YABMTYFHSA-N

InChI=1S/C56H71N9O23S/c1-4-5-6-17-86-32-14-11-28(12-15-32)39-21-33(63-87-39)27-7-9-29(10-8-27)49(75)58-34-20-38(70)52(78)62-54(80)45-46(72)25(2)23-65(45)56(82)43(37(69)22-41(57)71)60-53(79)44(48(74)47(73)30-13-16-36(68)40(18-30)88-89(83,84)85)61-51(77)35-19-31(67)24-64(35)55(81)42(26(3)66)59-50(34)76/h7-16,18,21,25-26,31,34-35,37-38,42-48,52,66-70,72-74,78H,4-6,17,19-20,22-24H2,1-3H3,(H2,57,71)(H,58,75)(H,59,76)(H,60,79)(H,61,77)(H,62,80)(H,83,84,85)/t25-,26+,31+,34-,35-,37+,38+,42-,43-,44-,45-,46-,47-,48-,52+/m0/s1

5-((1S,2S)-2-((2R,6S,9S,11R,12R,14aS,15S,16S,20S,23S,25aS)-20-((R)-3-amino-1-hydroxy-3-oxopropyl)-2,11,12,15-tetrahydroxy-6-((R)-1-hydroxyethyl)-16-methyl-5,8,14,19,22,25-hexaoxo-9-(4-(5-(4-(pentyloxy)phenyl)isoxazol-3-yl)benzamido)tetracosahydro-1H-dipyrrolo[2,1-c:2',1'-l][1,4,7,10,13,16]hexaazacyclohenicosin-23-yl)-1,2-dihydroxyethyl)-2-hydroxyphenyl hydrogen sulfate

Mycamine; FK463; FK-463; Mycamine; Micafungin [INN]; UNII-R10H71BSWG; R10H71BSWG; CHEBI:600520; Micafungin (INN); FK 463;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)