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Purity: ≥98%
Indotecan (LMP400; NSC724998) is a novel, potent and selective topoisomerase I inhibitor with IC50 values of 300, 1200, 560 nM for P388, HCT116, MCF-7 cell lines, respectively. It is undergoing Phase I trials and may have anticancer activity. Inhibitors of human nuclear type IB topoisomerase (Top1) are potent and commonly used anticancer drugs.
| Targets |
Topoisomerase I
Indotecan is a poison (inhibitor/stabilizer) of DNA topoisomerase IB (TopIB), specifically targeting the bisubunit TopIB of Leishmania infantum. |
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| ln Vitro |
The Indotecan IC50 values for L. inhibition (48 hours). It was 0.10 μM, 0.10 μM, and 57.16 μM for fantum promastigotes, isolated infected splenocytes, and uninfected splenocytes, respectively [2]. 30 minutes) inhibits L. DNA synthesis and creates covalent complexes of TopI-DNA. infantum cultures [2].
Indotecan exhibited potent activity against Leishmania infantum promastigotes with an IC50 of 0.10 ± 0.08 µM after 48 hours of exposure.[1] In an ex vivo model using murine splenic explants infected with L. infantum amastigotes, Indotecan showed an IC50 of 0.10 ± 0.05 µM.[1] The cytotoxicity of Indotecan against uninfected murine splenocytes (mammalian cells) was determined, yielding an IC50 of 57.16 ± 6.01 µM. This resulted in a high in vitro selectivity index (SI) of 571.6 (SI = IC50 uninfected splenocytes / IC50 infected splenocytes).[1] In a supercoiled plasmid DNA cleavage assay, Indotecan induced DNA cleavage complexes mediated by L. infantum TopIB (LiTopIB) in a pattern similar to camptothecin (CPT), confirming its role as a TopIB poison. Cleavage complex intensity was strong at 100 µM.[1] Indotecan inhibited the DNA relaxation activity of recombinant LiTopIB more potently than human TopIB. LiTopIB was inhibited at 80 nM indotecan, whereas 2.5 µM was required to inhibit the human enzyme.[1] In L. infantum promastigotes, Indotecan induced the formation of SDS-KCl-precipitable DNA-protein complexes (TopIB-DNA cleavage complexes) in a concentration-dependent manner, reaching up to 20% of total labeled DNA at 10 µM.[1] Indotecan at 1 µM arrested DNA synthesis in exponentially growing L. infantum promastigotes by more than 90% at all time points measured (2-24 hours), as assessed by [2-14C]thymidine incorporation assay.[1] A competition study showed that pre-treatment with 5 µM Indotecan did not prevent CPT-mediated TopIB-DNA stabilization, indicating its primary mechanism is poisoning rather than DNA intercalation. |
| ln Vivo |
The livers of mice with visceral leishmaniasis and their own livers are less parasitized when indocean (2.5 mg/kg; intraperitoneally every 2 days for 15 days) is administered [2].
In a murine model of visceral leishmaniasis, BALB/c mice infected with L. infantum were treated intraperitoneally with Indotecan at a dose of 2.5 mg/kg body weight every other day for 15 days (total of 8 doses). This treatment resulted in a drastic and statistically significant reduction (P < 0.001) of the parasite burden in both the spleen and liver, clearing more than 80% of the parasites compared to the vehicle-treated control group. |
| Enzyme Assay |
Supercoiled Plasmid DNA TopIB-mediated Cleavage Assay: Purified LiTopIB (at least 100 U) was incubated with 0.5 µg of supercoiled pBluescript SK(-) phagemid DNA in a reaction buffer containing Tris-HCl, MgCl2, DTT, EDTA, BSA, and KCl. Indotecan at various concentrations (in 1% DMSO) was added to the reaction mixture. After incubation at 25°C for 4 minutes, reactions were stopped with SDS and digested with proteinase K. DNA was extracted with phenol-chloroform and analyzed by electrophoresis on a 1% agarose gel containing ethidium bromide. Cleavage products (nicked DNA) were visualized to assess drug-induced stabilization of TopIB-DNA cleavage complexes.[1]
DNA Relaxation Assay: Recombinant LiTopIB (1 unit) was pre-incubated with Indotecan at 4°C for 15 minutes. Then, a reaction mixture containing supercoiled plasmid DNA, Tris-HCl buffer, MgCl2, EDTA, BSA, and KCl was added. After incubation at 25°C for 4 minutes, reactions were stopped with SDS and digested with proteinase K. The extent of supercoiled DNA relaxation was assessed by electrophoresis on a 1% agarose gel. A second electrophoresis in the presence of a low concentration of ethidium bromide was performed to separate nicked DNA from relaxed topoisomers. |
| Cell Assay |
Ex Vivo Splenic Explant Culture for Anti-leishmanial Activity and Cytotoxicity: Splenocytes were isolated from BALB/c mice infected with L. infantum (for activity) or uninfected mice (for cytotoxicity). Single-cell suspensions were seeded and treated with various concentrations of Indotecan for 48 hours. For infected explants, the viability of intracellular amastigotes was assessed by measuring the infrared fluorescence (at 708 nm) of an IFP1.4-expressing L. infantum strain using an infrared imaging system. For cytotoxicity on uninfected explants, cell viability was determined using the alamarBlue staining method according to the manufacturer's instructions. Dose-response curves were generated, and IC50 values were calculated using nonlinear fitting with statistical software.[1]
SDS-KCl Precipitation Assay for TopIB-DNA Complex Formation in Cells: L. infantum promastigotes were pre-labeled for 24 hours with [2-14C]thymidine. The labeled cells were then exposed to different concentrations of Indotecan for 30 minutes. Cells were pelleted and lysed with SDS in the presence of carrier DNA and EDTA. KCl was added to precipitate protein-DNA complexes. The precipitate was collected by filtration through glass fiber filters, washed, and air-dried. Radioactivity was measured by liquid scintillation counting. The percentage of DNA in cleavage complexes was calculated relative to the total incorporated radioactivity.[1] Thymidine Incorporation Assay for DNA Synthesis Inhibition: Exponentially growing L. infantum promastigotes were incubated with [2-14C]thymidine in the presence of 1 µM Indotecan or solvent control. Aliquots were taken at various time points (2, 4, 6, 8, 10, and 24 hours). Cells were collected on glass fiber filters, and DNA was precipitated with ice-cold trichloroacetic acid, followed by sequential washes. Incorporated radioactivity was quantified by liquid scintillation counting to assess the rate of DNA synthesis. |
| Animal Protocol |
Animal/Disease Models: Female balb/c (Bagg ALBino) mouse (4-6 weeks) were injected intravenously (iv) (iv)(iv) with posterior circulating parasites through the tail vein [2]
Doses: 2.5 mg/kg per injection Body weight Route of Administration: intraperitoneal (ip) injection once every 2 days for a total of 15 days (8 doses total) Experimental Results: A dramatic reduction in the number of transformed amastigotes recovered from target organs of drug-treated animals was observed. Mouse Model of Visceral Leishmaniasis: Female BALB/c mice (4-6 weeks old) were infected intravenously via the tail vein with 10^7 metacyclic promastigotes of L. infantum. Treatment: Beginning 14 days post-infection, mice were treated intraperitoneally every other day for a total of 15 days (8 doses in total). Indotecan was dissolved in DMSO and then diluted in sterile saline solution. The administered dose was 2.5 mg/kg body weight per injection. The control group received the vehicle (DMSO in saline) alone. Assessment: Five days after the last treatment, mice were euthanized. Spleens and livers were aseptically removed, weighed, and homogenized in Schneider's medium supplemented with 20% heat-inactivated FBS. The parasite burden in each organ was determined by a limiting-dilution assay. Tissue homogenates were serially diluted and cultured for 10 days, after which the presence of motile promastigotes was examined to determine the number of parasites per gram of wet tissue. |
| References |
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| Additional Infomation |
Indotecan is an indomethacin compound initially developed as an anti-tumor drug. This study demonstrates its potential as an anti-leishmaniasis drug. It is a non-camptothecin TopIB toxin that stabilizes the cleavable complex between Leishmaniae TopIB and DNA, leading to DNA breakage and inhibition of DNA synthesis. Indotecan exhibits higher selectivity for Leishmaniae amastigotes in infected spleen cells compared to mammalian cells and demonstrates significant parasite clearance in a mouse model of visceral leishmaniasis, highlighting its potential as a candidate drug for further development of treatments for this neglected tropical disease.
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| Molecular Formula |
C26H26N2O7
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|---|---|
| Molecular Weight |
478.493847370148
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| Exact Mass |
478.174
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| Elemental Analysis |
C, 65.26; H, 5.48; N, 5.85; O, 23.41
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| CAS # |
915303-09-2
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| Related CAS # |
915303-09-2 (Indotecan; LMP-400); 915360-05-3 ( Indimitecan, LMP776)
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| PubChem CID |
11533060
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| Appearance |
White to gray solid powder
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| LogP |
2.619
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
35
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| Complexity |
872
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C1C2C3C(C(N(C=2C2C1=CC1=C(C=2)OCO1)CCCN1CCOCC1)=O)=CC(OC)=C(OC)C=3
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| InChi Key |
FMFIFGLHVOZDEL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H26N2O7/c1-31-19-10-15-18(13-20(19)32-2)26(30)28(5-3-4-27-6-8-33-9-7-27)24-16-11-21-22(35-14-34-21)12-17(16)25(29)23(15)24/h10-13H,3-9,14H2,1-2H3
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| Chemical Name |
15,16-dimethoxy-20-(3-morpholin-4-ylpropyl)-5,7-dioxa-20-azapentacyclo[10.8.0.02,10.04,8.013,18]icosa-1(12),2,4(8),9,13,15,17-heptaene-11,19-dione
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| Synonyms |
NSC-724998; NSC724998; NSC 724998; LMP-400; LMP400; LMP 400; Indotecan
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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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
DMSO: < 1 mg/mL
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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 | 2.0899 mL | 10.4495 mL | 20.8991 mL | |
| 5 mM | 0.4180 mL | 2.0899 mL | 4.1798 mL | |
| 10 mM | 0.2090 mL | 1.0450 mL | 2.0899 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT01051635 | Completed | Drug: LMP400 Drug: LMP776 |
Neoplasms Lymphoma |
National Cancer Institute (NCI) |
January 25, 2010 | Phase 1 |
| NCT01794104 | Completed | Drug: LMP400 | Neoplasm Lymphoma |
National Cancer Institute (NCI) |
May 19, 2016 | Phase 1 |
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