Topoisomerase I; Camptothecins

Peak SN-38 concentrations occur within one hour following oral dosing, and at 1000 ng/mL, the percent unbound SN-38 lactone in murine plasma is 3.4 +/- 0.67%, while at 100 ng/mL, it is 1.18 +/- 0.14%. In comparison to nontumor-bearing animals, the SN-38 lactone AUCs of mice harboring human neuroblastoma xenografts are higher.[2]

In the reaction buffer, which is made up of 25 mM Tris-HCl (pH 7.5), 50 mM KC1, 5 mM MgCl₂, 0.25 mM EDTA disodium salt, 0.25 mM dithiothreitol, 15μg /mL bovine serum albumin, and 5% glycerol, one unit (the minimum amount for full relaxation of 0.5 μg SV40 DNA under the conditions of this study), 0.5 μL of the test compounds, and 0.5μg SV40 DNA are added successively to the reaction buffer. The 50 μL reaction mixture is then incubated for 10 minutes at 37°C. Finally, the reaction is stopped by treating the mixture for 30 more minutes at 37°C with 7.5 μL of a solution that contains 0.5 mg/mL proteinase K, 20 mM EDTA disodium salt, and 1% sodium dodecyl sulfate. The samples are combined with 5 μL of the loading buffer, which has 0.3% bromophenol blue, 31.3% sucrose, and 10 mM NaH₂PO₄. Form I (supercoiled) and form II (nicked) DNA are separated from relaxed (form Ir) DNA using electrophoresis on an 0.8% agarose gel at 50 mA and 20 V for 17 hours while 2 μg/mL chloroquine, 10 mM EDTA, 30 mM NaH₂PO₄, and 36 mM Tris-HCl (pH 7.8) are present. Following electrophoresis, 0.05% ethidium bromide staining is applied to the gel, and UV light (302 nm) photography is taken. A densitometer is used to measure the quantity of DNA.

The MTT assay is used to assess the sensitivity of SN-38 (NK012) in vitro. After seeding cells in 96-well plates, SN-38 (NK012) at various concentrations is added the next day. The medium is discarded after 48 hours of drug exposure, and the plates are then incubated for three hours in medium containing MTT (0.5 mg/mL). The formed formazan is dissolved by adding 20% sodium dodecyl sulphate, which has been acidified (0.02 M HCl). Cell viability is computed as a percentage relative to untreated cells using the optical density at 570 nm (with a background of 670 nm). The mean IC50 value ± standard deviation is found after three iterations of the experiments. The calculation of relative resistance for each resistant cell line involves dividing its mean IC50 value by the mean IC50 value of the corresponding parental cell line.

[1]. Cancer Res . 1991 Aug 15;51(16):4187-91.

[2]. Cancer Chemother Pharmacol, 1997, 40(3), 259-265.

[3]. Cancer . 2003 May 1;97(9 Suppl):2363-73.

[4]. Int J Nanomedicine . 2018 Dec 20:14:75-85.

SN-38 is a member of the class of pyranoindolizinoquinolines that is (4S)-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione bearing two additional ethyl substituents at positions 4 and 11 as well as two additional hydroxy substituents at positions 4 and 9. It is the active metabolite of irinotecan and is ~1000 times more active than irinotecan itself. It has a role as an apoptosis inducer, an EC 5.99.1.2 (DNA topoisomerase) inhibitor, a drug metabolite and an antineoplastic agent. It is a pyranoindolizinoquinoline, a delta-lactone, a tertiary alcohol and a member of phenols.
7-ethyl-10-hydroxycamptothecin (SN 38) is a liposomal formulation of the active metabolite of Irinotecan [DB00762], a chemotherapeutic pro-drug approved for the treatment of advanced colorectal cancer. SN 38 has been used in trials studying the treatment of Cancer, Advanced Solid Tumors, Small Cell Lung Cancer, Metastatic Colorectal Cancer, and Triple Negative Breast Cancer, among others.
7-Ethyl-10-hydroxycamptothecin has been reported in Apis cerana with data available.
A semisynthetic camptothecin derivative that inhibits DNA TOPOISOMERASE I to prevent nucleic acid synthesis during S PHASE. It is used as an antineoplastic agent for the treatment of COLORECTAL NEOPLASMS and PANCREATIC NEOPLASMS.

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Drug Indication
Investigated for use/treatment in colorectal cancer.

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Mechanism of Action
The entrapment of SN-38 in lipsomes results in a more stable and more soluble form of the drug. This allows for increased affinity of SN-38 to lipid membranes and improved delivery of the drug to tumor sites. SN-38 is a highly effective cytotoxic topoisomerase I inhibitor.

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Pharmacodynamics
SN-38 (7-ethyl-10-hydroxycamptothecin) is the active metabolite of Irinotecan (CPT-11). Irinotecan is a topoisomerase I inhibitor commercially available as Camptosar®. SN-38 has been found to be 200–2000 times more cytotoxic than CPT-11, but has not been used as an anticancer drug due to its poor solubility in pharmaceutically acceptable solvents and low affinity to lipid membranes. SN-38 also undergoes a reversible conversion to an inactive open lactone ring structure at physiological pH. LE-SN-38 is a novel lipsome based formulation containing liposomes of uniform size distribution (<200 nm). Drug entrapment efficiency of the formulation is>95%.

C22H20N2O5

392.4

392.137

C, 67.34; H, 5.14; N, 7.14; O, 20.39

86639-52-3

104842

Light yellow solid powder

1.5±0.1 g/cm3

810.3±65.0 °C at 760 mmHg

217 °C

443.8±34.3 °C

0.0±3.0 mmHg at 25°C

1.738

2.31

2

6

2

29

820

1

C(C1C2C=C(C=CC=2N=C2C3=CC4[C@@](C(OCC=4C(=O)N3CC=12)=O)(O)CC)O)C

FJHBVJOVLFPMQE-QFIPXVFZSA-N

InChI=1S/C22H20N2O5/c1-3-12-13-7-11(25)5-6-17(13)23-19-14(12)9-24-18(19)8-16-15(20(24)26)10-29-21(27)22(16,28)4-2/h5-8,25,28H,3-4,9-10H2,1-2H3/t22-/m0/s1

(19S)-10,19-diethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2,4(9),5,7,10,15(20)-heptaene-14,18-dione

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)

Solubility in Formulation 1: 2.5 mg/mL (6.37 mM) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 2.08 mg/mL (5.30 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: 2.08 mg/mL (5.30 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)