Topoisomerase I; Camptothecins
DNA topoisomerase I (IC50 values ranging from 0.01 to 0.1 μM in various tumor cell lines) [1][2][3]

In vitro activity: SN-38 is an irinotecan hydrochloride (CPT-11) biologically active metabolite. The greatest inhibition of DNA topoisomerase I is caused by SN-38, which is followed by CPT and CPT-11. Relaxed DNA is dose-dependently shifted in the direction of nicked DNA by CPT-11, while SN-38 and CPT have no effect on relaxed DNA position. DNA synthesis is inhibited by SN-38 both time- and dose-dependently. SN-38's respective IC50 values for DNA synthesis are 0.077 μM. SN-38 does not prevent protein synthesis from occurring, but its inhibitory effect on RNA synthesis is smaller than that of DNA synthesis. In P388 cells, SN-38 frequently caused single-strand breaks in the DNA.[1]

---

In human colon cancer cell lines (HT-29, HCT-116, SW480), SN-38 (NK012) exhibited potent concentration-dependent antiproliferative activity, with IC50 values between 0.02 and 0.08 μM. The antiproliferative effect was more pronounced with NK012 compared to free SN-38 due to enhanced cellular uptake [4]
- SN-38 (NK012) induced S-phase cell cycle arrest in colon cancer cells, accompanied by upregulation of p21 and downregulation of cyclin E, leading to inhibition of DNA replication [2][4]
- The drug triggered apoptosis in tumor cells, as evidenced by increased caspase-3 activation, PARP cleavage, and DNA fragmentation. NK012 showed higher apoptotic induction efficiency than free SN-38 at the same concentration [3][4]
- In human lung cancer A549 cells, SN-38 inhibited DNA topoisomerase I activity, stabilizing the enzyme-DNA cleavage complex and inducing DNA single-strand breaks, which contributed to its antitumor effect [1]
- NK012, as a SN-38-loaded nanoparticle formulation, released SN-38 in a sustained manner in vitro, maintaining effective drug concentrations for over 72 hours [4]

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 nude mice bearing human colon cancer HT-29 xenografts, intravenous administration of SN-38 (NK012) at doses of 10-30 mg/kg (based on SN-38 equivalent) significantly inhibited tumor growth, with tumor volume reduction rates of 60-80% after 4 weeks of treatment. NK012 showed superior antitumor efficacy compared to free SN-38 at the same dose [4]
- In murine models of lung cancer, SN-38 administered intravenously at 5-15 mg/kg exhibited dose-dependent tumor growth inhibition, with prolonged survival time in treated mice compared to control groups [1]
- NK012 demonstrated enhanced tumor targeting in vivo, with higher SN-38 accumulation in tumor tissues and lower distribution in normal organs (liver, kidney, spleen) compared to free SN-38 [4]
- In a phase I clinical study-related preclinical model, SN-38 (NK012) showed activity against refractory solid tumors, including colorectal and non-small cell lung cancer [3]

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.

---

DNA topoisomerase I activity assay: Purified human DNA topoisomerase I was incubated with supercoiled pBR322 DNA in reaction buffer at 37°C. SN-38 was added at serial concentrations (0.001-1 μM), and the mixture was incubated for 30 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 50°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The intensity of relaxed DNA bands was quantified to assess enzyme inhibition. SN-38 was found to stabilize the topoisomerase I-DNA cleavage complex, reducing the formation of relaxed DNA [1][2]

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.

---

Tumor cell antiproliferative assay: Colon cancer (HT-29, HCT-116) and lung cancer (A549) cells were seeded in 96-well plates at 3×10³ cells/well. Cells were treated with free SN-38 or NK012 (at SN-38 equivalent concentrations of 0.001-1 μM) and incubated at 37°C with 5% CO₂ for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay, and IC50 values were calculated from dose-response curves [2][4]
- Cell cycle analysis: HT-29 cells were treated with SN-38 (NK012) at 0.05 μM for 24-48 hours. Cells were harvested, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in G0/G1, S, and G2/M phases was determined to evaluate cell cycle arrest [2][4]
- Apoptosis assay: HCT-116 cells were treated with NK012 (0.03 μM SN-38 equivalent) for 48 hours. Cells were stained with annexin V-FITC and propidium iodide, then analyzed by flow cytometry to distinguish early (annexin V-positive/PI-negative) and late (annexin V-positive/PI-positive) apoptotic cells. Western blot analysis was performed to detect caspase-3 activation and PARP cleavage [3][4]
- Nanoparticle cellular uptake assay: HT-29 cells were incubated with fluorescently labeled NK012 for 1-4 hours. Cells were washed, fixed, and observed under a confocal laser scanning microscope to quantify intracellular SN-38 accumulation [4]

Mice: Mice of similar genetic background (>99% FVB) are used, including female wild-type, Slco1a/1b(−/−) (Oatp1a/1b knockout), Slco1a/1b(−/−);1B1(tg), and Slco1a/1b(−/−);1B3(tg) (liver-specific OATP1B1 and OATP1B3 humanized transgenic). The mice are aged 8 to 14 weeks. Mice receive an intravenous injection of 5 μL/g bodyweight of irinotecan (20 mg/mL in a water-based solution containing NaOH, lactic acid, and sorbitol) diluted with saline to a concentration of 10 mg/kg. To achieve a dosage of 1 mg/kg, SN-38 (NK012) is dissolved in DMSO (1 mg/mL) and given intravenously to mice at a rate of 1 μL/g body weight. The experiments end with isoflurane anesthesia, tissue collection, heparin-blood sampling via cardiac puncture, and cervical dislocation. Plasma is collected and kept at -30°C until analysis, while blood samples are centrifuged at 5,200 × g for 5 minutes at 4°C.

---

Colon cancer xenograft model: Nude mice were subcutaneously inoculated with HT-29 cells (5×10⁶ cells/mouse) to establish tumor xenografts. When tumors reached a volume of 100-150 mm³, mice were randomly divided into groups (n=6-8 per group). Free SN-38 or NK012 was dissolved in sterile saline and administered intravenously via the tail vein. Doses were 10, 20, or 30 mg/kg (based on SN-38 equivalent), with administration every 7 days for a total of 4 doses. Tumor volume and body weight were measured twice weekly. At the end of the experiment, tumors and major organs were collected for drug concentration analysis and histopathological examination [4]
- Lung cancer model: C57BL/6 mice were intravenously injected with Lewis lung cancer cells (2×10⁵ cells/mouse) to establish metastatic lung tumors. Mice were treated with SN-38 at 5, 10, or 15 mg/kg via intravenous injection once every 5 days for 3 cycles. Mice were euthanized 21 days after tumor cell inoculation, and lung metastatic nodules were counted to evaluate antitumor efficacy [1]
- Pharmacokinetic study: Rats were administered NK012 or free SN-38 intravenously at 20 mg/kg SN-38 equivalent. Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-administration. Plasma SN-38 concentrations were measured by HPLC, and pharmacokinetic parameters were calculated [4]

Absorption: Due to extensive first-pass metabolism, the oral bioavailability of SN-38 is poor (<10%); NK012 can achieve rapid systemic exposure by intravenous administration [3][4] - Distribution: NK012 enhances tumor distribution through enhanced permeability and retention (EPR) effects, and the tumor concentration of SN-38 is 3-5 times higher than that of free SN-38. Free SN-38 is widely distributed in normal tissues, with high concentrations in the liver and intestines [4] - Metabolism: SN-38 is metabolized in the liver by glucuronidation to inactive SN-38G, which is excreted via bile [3] - Excretion: Most SN-38 and its metabolites are excreted via bile (>70%), and a small amount is excreted via urine (<10%) [3] - Pharmacokinetic parameters: NK012 prolongs the half-life of SN-38 (t₁/₂ = 8-12 hours) compared to free SN-38 (t₁/₂ = 2-4 hours). The area under the curve (AUC) of SN-38 in the NK012 formulation was 2-3 times higher than that of free SN-38 [4]
- Plasma protein binding rate: SN-38 was 95-97% bound to plasma proteins [3]

Gastrointestinal toxicity: Free SN-38 can cause dose-dependent diarrhea (30-50% incidence at 15 mg/kg), while NK012 reduces gastrointestinal exposure, with the incidence of diarrhea decreasing to 10-15% at the same equivalent dose of SN-38 [3][4] - Myelosuppression: SN-38 (NK012) can cause mild to moderate dose-dependent myelosuppression, characterized by leukopenia and thrombocytopenia. The severity of NK012 and free SN-38 is comparable at equivalent doses [3] - Hepatotoxicity: High doses of free SN-38 (>30 mg/kg) can cause a mild increase in liver transaminases, while the increase in liver transaminases caused by NK012 is milder [4]

[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 belongs to the pyranoindoquinoline class of compounds, with the structure (4S)-pyrano[3',4':6,7]indo[1,2-b]quinoline-3,14-dione, containing two ethyl substituents at positions 4 and 11, and two hydroxyl substituents at positions 4 and 9. It is the active metabolite of irinotecan, with an activity approximately 1000 times greater than that of irinotecan. SN-38 possesses multiple functions, including inducing apoptosis, inhibiting EC 5.99.1.2 (DNA topoisomerase), acting as a drug metabolite, and as an antitumor drug. It is a pyranoindoquinoline compound belonging to the δ-lactone, tertiary alcohol, and phenolic compounds.
7-Ethyl-10-hydroxycamptothecin (SN 38) is a liposomal formulation of the active metabolite of irinotecan [DB00762], a pro-chemotherapeutic agent approved for the treatment of advanced colorectal cancer. SN 38 has been used in trials to treat various cancers, including cancer, advanced solid tumors, small cell lung cancer, metastatic colorectal cancer, and triple-negative breast cancer.
7-Ethyl-10-hydroxycamptothecin has been reported to be present in the Chinese honeybee (Apis cerana), and relevant data exist.
A semi-synthetic camptothecin derivative, it inhibits DNA topoisomerase I, thereby preventing S-phase nucleic acid synthesis. It is used as an antitumor drug for the treatment of colorectal and pancreatic tumors.

---

Drug Indications
Studied for the treatment of colorectal cancer.

---

Mechanism of Action
SN-38 is encapsulated in liposomes, making it more stable and soluble. This enhances the affinity of SN-38 for the lipid membrane and improves drug delivery to the tumor site. SN-38 is a highly potent cytotoxic topoisomerase I inhibitor.

---

Pharmacodynamics
SN-38 (7-ethyl-10-hydroxycamptothecin) is the active metabolite of irinotecan (CPT-11), a topoisomerase I inhibitor marketed under the brand name Camptosar®. SN-38 is 200-2000 times more cytotoxic than CPT-11, but it has not been used as an anticancer drug due to its poor solubility in pharmaceutical solvents and low affinity for the lipid membrane. Furthermore, SN-38 undergoes reversible conversion at physiological pH, forming an inactive open-ring lactone structure. LE-SN-38 is a novel liposomal formulation with a uniform liposome size distribution (<200 nm). The drug encapsulation efficiency of this formulation is >95%.

---

SN-38 is the active metabolite of irinotecan, a camptothecin derivative, with antitumor activity 100-1000 times higher than irinotecan [1][3]
- Mechanism of action: SN-38 binds to DNA topoisomerase I, stabilizing the enzyme-DNA cleavage complex, preventing DNA reconnection, and inducing DNA damage, ultimately leading to cell cycle arrest and apoptosis [1][2][3]
- NK012 is a polymer nanoparticle formulation loaded with SN-38, designed to improve solubility, enhance tumor targeting through the EPR effect, and reduce systemic toxicity [4]
- Clinical application potential: SN-38 (NK012) has shown activity against irinotecan-resistant tumors, including colorectal cancer and lung cancer. In preclinical studies, SN-38 has been used to treat breast cancer [3][4]
- Drug interactions: UGT1A1 inhibitors inhibit SN-38 Metabolism of these substances may increase their plasma concentration and toxicity[3]

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.

---

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.

---

View More

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.

---

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