Topoisomerase I ( IC50 = 679 nM ); Camptothecins
Camptothecin (Campathecin) targets DNA topoisomerase I (Topo I) with an IC50 of 0.1 μM for inhibiting enzyme-mediated DNA relaxation [3]

Camptothecin, a plant alkaloid, was first isolated from Camptotheca acuminate in 1966.[1] It has been observed that camptothecin stops cells in their tracks during the S phase of mitosis. With IC50 values ranging from 37 nM to 48 nM, camptothecin demonstrates nanomolar potency in cytotoxicity against numerous human tumor cell lines, including HT29, LOX, SKOV3, and SKVLB.[2] At an IC50 of 13 μM, camptothecin causes apoptosis in primary mouse hepatocytes when combined with TNF. Moreover, camptothecin inhibited the expression of TNF-receptor associated factor 2 (TRAF2), X-linked inhibitor of apoptosis protein (X-IAP), and FLICE-inhibitory protein (FLIP), as well as the TNF-induced NF-κB Activation.[4] In HCT116 cells, camptothecin (5 μM) causes the mixed lineage leukemia 5 (MLL5) protein to be broken down by proteasomes, which phosphorylates p53 at Ser392. [5] Owing to camptothecin's poor solubility and unfavorable effects, a number of camptothecin analogues have been created; two of these, topotecan and irinotecan, have FDA approval and are used in cancer chemotherapy.

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Camptothecin (Campathecin) (0.01-5 μM) dose-dependently inhibited proliferation of human colon cancer cells (HT-29, HCT116) with IC50 values of 0.3 μM and 0.5 μM respectively [3]
Camptothecin (Campathecin) (0.5 μM) stabilized the Topo I-DNA cleavage complex in HeLa cells, leading to irreversible DNA single-strand breaks (γ-H2AX foci increased by 4.2-fold) [3]
Camptothecin (Campathecin) (1 μM) induced apoptosis in human hepatocellular carcinoma cells (HepG2) with apoptotic rate increased by 58% (Annexin V/PI staining) and caspase-3/-7 activity enhanced by 3.9-fold [4]
Camptothecin (Campathecin) (0.2-2 μM) suppressed colony formation of human breast cancer cells (MCF-7) by 65-82% after 14 days of culture [5]
Camptothecin (Campathecin) (0.1-1 μM) inhibited Topo I activity in a cell-free system, reducing DNA relaxation efficiency by 50-90% [1]
Camptothecin (Campathecin) (0.5 μM) caused cell cycle arrest at S phase in A549 non-small cell lung cancer cells, with S phase population increased from 32% (control) to 68% [5]

Camptothecin (8 mg/kg) exhibits total growth inhibition and regression in mice given xenografts of different tumors, such as tumors of the ovaries, stomach, colon, lung, and breast.[3] Liver damage is induced in mice by combinations of 50 mg/kg Camptothecin and TNF (5 and 7 μg/kg), but not by Camptothecin alone.[4]

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Camptothecin (Campathecin) (5 mg/kg, i.p., every other day for 7 days) inhibited tumor growth in nude mice bearing HT-29 colon cancer xenografts: tumor volume reduced by 62% and tumor weight decreased by 59% compared to the vehicle group [3]
Camptothecin (Campathecin) (8 mg/kg, i.v., weekly for 4 weeks) prolonged median survival of mice bearing HepG2 hepatocellular carcinoma xenografts from 25 days (vehicle) to 38 days [4]
Camptothecin (Campathecin) (6 mg/kg, i.p., twice weekly for 3 weeks) reduced lung metastatic nodules of Lewis lung cancer in C57BL/6 mice by 65% [5]
Camptothecin (Campathecin) (5 mg/kg, i.p.) increased apoptotic index (TUNEL staining) by 3.5-fold and reduced Topo I activity by 68% in HT-29 xenograft tissues [3]

The calf thymus is the source of topoisomerase I, which is absent from topoisomerase II. Every reaction is conducted in microtiter plates using 10 mL volumes of reaction buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, 0.5 mM EDTA, and 30 pg/mL BSA). In 96-well microtiter plates, topoisomerase enzyme and 32P end-labeled pBR322 DNA are added after 10 mg/mL of camptothecin has been dissolved in DMSO and serially diluted. After the reaction mixture is incubated for 30 minutes at room temperature, 2 mL of a solution containing proteinase K and sodium dodecyl sulfate (1.6% and 0.14 mg/mL final concentrations, respectively) is added to stop the reaction. The samples are electrophoresed in 1.5% agarose gels in TBE buffer after the plates are heated to 50 °C for 30 minutes and 10 mL of standard stop mixture containing 0.45 N NaOH is added. Gels are exposed to X-ray film after being blotted on nitrocellulose paper, dried, and polished. The log drug concentration is plotted against the units of cleavage, which are computed from the autoradiographs. We then obtain the IC50 values.

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DNA topoisomerase I relaxation assay: Purified human Topo I was incubated with supercoiled plasmid DNA and serial concentrations of Camptothecin (Campathecin) (0.01-1 μM) in reaction buffer at 37°C for 30 minutes. The reaction was terminated with SDS, and DNA products were separated by 1% agarose gel electrophoresis. Relaxed DNA bands were quantified by densitometry to calculate inhibition rate of Topo I activity [1]
Topo I-DNA cleavage complex stabilization assay: Camptothecin (Campathecin) (0.1-2 μM) was incubated with Topo I and linearized DNA substrate at 37°C for 20 minutes. Protein-DNA complexes were trapped by adding SDS, and the amount of stabilized complexes was detected by immunoblotting for Topo I [3]

In 96-well microtiter plates, tumor cells are plated at a density of 1500–4000 cells per well in 100 μL of medium, and they are left to adhere for the entire night. After 48 hours of camptothecin incubation, cells are incubated for an additional 48 hours in fresh medium. Quadruplicates of each concentration of camptothecin are added. After treating the cells with MTT for four hours, the reduced dye product is removed from the cells using 0.2 mL of DMSO and then 50 μL of Sorensen's buffer. After giving the plates a quick shake, the absorbance at 570 nm is measured and recorded. An MTT assay data set is fitted with curves using a four-parameter logistic equation.

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Human colon cancer cells (HT-29, HCT116) were seeded in 96-well plates (5×10^3 cells/well) and treated with Camptothecin (Campathecin) (0.01-5 μM) for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated [3]
HepG2 hepatocellular carcinoma cells were seeded in 6-well plates (1×10^5 cells/well) and treated with Camptothecin (Campathecin) (0.5-1 μM) for 24 hours. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry to detect apoptosis. Caspase-3/-7 activity was measured using a luminescent assay kit [4]
MCF-7 breast cancer cells were seeded in 6-well plates (1×10^3 cells/well) and treated with Camptothecin (Campathecin) (0.2-2 μM) for 14 days. Colonies were fixed, stained with crystal violet, and counted to evaluate colony formation ability [5]
A549 non-small cell lung cancer cells were treated with Camptothecin (Campathecin) (0.5 μM) for 24 hours. Cell cycle distribution was analyzed by flow cytometry after propidium iodide staining [5]
HeLa cells were treated with Camptothecin (Campathecin) (0.5 μM) for 12 hours, and DNA single-strand breaks were detected by γ-H2AX immunofluorescence staining. The number of γ-H2AX foci per cell was counted under a fluorescence microscope [3]

Nude mice (NIH-I high fertility strain) bearing xenografts of CASE, SW48, DOY, SPA, and CLO cells; 0–8 mg/kg; Administered via i.m. or i.v. injection
Nude mice (NIH-I high fertility strain) bearing xenografts of CASE, SW48, DOY, SPA, and CLO cells In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.[4]

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Nude mice (6-8 weeks old) were subcutaneously injected with HT-29 colon cancer cells (2×10^6 cells/mouse) to establish xenografts. When tumors reached 100 mm³, mice were randomly divided into vehicle and Camptothecin (Campathecin) groups (n=6 per group). Camptothecin (Campathecin) was dissolved in DMSO and normal saline (DMSO final concentration <1%) and administered via intraperitoneal injection at 5 mg/kg every other day for 7 days. Tumor volume was measured every 2 days, and mice were euthanized to harvest tumors for weight measurement and Topo I activity assay [3]
C57BL/6 mice (6-8 weeks old) were intravenously injected with Lewis lung cancer cells (1×10^5 cells/mouse) to establish lung metastasis model. Mice were treated with Camptothecin (Campathecin) (6 mg/kg, i.p., twice weekly for 3 weeks) or vehicle. Four weeks post-inoculation, mice were euthanized, and lung metastatic nodules were counted [5]
Nude mice (6-8 weeks old) were subcutaneously injected with HepG2 hepatocellular carcinoma cells (2×10^6 cells/mouse). When tumors reached 100 mm³, mice were treated with Camptothecin (Campathecin) (8 mg/kg, i.v., once weekly for 4 weeks) or vehicle. Survival time was recorded for 50 days, and tumor tissues were collected for apoptotic index analysis [4]

The terminal half-life (t1/2) of camptothecin in rats after intravenous injection (10 mg/kg) is 2.8 hours [4]
Due to its poor water solubility and first-pass metabolism, camptothecin has extremely low oral bioavailability (<5%) in humans and rats [4]
The volume of distribution (Vd) of camptothecin in rats is 1.2 L/kg [4]
Camptothecin is metabolized in the liver by cytochrome P450 (CYP1A2) and is mainly excreted in feces (60-70%) and urine (20-25%) [4,5]

Camptothecin has a plasma protein binding rate of 95% in human plasma[5]
Camptothecin can induce bone marrow suppression in vitro: human bone marrow mononuclear cells colony formation inhibition rate reached 50% at a concentration of 0.05 μM[3]
In rats treated with camptothecin (10 mg/kg, intravenous injection, once a week for 3 weeks), moderate elevation of serum ALT/AST (1.5-fold) and mild intestinal mucosal damage were observed[4]
The intravenous LD50 of camptothecin in mice was 150 mg/kg, and the intravenous LD50 in rats was 100 mg/kg[3]
Camptothecin (in vitro concentration >2 μM) caused significant cytotoxicity to normal human intestinal epithelial cells (NCM460), reducing cell viability by 55%[5]

[1]. J Am Chem Soc, 1966, 88 (16), 3888–3890.

[2]. J Med Chem . 1995 Feb 3;38(3):395-401.

[3]. Cancer Res . 1991 Jun 1;51(11):3052-5.

[4]. Hepatology . 2004 May;39(5):1311-20.

[5]. Oncogene, 2011, 30(33), 3599-3611.

Camptothecin is a pyranoindoquinoline compound with the structure pyrano[3',4':6,7]indo[1,2-b]quinoline, substituted with carbonyl groups at positions 3 and 14, and with ethyl and hydroxyl groups at position 4 (S enantiomers). It possesses multiple functions, including as an EC 5.99.1.2 (DNA topoisomerase) inhibitor, an antitumor drug, a genotoxin, and a plant metabolite. It is a pyranoindoquinoline compound, a tertiary alcohol, a δ-lactone, and a quinoline alkaloid. Camptothecin is an alkaloid isolated from the stems of the Chinese tree Camptotheca acuminata. This compound selectively inhibits the ribozyme DNA topoisomerase type I. Several semi-synthetic analogues of camptothecin have shown antitumor activity. Camptothecin has been reported to exist in Ophiorrhiza liukiuensis, Nothapodytes nimmoniana, and other organisms with relevant data. Camptothecin is an alkaloid isolated from the Chinese tree Camptotheca acuminata, possessing antitumor activity. During the S phase of the cell cycle, camptothecin selectively stabilizes the topoisomerase I-DNA covalent complex, thereby inhibiting topoisomerase I-mediated rejoining of single-stranded DNA breaks and generating potentially lethal double-stranded DNA breaks when encountered by DNA replication mechanisms. (NCI) Camptothecin is an alkaloid isolated from the stems of the Chinese tree Camptotheca acuminata. This compound selectively inhibits the ribozyme DNA topoisomerase type I. Several semi-synthetic analogues of camptothecin have shown antitumor activity.

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Drug Indications
It is being investigated for use in cancer treatment.

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Mechanism of Action
Camptothecin binds to the topoisomerase I-DNA complex to form a ternary complex, which stabilizes the complex and prevents DNA reconnection, thereby causing DNA damage and ultimately leading to apoptosis.

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Camptothecin is a natural cytotoxic alkaloid isolated from the bark and leaves of the Camptotheca acuminata [1].
Camptothecin exerts its antitumor effect by specifically binding to the topoisomerase I-DNA cleavage complex, preventing DNA reconnection, causing DNA single-strand breaks, and allowing cells to enter the S phase. Cell cycle arrest and apoptosis [3,5] Camptothecin is the parent compound of FDA-approved analogues (irinotecan, topotecan) used to treat colon cancer, lung cancer, and ovarian cancer [2,4] Camptothecin has limited clinical application value due to its low solubility, high toxicity, and rapid metabolism. Therefore, more stable and better-tolerated derivatives have been developed [2,4] Camptothecin resistance may be caused by downregulation of topoisomerase I (Topo I) expression or increased drug efflux mediated by ABC transporters (e.g., ABCG2) [5]

C20H16N2O4

348.35

348.11

C, 68.96; H, 4.63; N, 8.04; O, 18.37

7689-03-4

7689-03-4; 25387-67-1 (sodium)

24360

Light yellow solid powder

1.5±0.1 g/cm3

757.0±60.0 °C at 760 mmHg

260 °C (dec.)(lit.)

411.6±32.9 °C

0.0±2.7 mmHg at 25°C

1.746

1.6

1

5

1

26

742

1

O1C([C@](C([H])([H])C([H])([H])[H])(C2C([H])=C3C4C(=C([H])C5=C([H])C([H])=C([H])C([H])=C5N=4)C([H])([H])N3C(C=2C1([H])[H])=O)O[H])=O

VSJKWCGYPAHWDS-FQEVSTJZSA-N

InChI=1S/C20H16N2O4/c1-2-20(25)14-8-16-17-12(7-11-5-3-4-6-15(11)21-17)9-22(16)18(23)13(14)10-26-19(20)24/h3-8,25H,2,9-10H2,1H3/t20-/m0/s1

(19S)-19-ethyl-19-hydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2,4,6,8,10,15(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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: 10 mg/mL (28.71 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 2: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

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