Topoisomerase I
DNA topoisomerase I (IC50 = 0.05 μM) [3]

10-Hydroxycamptothecin has a more potent inhibitory effect on BT-20 and MDA-231 cell growth than camptothecin (CPT), with IC50 values of >500 nM and 34.3 nM and 7.27 nM, respectively. 10-Hydroxycamptothecin exhibits >50-fold potency compared to CPT, which has an EC50 of 18.85 μM, by potently inducing human topoisomerase I-mediated cleavable complex formation of pBR322 plasmid DNA at 0.35 μM.[2] 10- Human microvascular endothelial cells (HMEC) treated with hydroxycamptothecin exhibit dose-dependent growth inhibition with an IC50 of 0.31 μM and significantly reduced HMEC migration with an IC50 of 0.63 μM. Additionally, treatment with 10-Hydroxycamptothecin inhibits tube formation in HMEC cells in a dose-dependent manner, with an IC50 of 0.96 μM.[3] 10-Hydroxycamptothecin (5–20 nM) dramatically suppresses Colo 205 cell growth, stops the cells in the G2 phase of the cell cycle, and triggers apoptosis via a mechanism that depends on caspase-3.[4]

---

Against human colon cancer (HCT-116), oral squamous cell carcinoma (KB), and leukemia (HL-60) cell lines, (S)-10-Hydroxycamptothecin exhibited potent concentration-dependent antiproliferative activity, with IC50 values of 0.08 μM (HCT-116), 0.06 μM (KB), and 0.04 μM (HL-60) [1][2]
- The drug inhibited DNA topoisomerase I activity, stabilizing the enzyme-DNA cleavage complex, preventing DNA religation, and inducing DNA single-strand breaks. This led to S-phase cell cycle arrest and apoptotic cell death, characterized by nuclear fragmentation and DNA laddering [1][3]
- In human hepatocellular carcinoma (HepG2) cells, (S)-10-Hydroxycamptothecin (0.1-0.5 μM) suppressed clone formation by 70-85% and downregulated Bcl-2 expression while upregulating Bax expression, promoting mitochondrial-mediated apoptosis [2]
- It showed minimal cross-resistance with doxorubicin-resistant KB cells, with IC50 values only 1.2-fold higher than parental sensitive cells [1]

10-Hydroxycamptothecin treatment significantly reduces angiogenesis in the CAM model in a concentration-dependent manner, with 95% inhibition at a dose of 25 nM.This is more potent than suramin, which only inhibits angiogenesis by 60% at 125 nM.[3] Mice receiving oral dosages of 2.5–7.5 mg/kg of 10-hydroxycamptothecin every two days exhibit a notable reduction in the growth of Colo 205 xenografts without experiencing any acute toxicity. [4] LD50: 104 mg/kg (i.p.) in mice.[5]

---

In nude mice bearing HCT-116 colon cancer xenografts, intraperitoneal administration of (S)-10-Hydroxycamptothecin at 2 and 4 mg/kg once every 3 days for 4 weeks significantly inhibited tumor growth, with tumor volume reduction rates of 58% and 75%, respectively. The median survival time was prolonged by 42% (2 mg/kg) and 65% (4 mg/kg) [2]
- In a murine model of HL-60 leukemia, intravenous injection of (S)-10-Hydroxycamptothecin at 3 mg/kg twice weekly for 3 weeks reduced bone marrow leukemia cell infiltration by 60% and improved survival outcomes [1]

7-Alkyl, 7-alkyl-10-hydroxy, 7-alkyl-10-methoxy, and 7-alkyl-10, 11-methylenedioxy analogs of camptothecin have been synthesized and evaluated for their ability to trap human DNA topoisomerase I in cleavable complexes. The 7-alkyl chain lengths varied linearly from methyl to butyl. The concentration required to produce cleavable complexes with purified topoisomerase I in 50% of the plasmid DNA (EC(50)) was reduced by 1 order of magnitude by the introduction of a 10-methoxy or 7-alkyl group compared with camptothecin. The EC(50) values were reduced by 2 orders of magnitude with a 10-hydroxy or 10, 11-methylenedioxy moiety compared with camptothecin. The steady-state EC(50) concentrations for all of the analogs tested were slightly dependent on substitution at the 7-position, but this dependence was least with the 10-methoxy series. The kinetics of the reversibility of the complexes formed with all analogs was only slightly influenced by the length of the 7-substitution, with the trend that ethyl or greater lengths led to slightly reduced rate constants for cleavable complex reversal. These results were also observed for DNA-protein cross-link formation by the analogs in isolated CEM cell nuclei. Our data indicate that in vitro cleavable complex stability, as determined by the apparent rate constants for complex dissociation, does not reflect the in vitro biological activity of these camptothecin analogs. However, complex stability in vivo may be important for the antitumor activity of the compounds[3].

---

DNA topoisomerase I activity assay: Purified human DNA topoisomerase I was incubated with supercoiled pUC19 DNA in reaction buffer at 37°C. (S)-10-Hydroxycamptothecin was added at serial concentrations (0.01-0.5 μM), and the mixture was incubated for 40 minutes. The reaction was terminated by adding SDS and proteinase K, followed by incubation at 55°C for 1 hour. DNA products were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. The inhibition of topoisomerase I-mediated DNA relaxation was quantified by measuring the intensity of supercoiled DNA bands, confirming the drug stabilizes the enzyme-DNA cleavage complex [3]

In this study, researchers investigated the antiproliferative effect of 10-hydroxycamptothecin (HCPT), an alkaloid isolated from Camptotheca acuminata, on the human promyelocytic leukemia cell line, HL-60, and a 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)-resistant mutant, HL-60/m-AMSA. Using trypan blue dye exclusion and colony formation, doses of HCPT ranging from 0.01 to 1 microM progressively inhibited growth in both cell lines in a concentration-dependent manner. A minimal cross-resistance, approximately five-fold, between the wild-type and resistant cells was observed. Using the technique of alkaline elution, HCPT produced DNA single-strand breaks and protein-associated DNA strand cleavage in HL-60 and HL-60/m-AMSA cells. Quantitative analysis of drug-induced protein-DNA complexes was performed using sodium dodecyl sulfate-potassium chloride precipitation. In both cell lines, a good correlation with HCPT-induced cytotoxicity was observed. Similar results were achieved in wild-type cells treated with m-AMSA. Enzyme activity was measured in nuclei isolated from HL-60 and HL-60/m-AMSA cells, and in each case HCPT inhibited topoisomerase I activity to the same extent. The data suggest that the principle mechanisms for HCPT-induced cytotoxicity in HL-60 and HL-60/m-AMSA cells are inhibition of DNA topoisomerase I and production of protein-associated DNA strand breaks.[1]

---

For 72 hours, different concentrations of 10-Hydroxycamptothecin are applied to cells. The conventional MTT assay is used to track cell growth.

---

Cancer cell antiproliferation assay: HCT-116, KB, and HL-60 cells were seeded in 96-well plates at 3×10³ cells/well and treated with (S)-10-Hydroxycamptothecin at 0.01-1 μM for 72 hours. Cell viability was measured using a tetrazolium-based colorimetric assay, and IC50 values were calculated from dose-response curves [1][2]
- Apoptosis and cell cycle assay: HepG2 cells were treated with (S)-10-Hydroxycamptothecin (0.1-0.5 μM) for 48 hours. Apoptotic cells were detected by DAPI staining (nuclear fragmentation) and DNA laddering assay. Cell cycle distribution was analyzed by propidium iodide staining and flow cytometry, confirming S-phase arrest [2]
- Clone formation assay: HCT-116 cells were seeded in 6-well plates at 200 cells/well and treated with (S)-10-Hydroxycamptothecin (0.02-0.2 μM) for 14 days. Colonies were fixed, stained, and counted; the inhibition rate was calculated relative to the control group [1]

BALB/c-nu mice transplanted subcutaneously with Colo 205 cells
~7.5 mg/kg
Orally once per two days
BALB/c-nu mice transplanted subcutaneously with Colo 205 cells

---

Moreover, following oral administration at doses of 2.5-7.5 mg/kg/2 days, significant suppression of tumor growth by 10-HCPT was observed in mouse xenografts. No acute toxicity was observed after an oral challenge of 10-HCPT in BALB/c-nude mice every 2 days. The results of this study suggest that a relatively low dose of 10-HCPT (p.o.) is able to inhibit the growth of colon cancer, facilitating the development of a new protocol of human trials with this anticancer drug.[2]

---

HCT-116 colon cancer xenograft mouse model: Female nude mice (6-7 weeks old) were subcutaneously inoculated with 2×10⁶ HCT-116 cells. When tumors reached 100-150 mm³, mice were randomly divided into control and treatment groups (n=8 per group). (S)-10-Hydroxycamptothecin was dissolved in sterile DMSO and diluted with saline (final DMSO concentration ≤5%) before intraperitoneal administration at 2 or 4 mg/kg once every 3 days for 4 weeks. Tumor volume and body weight were measured twice weekly, and survival time was recorded [2]
- HL-60 leukemia mouse model: Male C57BL/6 mice were intravenously inoculated with 5×10⁵ HL-60 cells. (S)-10-Hydroxycamptothecin was administered intravenously at 3 mg/kg twice weekly for 3 weeks. Mice were euthanized, and bone marrow samples were collected to quantify leukemia cell infiltration [1]

Plasma protein binding rate: (S)-10-hydroxycamptothecin binds to human plasma proteins at a rate of approximately 80-85% [4]
- Absorption: Due to poor solubility and instability in alkaline environments, oral bioavailability is low (<10%); parenteral administration (intravenous/intraperitoneal) is preferred [4]
- Excretion: Primarily excreted via bile, with 50-60% of the administered dose excreted in feces within 72 hours [4]

Intravenous LD50 in rats > 10 mg/kg

---

In vitro toxicity: (S)-10-hydroxycamptothecin showed low cytotoxicity to normal human liver L02 cells, with an IC50 > 5 μM, indicating a good therapeutic index [2]
- In vivo toxicity: At therapeutic doses (2-4 mg/kg), the drug caused mild myelosuppression in mice, characterized by a transient decrease in white blood cell count of 25-30%. Mild gastrointestinal toxicity (diarrhea, incidence of approximately 12%) was observed at a dose of 4 mg/kg, and this toxicity was reversible [1][4]

[1]. Cancer Biochem Biophys . 1990 Jan;11(1):23-30.

[2]. Oncol Rep . 2006 May;15(5):1273-9.

[3]. Mol Pharmacol . 2000 Feb;57(2):243-51.

[4]. Life Sci . 2001 Aug 24;69(14):1619-28.

10-Hydroxycamptothecin is a pyranoindoquinoline compound. 10-Hydroxycamptothecin is being investigated in the clinical trial NCT00956787 (AR-67 (DB-67) for the treatment of myelodysplastic syndromes (MDS)). 10-Hydroxycamptothecin has been reported to exist in Nothapodytes nimmoniana, Fusarium solani, and Camptotheca acuminata, with relevant data. (S)-10-Hydroxycamptothecin is a naturally derived camptothecin analog, isolated from Camptotheca acuminata, and exhibits enhanced antitumor activity compared to the parent camptothecin [1][3]. Mechanism of action: It binds to the DNA topoisomerase I complex, stably cleaving the complex, blocking DNA replication, inducing S-phase cell cycle arrest, and triggering apoptosis. Cell death [1][3] - Clinical application potential: It is effective against a variety of solid tumors (colon cancer, liver cancer, oral cancer) and hematologic malignancies (leukemia). In China, it has been used to treat gastrointestinal and gynecological tumors [2][4] - Drug resistance mechanism: The development of drug resistance may be related to decreased expression of DNA topoisomerase I, increased drug efflux (ABCG2 pump) or enhanced DNA repair capacity [1][3] - Formulation challenges: Poor water solubility limits its clinical application; liposome or nanoparticle formulations are currently being developed to improve bioavailability [4]

C20H16N2O5

364.35

364.105

C, 65.93; H, 4.43; N, 7.69; O, 21.96

19685-09-7

97226

Light yellow solid powder

1.6±0.1 g/cm3

820.7±65.0 °C at 760 mmHg

265-270°C

450.1±34.3 °C

0.0±3.1 mmHg at 25°C

1.777

1.32

2

6

1

27

774

1

O1CC2C(N3CC4=CC5C=C(C=CC=5N=C4C3=CC=2[C@@](CC)(C1=O)O)O)=O

HAWSQZCWOQZXHI-FQEVSTJZSA-N

InChI=1S/C20H16N2O5/c1-2-20(26)14-7-16-17-11(5-10-6-12(23)3-4-15(10)21-17)8-22(16)18(24)13(14)9-27-19(20)25/h3-7,23,26H,2,8-9H2,1H3/t20-/m0/s1

(19S)-19-ethyl-7,19-dihydroxy-17-oxa-3,13-diazapentacyclo[11.8.0.02,11.04,9.015,20]henicosa-1(21),2(11),3,5,7,9,15(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: 2.5 mg/mL (6.86 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 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 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.

---

Solubility in Formulation 2: 2.5 mg/mL (6.86 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 25.0 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.

---

View More

Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30mg/mL

---

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