Topoisomerase I

9-Aminocamptothecin cytotoxicity rises with increasing drug concentrations and longer exposure times in human breast (MCF-7), bladder (MGH-U1), and colon (HT-29) cancer cell lines. Unless the concentration of 9-Aminocamptothecin surpasses a threshold of 2.7 nm, minimal cell killing is also observed[1]. After 96 hours of medication exposure, PC-3, PC-3M, DU145, and LNCaP cells are inhibited by 9-Aminocamptothecin with IC50 values of 34.1, 10, 6.5, and 8.9 nM, respectively[2].

9-Aminocamptothecin (9-Amino-CPT) suppresses tumor growth at the lowest oral dose of 0.35 mg/kg/day; tumor regression is caused by s.c. administration (4 mg/kg/week) and higher oral doses of 0.75 and 1 mg/kg/day. All dosages of 9-Aminocamptothecin are well tolerated, and no group has experienced toxic death or weight loss greater than 10%[2]. A full remission is induced in 55% of SCID mice engrafted with human myeloid leukemia by 9-Aminocamptothecin. Both the intravenous and oral routes work equally well. The evaluation of 9-Aminocamptothecin as an antileukemic agent in a phase I trial involving AML patients is supported by the results obtained from this pre-clinical model[3].

The clonogenic assay is used to evaluate the cytotoxicity of 9-amino-CPT (9-amino-20(S)-camptothecin). 100–250 cells are inoculated in triplicate onto 60 15-mm dishes holding 5 mL of medium after exponentially growing cells are resuspended in media and the number of cells is ascertained using an electronic counter. 5 μL of 9-Aminocamptothecin stock solutions are added to the dishes to reach final concentrations of 137, 274 nM, 0.27, 1.37, 2.74, 13.7, 27.4, and 0.27 nM after an overnight incubation. Fresh medium is added to the dishes and the medium is removed by aspiration after 4, 8, 12, 24, 48, 72, and 240 hours of exposure. The ratio of colons in the drug-treated sample to those in the control (DMSO vehicle-treated) sample is used to calculate the percentage of survival at each drug concentration and exposure time[1].

Mice: On the seventh day following the KBM-3 cell inoculation, 9-Aminocamptothecin treatment is initiated. The following treatment is given to five groups of five mice each, with an average weight of 22 g, four days a week for three weeks: 1) PBS is IV injected into group 1 control mice; 2) group 2 mice receive 1.33 mg/kg Group 3 mice are given 1.33 mg/kg of 9-Aminocamptothecin IV. 4) Group 4 mice receive 2.0 mg/kg of 9-Aminocamptothecin IV; 5) Group 5 mice receive 2.0 mg/kg of 9-Aminocamptothecin orally by gavage. Gavage of 9-Aminocamptothecin orally[3]. The intravenous (i.v.) injection of the human acute myelogenous leukemia cell line KBM-3 into severe combined immune deficient (SCID) mice results in disseminated multi-organ human disease involvement in these animals which leads to their death over a defined period of time. We utilized this model of human leukemia to investigate the in vivo therapeutic efficacy of the topoisomerase I inhibitor 9-aminocamptothecin (9-AC) given by two different routes. Mice injected with KBM-3 were divided into five groups. Group 1 received only diluent and served as control. The four remaining groups were treated with 9-AC four days a week for three consecutive weeks as follows: group 2 received 1.33 mg/kg/dose, i.v.; group 3, 1.33 mg/kg/dose, orally (p.o.); group 4, 2.0 mg/kg/dose i.v. and group 5, 2.0 mg/kg/dose p.o.. All animals in the control group died from disseminated human leukemia by day 64 from grafting, with a median survival of 59 days. Eleven out of 20 treated mice survived with no evidence of disease and were sacrificed at the termination of the experiment on day 128. PCR-assisted tissue analysis for the presence of human DNA showed no evidence of human leukemia. In conclusion, 9-AC is an active agent in SCID mice engrafted with human myelogenous leukemia and should be explored in phase I-II trials. Oral and intravenous routes are equally effective.[3]

[1]. Pharmacological determinants of 9-aminocamptothecin cytotoxicity. Clin Cancer Res. 2001 Jan;7(1):168-74.

[2]. 9-Aminocamptothecin: a topoisomerase I inhibitor with preclinical activity in prostate cancer. Clin Cancer Res. 1997 Feb;3(2):287-94.

[3]. Activity of oral and intravenous 9-aminocamptothecin in SCID mice engrafted with human leukemia. Leuk Lymphoma. 1998 Dec;32(1-2):159-64.

9-Aminocamptothecin is a pyranoindoquinoline compound. Aminocamptothecin has been used in clinical trials for the treatment of various cancers, including lymphoma, gastric cancer, ovarian cancer, esophageal cancer, and ovarian tumors. Aminocamptothecin is a water-insoluble camptothecin derivative. It binds to topoisomerase I, thereby inhibiting the repair of single-stranded DNA breaks. Because the terminal lactone ring required for aminocamptothecin to exert its antitumor activity spontaneously opens to an inactive carboxyl group under physiological conditions, long-term administration is necessary to achieve effective cytotoxicity. (NCI04) See also: 10-Aminocamptothecin (note moved to). Camptothecins are a class of anticancer drugs with a unique mechanism of action: toxicity to eukaryotic DNA topoisomerase I. 9-Aminocamptothecin (9-AC) is a potent, water-insoluble derivative of camptothecin and is currently undergoing clinical trials. This study determined the kinetics of the active derivative 9-AC lactone in cell culture medium and investigated the cytotoxicity of 9-AC against human breast cancer (MCF-7), bladder cancer (MGH-U1), and colon cancer (HT-29) cell lines. The relationship between cytotoxicity, drug concentration, and exposure time was then explored. For all three cell lines, the cytotoxicity of 9-AC increased with increasing drug concentration and exposure time. However, cytotoxicity was limited when exposure time was less than 24 hours, with cell killing rates below 1 log unit even at high drug concentrations. Cell killing was barely observed unless the 9-AC concentration exceeded the threshold of 2.7 nM. A fixed model of the survival versus area under the drug concentration-time curve applicable to all three cell lines could not be established. However, data from multiple exposure time experiments for the three cell lines fit well with the pharmacodynamic model C(n)t = k(r², 0.90–0.99), where C is the drug concentration, n is the drug concentration coefficient, and t is the exposure time. For these three cell lines, to kill one log unit of cells, 0.30 < n < 0.85, indicating that exposure time is more important than drug concentration. Our data support the use of 9-AC by infusion for 24 hours or longer in clinical studies, provided that target plasma concentrations can be achieved. [1] 9-Aminocamptothecin (9-AC) is a topoisomerase I inhibitor currently being developed as an antitumor drug. These preclinical studies aimed to evaluate the activity of 9-AC against prostate cancer, a malignant tumor that is insensitive to most clinical cytotoxic drugs. The activity of 9-AC against one hormone-sensitive (LNCaP) and three hormone-resistant (PC-3, PC-3M and DU145) human prostate cancer cell lines was first tested in vitro. Ninety-six hours after drug exposure, the concentrations (IC50) required to inhibit cell viability to 50% of the control were: 34.1 nM for PC-3 cells, 10 nM for PC-3M cells, 6.5 nM for DU145 cells, and 8.9 nM for LNCaP cells. Because 9-AC is known to undergo rapid hydrolysis, we measured the concentration of 9-AC lactone in the tissue culture medium and found that its half-life was 20 minutes, with only 15% of the drug remaining in the lactone form at steady state. Therefore, the IC50 value calculated from a single dose may be overestimated. Subsequently, we tested the activity of the 9-AC colloidal dispersion against PC-3 tumors implanted in the flanks of nude mice. 9-AC was administered via daily gavage (except weekends) or subcutaneous injection twice weekly for a total of 3 weeks. The lowest oral dose (0.35 mg/kg/day) inhibited tumor growth, while higher oral doses (0.75 and 1 mg/kg/day) and subcutaneous injection (4 mg/kg/week) led to tumor regression. 9-AC was well tolerated at all doses, and no toxic death or weight loss exceeding 10% was observed in any group. Finally, we believe that the 9-AC activity observed in the mouse xenograft model may be partly attributed to the relatively acidic tumor microenvironment, which favors the formation of more active lactones. This hypothesis was confirmed by the simultaneous determination of 9-AC lactone concentrations in plasma and tumor. In conclusion, these studies suggest that 9-AC should be submitted to clinical trials in patients with prostate cancer. [2]

C20H17N3O4

363.36668

363.121

C, 66.11; H, 4.72; N, 11.56; O, 17.61

91421-43-1

72402

Light yellow to brown solid powder

1.6±0.1 g/cm3

819.6±65.0 °C at 760 mmHg

449.5±34.3 °C

0.0±3.1 mmHg at 25°C

1.771

0.44

2

6

1

27

775

1

C([C@]1(C(=O)OCC2C(N3CC4C=C5C(N)=CC=CC5=NC=4C3=CC1=2)=O)O)C

FUXVKZWTXQUGMW-FQEVSTJZSA-N

InChI=1S/C20H17N3O4/c1-2-20(26)13-7-16-17-10(6-11-14(21)4-3-5-15(11)22-17)8-23(16)18(24)12(13)9-27-19(20)25/h3-7,26H,2,8-9,21H2,1H3/t20-/m0/s1

(19S)-8-amino-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

9AC; 9aminoCPT; 9-amino-20-camptothecin; 9-amino-camptothecin; 9-AC9-amino-CPT; 9-amino-20(S)-camptothecin; 9aminocamptothecin; Aminocamptothecin

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)

DMSO: ~3.3 mg/mL (~9.2 mM)

Solubility in Formulation 1: ≥ 0.33 mg/mL (0.91 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 3.3 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 2: ≥ 0.33 mg/mL (0.91 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 3.3 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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Solubility in Formulation 3: ≥ 0.33 mg/mL (0.91 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 3.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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