Camptothecins/DNA Topoisomerase I; Drug-linker conjugate for ADC

It is known that 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11), a semisynthesized derivative of camptothecin (CPT), has a potent antitumor activity in vivo, but 7-ethyl-10-hydroxycamptothecin (SN-38), a metabolite of CPT-11, shows much stronger cytotoxicity in vitro than CPT-11. In this study, we demonstrated that the relaxation of SV40 DNA plasmids by type I DNA topoisomerase prepared from P388 murine leukemia cells was inhibited by 50% by SN-38 at approximately 1 microM, although CPT-11 at 1 mM slightly inhibited the relaxation. SN-38 and CPT showed strong, time-dependent inhibitory activity against DNA synthesis of P388 cells. However, CPT-11 weakly inhibited DNA synthesis independently of time with coincident inhibition of the total thymidine uptake by the cells. By alkaline and neutral elution assays, it was demonstrated that SN-38 caused much more frequent DNA single-strand breaks in P388 cells than did CPT-11. The same content of SN-38 and a similar frequency of single-strand breaks were detected in the cells treated with SN-38 at 0.1 microM or with CPT-11 at 100 microM. Therefore, single-strand breaks by CPT-11 seem to be due to SN-38 produced from CPT-11 in cells. These results indicate that CPT-11 itself possesses a marginal antiproliferative effect but that SN-38 plays an essential role in the mechanism of action of CPT-11[2].

[1]. Characterization of DNA topoisomerase I in three SN-38 resistant human colon cancer cell lines reveals a newpair of resistance-associated mutations. J Exp Clin Cancer Res. 2016 Mar 31;35:56.

[2]. Intracellular roles of SN-38, a metabolite of the camptothecin derivative CPT-11, in the antitumor effect of CPT-11. Cancer Res. 1991;51(16):4187-4191.

DNA topoisomerase I (Top1) is a DNA helicase and a specific target of camptothecin-based chemotherapy drugs. Irinotecan, one such drug, exerts its effect through its active metabolite SN-38 and is used to treat metastatic colorectal cancer. However, irinotecan resistance is a major clinical challenge. Since molecular alterations to Top1 can lead to irinotecan resistance, we characterized Top1 in three human colon cancer cell lines that acquired SN-38 resistance. Methods: We constructed three SN-38-resistant cell lines (resistance increased 20-67-fold) and compared them with wild-type parental cells. Comparative metrics included: Top1 gene copy number and sequence, Top1 expression (mRNA and protein), Top1 enzymatic activity in the presence and absence of the drug, and the formation of the Top1-DNA cleavage complex in drug-treated cells. We validated Top1 mutations using mutation-specific primers via PCR. In addition, cross-resistance to two indomethacin topoisomerase I (Top1) targeted drugs (NSC 725776 and NSC 743400) and two topoisomerase II (Top2) targeted drugs (epirarubicin and etoposide) was investigated. Results: Two of the three SN-38 resistant cell lines carried abnormal TOP1 gene copy numbers: an increased TOP1 gene copy number and a deletion on chromosome 20, respectively. One resistant cell line carried a pair of previously unreported TOP1 mutations (R364K and G717R) located near the drug-binding site. The expression level of mutant TOP1 was significantly higher than that of wild-type TOP1. No decrease in Top1 expression or activity was observed, or only a very small decrease, in the absence of the drug. In all three SN-38 resistant cell lines, Top1 activity remained maintained even in the presence of high concentrations of SN-38. No cross-resistance or only partial cross-resistance was observed with etoposide and epirubicin, respectively. SN-38 resistant cells carrying wild-type TOP1 remained sensitive to NSC 743400, while cells carrying mutant TOP1 showed complete cross-resistance to both indomethacin drugs. The formation of Top1-DNA cleavage complexes after drug treatment supported the other findings. Conclusion: This study further enriches our understanding of the resistance mechanism to Top1-targeted chemotherapy drugs. Importantly, this study identified two previously unreported TOP1 mutations and highlighted that cross-resistance to novel indomethacin drugs depends on the specific molecular mechanism of SN-38 resistance. [1]

C32H31N3O8

585.60

585.211

C, 65.63; H, 5.34; N, 7.18; O, 21.86

1473403-87-0

146673122

White to yellow solid powder

2.3

1

9

10

43

1300

1

O=C1C=CC(=O)N1CCCCCC(O[C@@]1(C(OCC2C(N3CC4=C(CC)C5=CC(=CC=C5N=C4C3=CC=21)O)=O)=O)CC)=O

ACRMADURFCYBAU-YTTGMZPUSA-N

InChI=1S/C32H31N3O8/c1-3-19-20-14-18(36)9-10-24(20)33-29-21(19)16-35-25(29)15-23-22(30(35)40)17-42-31(41)32(23,4-2)43-28(39)8-6-5-7-13-34-26(37)11-12-27(34)38/h9-12,14-15,36H,3-8,13,16-17H2,1-2H3/t32-/m0/s1

[(19S)-10,19-diethyl-7-hydroxy-14,18-dioxo-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)-heptaen-19-yl] 6-(2,5-dioxopyrrol-1-yl)hexanoate

MC-SN38; 1473403-87-0; AKOS040756843; [(19S)-10,19-diethyl-7-hydroxy-14,18-dioxo-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)-heptaen-19-yl] 6-(2,5-dioxopyrrol-1-yl)hexanoate;

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 : 140 mg/mL (239.07 mM)

Solubility in Formulation 1: ≥ 5.75 mg/mL (9.82 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 57.5 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: 5.75 mg/mL (9.82 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 57.5 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.)