Topotecan HCl (formerly known as NSC609699, Nogitecan, NSC-609699, SKFS-104864A; trade name: Hycamtin), an FDA approved drug for cancer treatment, is a topoisomerase I inhibitor with potent antineoplastic activity. In cell-free assays, it inhibits topoisomerase I in MCF-7 Luc and DU-145 Luc cells with IC50 values of 13 nM and 2 nM, respectively. A semisynthetic derivative of camptothecin with antitumor properties is topotecan. Topotecan selectively stabilizes topoisomerase I-DNA covalent complexes during the S phase of the cell cycle. This prevents topoisomerase I-mediated single-strand DNA breaks from religating and creates potentially fatal double-strand DNA breaks when the DNA replication machinery comes into contact with the complexes.

Physicochemical Properties

Molecular Formula	C23H23N3O5.HCL
Molecular Weight	457.91
Exact Mass	421.163
Elemental Analysis	C, 60.33; H, 5.28; Cl, 7.74; N, 9.18; O, 17.47
CAS #	119413-54-6
Related CAS #	123948-87-8; 119413-54-6(HCl); 1044663-62-8 (Topotecan HCl hydrate)
PubChem CID	60699
Appearance	Yellow solid powder
Density	1.5±0.1 g/cm3
Boiling Point	782.9±60.0 °C at 760 mmHg
Melting Point	213-218ºC
Flash Point	427.3±32.9 °C
Vapour Pressure	0.0±2.8 mmHg at 25°C
Index of Refraction	1.734
LogP	1.08
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	3
Heavy Atom Count	32
Complexity	867
Defined Atom Stereocenter Count	1
SMILES	O1CC2C(N3CC4=CC5C(CN(C)C)=C(C=CC=5N=C4C3=CC=2[C@@](CC)(C1=O)O)O)=O
InChi Key	DGHHQBMTXTWTJV-BQAIUKQQSA-N
InChi Code	InChI=1S/C23H23N3O5.ClH/c1-4-23(30)16-8-18-20-12(9-26(18)21(28)15(16)11-31-22(23)29)7-13-14(10-25(2)3)19(27)6-5-17(13)24-20;/h5-8,27,30H,4,9-11H2,1-3H3;1H/t23-;/m0./s1
Chemical Name	(19S)-8-[(dimethylamino)methyl]-19-ethyl-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;hydrochloride
Synonyms	NSC609699; SKF-104864-A; NSC 609699; SKF 104864 A; NSC-609699; SKF S104864A; Nogitecan HCl; SKFS 104864A; SKF104864A; TOPO. Hycamtamine; Hycamtin Hydrochloride; Nogitecan Hydrochloride; Topotecan; Nogitecan Hydrochloride; Hycamtin; Nogitecan hydrochloride; Topotecan (Hydrochloride); Topotecan monohydrochloride; Evotopin; Trade name: Hycamtin
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Topo I (DU-145 Luc cells) ( IC50 = 2 nM ); Topo I (MCF-7 Luc cells) ( IC50 = 13 nM )
ln Vitro	In vitro activity: Topotecan is observed to have stronger drug activity for MCF-7 Luc and DU-145 Luc cells. [1] By stabilizing the covalent complex between topoisomerase I and DNA and inhibiting the religation of enzyme-linked single-strand DNA breaks, topotecan causes cytotoxicity during DNA replication. In radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, topotecan stabilizes topoisomerase I/DNA cleavable complexes, induces rapid apoptotic cell death despite high-level expression of bcl-2 protein, and dose-dependently suppresses the clonogenic growth of ALL cells. [2]
ln Vivo	Measuring tumor growth and regression with calipers and luminescent imaging, animals given DU-145 Luc cells intraperitoneally and treated with topotecan showed notable results. Topotecan-treated group's correlation coefficient is 0.93, while the control group's is 0.75. For both untreated and Topotecan-treated mice injected intraperitoneally (i.p.) with MCF-7 Luc cells, tumor growth and regression can be quantified through luminescent imaging.[1] In mice with severe combined immune deficiency (SCID), a model for human ALL with a poor prognosis, topotecan demonstrated strong antileukemic activity. When SCID mice were exposed to doses of humaln leukemia cells at systemic drug exposure levels, topotecan significantly increased their event-free survival.[2] Topotecan treatment significantly up-regulates the expression of TRAIL R2, which is preferentially expressed by gliomas.[3]
Enzyme Assay	Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia[2].
Cell Assay	Topotecan is first diluted to 6 μg/mL in cultured medium after being dissolved in sterile water to a stock concentration of 1 mg/mL. The final volume of each opaque, white tissue culture-treated microplate is achieved by serially diluting the solution 1:4 until it is 0.1 mL/well. 100 μL of cells are added to each well. MCF-7 Luc and DU-145 Luc cells are resuspended in 3×104 cells/mL in DMEM with high glucose containing 10% FBS and 0.5 mg/mL Geneticin. Plates are incubated for four days at 37 °C with 5% CO2 and 95% humidity. Each well receives 0.05 mL of 0.1 M HEPES buffer (pH 7.9) containing 50 μg/mL D-luciferin after incubation. The culture microplate is measured in a molecular light imager and a microplate luminometer after being incubated at room temperature for ten minutes. The microplate luminometer's results are computed using maximum inhibition control wells that contain an ATP inhibitor and no inhibition control wells that do not contain an exogenous drug. The values acquired with a 5-minute luminescent imager are used in a similar manner to calculate the results for the molecular light imager.
Animal Protocol	Mice: We used SK-N-BE, SH-SY5Y, KHOS, and RH30 for subcutaneous xenograft studies. The inguinal fat pad of each nonobese diabetic/severe combined immune deficient (NOD/SCID) mouse is subcutaneously implanted with 1×106 cells. The animals are divided into 4 groups at random and given oral gavage treatment every day once the tumors have grown to a diameter of 0.5 cm. The animals are divided into four groups: combination (TP + PZ; 1 mg/kg Topotecan Hydrochloride + 150 mg/kg Pazopanib), LDM Topotecan (1 mg/kg Topotecan), and Pazopanib (PZ; 150 mg/kg Pazopanib). In the KHOS osteosarcoma model, PZ is substituted with a weekly oral dose of either Pulse TP (15 mg/kg Topotecan) or pulse Topotecan in order to compare the two. Tumors with a diameter greater than 2.0 cm or animals exhibiting symptoms of morbidity are the endpoint criteria. Up until the endpoint or sacrifice, the tumor sizes are measured every day. Using calipers, the long (D) and short (d) diameters are measured. To compute tumor volume (cm3), use the formula V=0.5×D×d2. The animals are sacrificed by cervical dislocation when the treatment's endpoint is reached.
ADME/Pharmacokinetics	Absorption, Distribution and Excretion Renal clearance is an important determinant of topotecan elimination. In a mass balance/excretion study in 4 patients with solid tumors, the overall recovery of total topotecan and its N-desmethyl metabolite in urine and feces over 9 days averaged 73.4 ± 2.3% of the administered IV dose. Fecal elimination of total topotecan accounted for 9 ± 3.6% while fecal elimination of N-desmethyl topotecan was 1.7 ± 0.6%. The pharmacokinetics of topotecan have been extensively studied in patients with normal renal function and there is one study of patients with mild to moderate renal insufficiency. However, the effect of hemodialysis on topotecan disposition has not been reported. The objective of this study was to characterize the disposition of topotecan in a patient with severe renal insufficiency receiving hemodialysis. Topotecan lactone disposition was characterized in a patient on and off hemodialysis. The topotecan lactone clearance determined after administration of topotecan alone and with hemodialysis was 5.3 L/hr per sq m vs 20.1 L/hr per sq m respectively. At 30 min after the completion of hemodialysis, the topotecan plasma concentration obtained was greater than that measured at the end of hemodialysis (i.e. 8.0 ng/mL vs 4.9 ng/mL), suggesting a rebound effect. The topotecan terminal half-life off dialysis was 13.6 hr, compared with an apparent half-life determined during hemodialysis of 3.0 hr. These results demonstrate that topotecan plasma clearance while on hemodialysis increased approximately fourfold. Hemodialysis may be an effective systemic clearance process for topotecan and should be considered in selected clinical situations (e.g. inadvertent overdose, severe renal dysfunction). In lactating rats receiving IV topotecan at a dosage of 4.72 mg/sq m, high concentrations of the drug (i.e., up to 48 times higher than plasma concentrations) were distributed into milk. It is not known whether topotecan is distributed into human milk. Following oral administration, about 57% of topotecan (administered daily for 5 days) is excreted in urine as unchanged drug (20%) and as the N-desmethyl metabolite (2%).47 Approximately 33% of the oral dose of topotecan was eliminated in feces as total topotecan and approximately 2% as N-desmethyl topotecan. Following IV administration, about 74% of a topotecan dose is excreted, mostly unchanged in urine (51%) and feces (18%) within 9 days; excretion of N-desmethyl topotecan in urine is approximately 3% and in feces is approximately 2%. O-Glucuronide metabolites of topotecan and N-desmethyl topotecan also have been detected in urine following oral and IV (less than 2% of the administered IV dose) administration of the drug. No substantial gender-related differences in pharmacokinetics were reported in patients receiving oral topotecan. The average plasma clearance of IV topotecan was 24% higher in males than in females, mainly because of difference in body size. For more Absorption, Distribution and Excretion (Complete) data for Topotecan (6 total), please visit the HSDB record page. Metabolism / Metabolites Topotecan undergoes a reversible pH dependent hydrolysis of its lactone moiety; it is the lactone form that is pharmacologically active. Topotecan undergoes a reversible pH-dependent hydrolysis of its lactone moiety; it is the lactone form that is pharmacologically active. At pH =4, the lactone is exclusively present, whereas the ring-opened hydroxy-acid form predominates at physiologic pH. In vitro studies in human liver microsomes indicate topotecan is metabolized to an N-demethylated metabolite. The mean metabolite:parent AUC ratio was about 3% for total topotecan and topotecan lactone following IV administration. Biological Half-Life 2-3 hours The pharmacokinetics of topotecan have been evaluated in cancer patients following doses of 0.5 to 1.5 mg/sq m administered as a 30-minute infusion. Topotecan exhibits multiexponential pharmacokinetics with a terminal half-life of 2 to 3 hours. Topotecan has a terminal half-life of 3-6 hours following oral administration and 2-3 hours following IV administration of the drug. ... The objective of this study was to characterize the disposition of topotecan in a patient with severe renal insufficiency receiving hemodialysis. ... The topotecan terminal half-life off dialysis was 13.6 hr, compared with an apparent half-life determined during hemodialysis of 3.0 hr. ...
Toxicity/Toxicokinetics	Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation Most sources consider breastfeeding to be contraindicated during maternal high-dose antineoplastic drug therapy. The manufacturer recommends that women not breastfeed during treatment with topotecan and for 1 week after the last dose. Chemotherapy may adversely affect the normal microbiome and chemical makeup of breastmilk. Women who receive chemotherapy during pregnancy are more likely to have difficulty nursing their infant. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date.
References	[1]. Anticancer Drugs . 2003 Aug;14(7):569-74. [2]. Blood . 1995 May 15;85(10):2817-28. [3]. J Neurooncol . 2005 Jan;71(1):19-25. [4]. J Nucl Med . 2012 Jul;53(7):1146-54. [5]. Cancer Chemother Pharmacol . 1998;41(5):385-90.
Additional Infomation	Topotecan Hydrochloride is the hydrochloride salt of a semisynthetic derivative of camptothecin with antineoplastic activity. During the S phase of the cell cycle, topotecan selectively stabilizes topoisomerase I-DNA covalent complexes, inhibiting religation of topoisomerase I-mediated single-strand DNA breaks and producing potentially lethal double-strand DNA breaks when complexes are encountered by the DNA replication machinery. Camptothecin is a cytotoxic quinoline-based alkaloid extracted from the Asian tree Camptotheca acuminata. An antineoplastic agent used to treat ovarian cancer. It works by inhibiting DNA TOPOISOMERASES, TYPE I. See also: Topotecan (has active moiety). Drug Indication Hycamtin capsules are indicated as monotherapy for the treatment of adult patients with relapsed small cell lung cancer (SCLC) for whom re-treatment with the first-line regimen is not considered appropriate. Topotecan is indicated for the treatment of patients with metastatic carcinoma of the ovary after failure of first-line or subsequent therapy. Hycamtin capsules are indicated as monotherapy for the treatment of adult patients with relapsed small cell lung cancer (SCLC) for whom re-treatment with the first-line regimen is not considered appropriate. Topotecan monotherapy is indicated for the treatment of: - patients with metastatic carcinoma of the ovary after failure of first-line or subsequent therapy- patients with relapsed small cell lung cancer (SCLC) for whom re-treatment with the first-line regimen is not considered appropriate (see section 5. 1). Topotecan in combination with cisplatin is indicated for patients with carcinoma of the cervix recurrent after radiotherapy and for patients with Stage IVB disease. Patients with prior exposure to cisplatin require a sustained treatment free interval to justify treatment with the combination (see section 5. 1). Topotecan monotherapy is indicated for the treatment of patients with relapsed small-cell lung cancer (SCLC) for whom re-treatment with the first-line regimen is not considered appropriate. , , Topotecan in combination with cisplatin is indicated for patients with carcinoma of the cervix recurrent after radiotherapy and for patients with stage IVB disease. Patients with prior exposure to cisplatin require a sustained treatment-free interval to justify treatment with the combination. , Topotecan monotherapy is indicated for the treatment of patients with relapsed small cell lung cancer [SCLC] for whom re-treatment with the first-line regimen is not considered appropriate. , , Topotecan in combination with cisplatin is indicated for patients with carcinoma of the cervix recurrent after radiotherapy and for patients with Stage IVB disease. Patients with prior exposure to cisplatin require a sustained treatment free interval to justify treatment with the combination. , Topotecan monotherapy is indicated for the treatment of: , , , patients with metastatic carcinoma of the ovary after failure of first line or subsequent therapy; , patients with relapsed small cell lung cancer [SCLC] for whom re-treatment with the first-line regimen is not considered appropriate. , , , Topotecan in combination with cisplatin is indicated for patients with carcinoma of the cervix recurrent after radiotherapy and for patients with Stage IVB disease. Patients with prior exposure to cisplatin require a sustained treatment free interval to justify treatment with the combination. , Topotecan monotherapy is indicated for the treatment of patients with relapsed small cell lung cancer (SCLC) for whom re-treatment with the first-line regimen is not considered appropriate. Topotecan in combination with cisplatin is indicated for patients with carcinoma of the cervix recurrent after radiotherapy and for patients with Stage IVB disease. Patients with prior exposure to cisplatin require a sustained treatment free interval to justify treatment with the combination. Topotecan is indicated for the treatment of patients with metastatic carcinoma of the ovary after failure of first-line or subsequent therapy.

Solubility Data

Solubility (In Vitro)

DMSO: 92~100 mg/mL (200.9~218.4 mM) Water: <1 mg/mL Ethanol: ~92 mg/mL (~200.9 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.46 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 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 (4.54 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 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. Solubility in Formulation 3: ≥ 2.08 mg/mL (4.54 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 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. Solubility in Formulation 4: Saline: 30 mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.1838 mL	10.9192 mL	21.8384 mL
	5 mM	0.4368 mL	2.1838 mL	4.3677 mL
	10 mM	0.2184 mL	1.0919 mL	2.1838 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.