hepatocellular carcinoma. Cancer Treat Rep 71:1213-1216, 1987 (57)




AL: Cyclosporin A corrects daunorubicin resistance in Ehrlich ascites carcinoma. Br J Cancer 54:235-238, 1986 (32)


AL: Verapamil (V) and quinine (Q) chemosensitize myeloma cells to doxorubicin (DOX) and vincristine (VCR) in vitro and are capable of reversing clinical relapses in multiple myeloma (MM). Blood [Suppl] 74:197, 1989 (33)


AL: The use of R-verapamil (R-VPM) is superior to racemic VPM in breaking multidrag resistance (MDR) of malignant cells. Proc Am Assoc Cancer Res 30:503, 1989 (34)


Phase I trial of multidrug resistance modulation with cyclosporin A. Proc Am Assoc Cancer Res 32:195, 1991 (35)




Determination of tamoxifen and biologically active metabolites in human breast tumours and plasma. Eur J Cancer Clin Oncol 17:1183-1189, 1981 (37)


E, ET AL: Serum tamoxifen concentrations in the athymic nude mouse after three methods of administration. Cancer Chemother Pharmacol 20:316-318, 1987 (38)







Modulation of vinblastine [VB] cytotoxicity by dipyridamole [DP] versus tamoxifen [TAM]: An in vivo preclinical study by subrenal capsule assay [SRCA] of human renal carcinoma [HRCC]. Proc Am Assoc Cancer Res 31:376, 1990 (39)


ET AL: Triphenylethylenes: A new class of protein kinase C inhibitors. JNCI 76:12431246, 1986 (40)


RO: Calcium channel antagonist properties of antineoplastic antiestrogen. Tamoxifen in the PC12 neurosecretory cell line. Cancer Res 47:70-74, 1987 (41)


AL: Cellular localization of the multidrugresistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci U S A 84:7735-7738, 1987

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Howard A. Burris III, Axel-R. Hanauske, Randall K. Johnson, Martha H. Marshall, John G. Kuhn, Susan G. Hilsenbeck, Daniel D. Von Hoff*

Background: Topotecan [(S)-9dimethylaminomethyl(10-hydroxycamptothecin), NSC 609699, SK&F 104864A], a semisynthetic analogue of the natural product camptothecin, is a cell cycle-specific drug that exerts antineoplastic activity through inhibition of topoisomerase I. Currently, topotecan is undergoing phase I and early phase II clinical trials. The dose-limiting toxicity for topotecan is myelosuppression. Purpose: Our purpose was to determine Topotecan [(S)-9-dimethylaminoplasma concentrations and exposure rnethyl(lO-hydroxycamptothecin), NSC times necessary for optimal clinical 609699, SK&F 104864A] is a semiactivity and tumor types that may be responsive in phase II clinical studies of topotecan. Methods: A soft-agar cloning system assay was used to determine the in vitro effects of topotecan against cells from biopsy Received October 2, 1991; revised August 12, specimens of colorectal, breast, lung, 1992; accepted September 16, 1992. Supported in part by grants from SmithKJine ovarian, renal cell, and gastric can- Beecham Pharmaceuticals, King of Prussia, Pa., cers and cancers of unknown pri- and the Cancer Therapy and Research Foundamary origin. We studied 141 freshly tion of South Texas, San Antonio. H. A. Burris III, Hematology-Oncology Servexplanted tumor specimens, using ice, Brooke Army Medical Center, Fort Sam 1-hour exposure to topotecan, and 80 Houston, Tex. A.-R. Hanauske, Technische Universitat were studied using continuous exMunchen, Abteilung Hamatologie und posure. A decrease in tumor colony Onkologie, KJinikum rechts der Isar, Munich, formation resulting from drug ex- Federal Republic of Germany. R. K. Johnson, SmithKJine Beecham, King of posure was considered an in vitro Prussia, Pa. response if survival of colonies was M. H. Marshall, J. G. Kuhn, S. G. Hilsenbeck, up to 50% of that in controls. D. D. Von Hoff, The University of Texas Health Results: With 1-hour exposure, in Science Center at San Antonio and Cancer Therapy and Research Center, San Antonio. vitro responses were seen in 10% •Correspondence to: Daniel D. Von Hoff, and 25% of assessable tumor speci- M.D., Section of Drug Development, Division of mens at final topotecan concentra- Oncology, Department of Medicine, The University of Texas Health Science Center at San tions of 1.0 and 10.0 (ig/mL, Antonio, 7703 Floyd Curl Dr., San Antonio, TX respectively. With continuous ex- 78284-7884. Journal of the National Cancer Institute

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AL: Bilirubin: A physiological substrate for the multidrug transporter. Proc Am Assoc Cancer Res 32:426, 1991

Activity of Topotecan, a New Topoisomerase I Inhibitor, Against Human Tumor Colony-Forming Units In Vitro

posures at concentrations of 0.1 and 1.0 (ig/mL, in vitro response rates were 34% and 76%, respectively. Specific activity was seen against colorectal, breast, non-small-cell lung, ovarian, and renal cell cancers, with responses observed in 27%, 25%, 32%, 39%, and 83%, respectively, of assessable tumor specimens after continuous exposure to 0.1 \LglmL topotecan. A subset of tumor specimens resistant to doxorubicin or fluorouracil was sensitive to topotecan, and the difference in sensitivity was statistically significant. In addition, some of the tumor specimens resistant to cyclophosphamide and etoposide were also sensitive to topotecan. Conclusions: Topotecan appears to. be active in vitro against a variety of human tumors, including a subgroup resistant in vitro to standard antineoplastic agents. If plasma levels of 0.1 p.g/mL can be achieved for prolonged periods of time in ongoing clinical trials, topotecan should have substantial clinical activity. Implications: Further clinical development of topotecan is warranted. [J Natl Cancer Inst 84:18161820, 1992]

Vol. 84, No. 23, December 2, 1992

as to pinpoint tumor types that may be responsive in phase II clinical trials.

Materials and Methods Compounds Topotecan was provided by SmithKline Beecham Pharmaceuticals (King of Prussia, Pa.). Final concentrations for 1-hour-exposure experiments were 1.0 ^.g/mL and 10 jig/mL. For continuous-exposure experiments, final concentrations were 0.1 ng/mL and 1.0 (Ag/mL. Stock solutions were prepared in distilled water (used as a solvent control) and stored at -20 °C in 1-mL aliquots prior to use.

Drug Stability Experiments To determine the stability of topotecan in the soft-agar cloning system, control experiments were performed. Topotecan was added to six petri dishes without tumor cells, and we determined pH levels in the petri dishes immediately and again after incubation at 37 °C under 5% CO 2 and 100% humidity for 2, 6, 24, 48, and 72 hours. The topotecan lactone form was identified by high-pressure liquid chromatography at the same time points (2).

Human Tumor Cloning System After obtaining written informed consent from the patients in accordance with federal and institutional guidelines, we collected tumor biopsy specimens by sterile standard procedures as part of routine clinical procedures. For transport to the laboratory, biopsy specimens of solid tumors were stored in McCoy's 5A medium containing newborn calf serum, 10 mM HEPES, 90 U/mL penicillin, and 90 u,g/mL streptomycin (all from GIBCO BRL, Grand Island, N.Y.). Preservative-free heparin (10 U/mL; O'Neill, .Johns and Feldman, St. Louis, Mo.) was added immediately after collection of fluids to prevent coagulation. Solid tumor specimens were minced and repeatedly passed through metal meshes with widths of 40 \im (E. C. Apparatus, St. Petersburg, Fla.) to obtain a single-cell suspension. Effusions were centrifuged at 150g for 5-7 minutes and passed through 25-gauge needles to obtain single-cell suspensions. Cells were suspended in McCoy's 5A medium containing 5% horse serum and 10% fetal calf serum (both from Hyclone, Logan, Utah) and 2 mM sodium pyruvate, 1 mM glutamine, 90 U/mL penicillin, 90 jig/mL streptomycin, and 35 ng/mL L-serine (all from GIBCO BRL). The human tumor cloning assay was performed with the two-layer system described by Hamburger and Salmon (//), with several modifications. Base layers contained 0.5% agar (Difco Laboratories, Detroit, Mich.) in the mixture of McCoy's 5A medium described above, 0.6% soy broth (Difco Laboratories), and 100 ng/mL asparagine (GIBCO BRL). Cells were plated at a density of 5 X 105 per dish in

35-mm petri dishes (Corning Science Products, Corning, N.Y.) in a mixture of 0.3% agar in Connaught Medical Research Laboratories medium 1066 (Irvine Scientific, Irvine, Calif.) containing 15% horse serum, 2% fetal calf serum, 5 mg/100 mL vitamin C, 90 U/mL penicillin, 90 ng/mL streptomycin, 0.1 mM nonessential amino acids, 2 mM glutamine (all from GIBCO BRL), 2 U/mL insulin (Iletin IR; Eli Lilly, Indianapolis, Ind.), 2 u,g/mL transferrin, and 4 ng/mL hydrocortisone (both from Sigma Chemical Co., St. Louis, Mo.). Immediately before cells were plated, HEPES (GIBCO BRL; 10 mM final concentration) and sodium pyruvate (2 mM final concentration) were added. All determinations were performed in triplicate. Each experiment included a control with orthosodiumvanadate (0.01 M; Sigma Chemical Co.) to assure the presence of a good single-cell suspension (positive control) (12). Plates were incubated at 37 °C under 5% CO 2 and 100% humidity. After 14 days, colonies were counted with an inverted microscope. An experiment was considered assessable when the distilled water control (without topotecan) had 20 or more colonies per plate and when the positive control (orthosodiumvanadate) showed inhibition of colony formation of at least 70%. A decrease in tumor cell colony formation resulting from exposure to topotecan was considered to be a positive response if survival of colonies was 50% or less than that of controls. Tumor cells were exposed to the drug for 1 hour, and the cells were washed twice with McCoy's 5A medium and 10% heat-inactivated fetal calf serum. For the experiment that involved continuous exposure to topotecan, the drug was added directly to the cells, and both topotecan and the cells were immediately plated in the petri dishes. The cells were not washed.

Statistical Analyses Data were expressed as means and standard deviations of triplicate determinations per datapoint. We calculated percent survival by expressing the average number of tumor colony-forming units per petri dish from topotecan-treated cells as percent of the average number of tumor colony-forming units per petri dish from untreated controls. All studies were performed with triplicate petri plates. Statistical analyses to compare in vitro responses at various concentrations and to compare 1-hour exposure data to continuous exposure data were performed using the chi-squared test and the McNemar Test.

Results A total of 348 freshly explanted tumor specimens were studied using a 1-hour exposure schedule at topotecan concentrations of 1 n-g/mL and 10 p-g/mL. Of these specimens, 141 (41%) showed adequate growth in diluent controls. The histologic types of tumors tested are summarized in Table 1. The REPORTS 1817

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synthetic analogue of the natural product camptothecin. A previous study has indicated that camptothecin and its analogues exert their cytotoxic effects by inhibition of topoisomerase I, an enzyme important for relieving torsional strain during replication of DNA. Camptothecin derivatives stabilize the complex between topoisomerase I and DNA. As a result of stabilization of that complex, single-stranded breaks of the DNA occur (1). Topotecan can exist as both a closed lactone form and as an open-ringed carboxylate form. The inactive openringed form is generated by pHdependent hydrolysis at more basic pH levels, including physiologic values (2). Only the closed lactone form of topotecan has antitumor activity (2). Camptothecin has been reported to induce objective partial responses in a small percentage of patients with colorectal, non-small-cell lung, and gastric cancers, as well as in patients with melanoma and acute myelogenous leukemia. However, clinical phase II trials of camptothecin were abandoned because the drug caused unpredictable and severe myelosuppression, gastrointestinal toxicity, and hemorrhagic cystitis (3-5). Compared with camptothecin, topotecan has a higher water solubility that is due to a chemically stable basic side chain. This modification has resulted in decreased toxicity in animal models (2,6). Topotecan has been observed to have considerable activity against tumors implanted subcutaneously, intravenously, or intraperitoneally in animal models (7). Equivalent activity has been noted when the agent was administered by different routes, including oral, intravenous, intraperitoneal, or subcutaneous (7). Topotecan has been undergoing phase I clinical testing. The dose-limiting toxicity in patients has been myelosuppression (2,8-10). The drug is just now entering phase II clinical testing. The purpose of our present study was to determine the in vitro effects of topotecan against a variety of human tumors, with the use of a soft-agar cloning system. This system may help to define plasma concentrations and treatment schedules required for optimal clinical antitumor activity as well

Table 1. Tumor types studied with topotecan

Tumor type Colorectal Breast Lung, non-small-cell Ovarian Renal cell Gastric Unknown primary Other Total

1-hour incubation: No. assessable*/No. attempted (%) 13/41 26/51 30/88 37/52 6/14 5/15 3/20 21/67 141/348

Continuous incubation: No. assessable'/No. attempted (%]

(32) (51) (34) (71) (43) (33) (15) (31) (41)

11/32 12/28 22/74 18/29 6/14 2/11 2/15 7/38 80/241

(34) (43) (30) (62) (43) (18) (13) (18) (33)

•Mean of 20 or more colonies in control plates and positive control 0.3 or less times control.


doxorubicin (P = .0009) or fluorouracil (P = .002). Some of the tumor specimens resistant to cyclophosphamide and etoposide were also sensitive to topotecan, although this difference was not statistically significant (possibly because of the small sample size). As is also shown in Table

Table 2. In vitro antitumor activity of topotecan after continuous exposure or after 1-hour exposure* Continuous incubation

1-hour incubation Tumor type

1.0 (Ag/mL

10.0 ji.g/mL

0.1 jig/mL

Colorectal Breast Lung, non-small-cell Ovarian Renal cell Other Total

0/13 4/26 2/30 2/37 1/6 5/29 14/141

3/13 9/26 6/30 7/37 1/6 9/28 35/140

3/11 3/12 7/22 7/18 5/6 2/11 27/80

(0) (15) (7) (5) (17) (17) (10)

(23) (35) (20) (19) (17) (32) (25)

(27) (25) (32) (39) (83) (18) (34)

1.0 |xg/mL 8/11 9/12 19/22 11/18 6/6 8/11 61/80

(73) (75) (86) (61) (100) (73) (76)

•Values in columns = number of specimens inhibited (i.e., colony formation ^0.5 times that of the control)/number of specimens assessable (%).

Table 3. Comparison of the antitumor activity in tumor specimens with continuous exposure to topotecan or conventional antineoplastic agents Topotecan (0.1 (jLg/mL) No. sensitive

No. resistant


Doxorubicin (0.4 ng/mL) No. sensitive* No. resistant

4 11

0 28


Fluorouracil (6.0 ng/mL) No. sensitive* No. resistant

0 12

1 23


Cyclophosphamide (3.0 ng/mL) No. sensitive* No. resistant

5 5

1 15


Etoposide (3.0 ng/mL) No. sensitive* No. resistant

1 6

1 10


•Colony formation 0.5 or less times control. fMcNemar's test.

Journal of the National Cancer Institute

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major subgroups of tumors tested were colorectal, breast, non-small-cell lung, ovarian, renal cell, and gastric cancers, as well as cancer of unknown primary origin. In vitro activity against tumor colony-forming units was noted in 14 (10%) of 141 specimens at 1.0 p-g/mL and in 35 (25%) of 140 specimens at 10.0 (j.g/mL (P = .0009) (Table 2). For continuous-exposure experiments, topotecan was studied at 0.1 (ig/mL and 1.0 (xg/mL in a total of 241 tumor specimens. The major tumor types were identical with those studied in 1-hour incubation experiments (Table 1). Eighty (33%) specimens had adequate growth in control plates. There was a statistically significant decrease (P2a. Conclusions: The magnitude of the change in 5-FU catabolism is similar to the magnitude of the decrease in 5-FU clearance in our previous study. These observations suggest that changes in 5-FU catabolism during therapy with IFN a-2a, 5-FU, and LV may account for the decreased 5-FU clearance. [J Natl Cancer Inst 84:18201825, 1992]

Activity of topotecan, a new topoisomerase I inhibitor, against human tumor colony-forming units in vitro.

Topotecan [(S)-9-dimethylaminomethyl(10-hydroxy-camptothecin), NSC 609699, SK&F 104864A], a semisynthetic analogue of the natural product camptothecin...
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