GYNECQLOGIC
ONCOLOGY
47, 186-190 (1992)
Comparison of U-73,975 and Cisplatin Cytotoxicity in Fresh Cervical and Ovarian Carcinoma Specimens with the ATP-Chemosensitivity Assay’ RANDALL D. HIGHTOWER, M.D.,2 BERND-UWE UNTCH, M.D.,3 ROBERTOANGIOLI, M.D.,4
SEVIN, M.D., PH.D., JAMES P. PERRAS,PH.D., MICHAEL REINALD~ RAMOS, B.S., AND HERVY AVERETTE, M.D.
Divkion of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Miami School of Medicine, Miami, Florida 33101 Received January 22, 1992
U-73,975 (U-73), a closely related synthetic analogueof the antitumor agent CC-1065, acts by binding tightly in the minor grooveof DNA. A comparisonwasmadebetweenthe cytotoxicity of U-73 and cisplatin (DDP) on 11 fresh cervical and 7 fresh ovarian carcinoma specimens.The ATP-chemosensitivity assay as previously described(Sevin et al. Gynecd. Oncol. 31, 191204, 1988)was used to determinethe cytotoxic effect of U-73 and DDP. IC 50swerecalculatedusing regressionanalysis.The meanIC 50sfor U-73 and DDP were519pg/ml and 2918rig/ml, respectively,for the cervical carcinomaspecimensand 324pg/ml and 2649 rig/ml, respectively, for the ovarian carcinoma specimens. Signilkance comparing U-73 and DDP for cervical and ovarian tissuewas demonstratedwith P < 0.001. U-73 was4000 timesascytotoxic per unit of massas DDP on cervical carcinoma comparedto over 8000 times for ovarian carcinoma. Basedon thesein vitro data, U-73 appearsto bea very promisingantitumor agent for cervical and ovarian carcinoma. 0 1992 Academic press, hf.
INTRODUCTION u-73,975
antitumor organism,
(U-73) is a new synthetic analog of the potent agent CC-1065 [l-6]. Isolated from the soil Streptomyces zelensis, in 1978, CC-1065 has
Presented at the 23rd Annual Meeting of the Society of Gynecologic Oncologists, San Antonio, TX, March 15-18, 1992. ’ This article was written while Randall D. Hightower, lieutenant commander in the U.S. Navy, was a fellow at the University of Miami/Jackson Memorial Hospital training in Gynecologic Oncology. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. ’ To whom correspondence should be addressed at Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, (D52), University of Miami School of Medicine, P.O. Box 016960, Miami, FL, 33101. Fax: (305) 325-1469. 3 Supported in part by the Grimmke Foundation, Germany. 4 Supported in part by Consorzio Interuniversitario Nazionale per la Bio-Oncologia (CINBO), Italy.
been shown to inhibit DNA synthesis and exhibits nonintercalative binding in the minor groove of doublestranded DNA in the adenine-thymidine regions followed by covalent bonding with the N-3 adenine [l-4,7-11]. Its spiral structure was found to promote preferential and selective binding to specific DNA sequences [12,13]. The adjacent cyclopropyl group can then initiate the alkylating reactions and form covalent bonds to cellular DNA [1,2,12]. Given intravenously to healthy mice, it was discovered that CC-1065 caused a delayed fatal hepatotoxicity [1,2,14]. Subsequently, the segment of CC-1065 incorporating the alkylating conjugated cyclopropyl ring was synthesized [1,13-151. U-73 was thus developed which resulted in the elimination of the fatal hepatotoxicity while maintaining similar binding and alkylating action and antitumor efficacy [1,14-161. U-73 appears to only attack double-stranded DNA and specific adenines [l]. This compound was synthesized and studied by Upjohn of Kalamazoo, MI [2]. The efficacy of U-73 has been evaluated with a number of human solid tumor xenografts. When mice received a single intravenous injection after tumor implantation, U-73 was highly active against clear cell Cak.-1 carcinoma xenograft and yielded 97 to 100% inhibition of tumor growth measured by tumor weight (TGI). When mice received multiple intravenous injections, U-73 was also highly active against both colon Cx- and Lx-l tumor xenografts and produced a greater than 90% TGI [6]. U-73 is therapeutically highly active against a broad spectrum of tumors including L1210 leukemia, colon 38, and pancreas 02 tumors [5]. This structurally simplified analog showed activity superior to that of CC-1065 against a number of experimental tumors after a single intravenous injection [15]. Chemotherapy for carcinoma of the cervix has been generally reserved for patients with recurrent or advanced disease. Cisplatin (DDP) is the most extensively studied
186 00!%8258/!42
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
COMPARISON
OF U-73,975 AND CISPLATIN
single agent in squamous cell carcinoma of the cervix [21]. The standard treatment for ovarian malignancy includes cytoreductive surgery followed by adjuvant chemotherapy with one or a combination of several agents, commonly including DDP [17]. Thus, DDP was the single agent chosen for comparison in these gynecologic malignancies. The cytotoxicity of U-73 was compared to DDP on freshly obtained cervical and ovarian carcinoma specimen using the ATP-chemosensitivity assay in this in vitro study. MATERIALS
AND METHODS
Tumor tissue specimens were obtained from 17 patients who underwent either a radical hysterectomy or exploratory laparotomy for staging of cervical carcinoma or exploratory laparotomy with cytoreduction for ovarian carcinoma at the University of Miami/Jackson Memorial Medical Center between January and June 1991. Eleven of these specimens represented cervical carcinoma and seven ovarian carcinoma. Two of the ovarian carcinoma tissues obtained were from patients with recurrent disease. One cervical specimen represented a metastatic node in a patient with cervical carcinoma. The specimens were immediately brought to the lab for processing. On arrival at the laboratory the Tumor preparation. cervical tumor was pretreated for 20 min with 100 IU/ml penicillin (5 x ), streptomycin 100 pg/ml (5 x ), and fungizone (5 x ) to sterilize the surface of the tumor. The ovarian tumor specimens received no pretreatment since they had been obtained from a sterile environment. The tumors were then minced into fine pieces with iris scissors and the resulting suspension incubated overnight in McCoy’s media with 5% fetal bovine serum (FBS), penicillin (2 X ), streptomycin (2 X ), 0.2 mg/ml collagenase II (Worthington), 2.25 PUK/ml pronase (Calbiochem), and 1400 U/ml DNase (Calbiochem). After enzyme treatment the cell suspension was separated from the tissue fragments and then suspended in enriched CMRL media with 15% FBS. This suspension contained both single cells and small clusters of up to 30 cells. Approximate cell counts were obtained using trypan blue exclusion. The cells were then diluted to a cell concentration of 100,000 viable cells/ml for plating into 24-well plates. These cells were plated by distributing the suspension in 0.5-ml aliquots over an underlayer of 0.5% agar in McCoy’s media containing 15% FBS. Drugs. DDP was tested using the reported peak plasma concentration (PPC) as the reference value [18]: cisplatin (Bristol Myers) 2.5 pg/ml. The PPC for U-73 has not been determined as U-73 is currently undergoing phase I trials. The PPC was arbitrarily chosen as 1 rig/ml as determined by preliminary data and manufacturer’s suggestions. All agents were freshly dissolved in Hanks’
ON CERVIX
AND OVARY
187
balanced salt solution (HBSS). U-73 was kindly provided by the Upjohn Company. A TP chemosensitivity assay. Triplicate wells were used for each of the five drug concentrations for each drug tested (0.1X, 0.2X, 0.5X, 1X, 5X PPC). The cells were plated at 50,000 ceils per well followed by continuous drug exposure. A 6-day continuous exposure was used because of the difficulty in manipulating fresh tumor cells without affecting the viability of the cells in order to remove the drug. Twenty microliters of each drug was added to the appropriate wells at a concentration sufficient to obtain the proper final concentration. Dose-response curves were obtained for the fractional intracellular ATP, representing cell survival, relative to untreated controls on Day 6 by extracting the ATP from the cells in situ with 4% trichloroacetic acid. ATP luminescence was determined as previously described [19]. Cytostatic drugs do not influence the bioluminescence reaction at reasonable concentrations, but very high levels (mmol/liter) of colored drugs, such as doxorubicin, quench the light reaction [20]. U-73 is a colorless compound. To ensure that U-73 did not absorb the luminescence signal, two standard curves were obtained. The standard curves were obtained after serially diluting a known quantity of ATP with media and evaluating the bioluminescence of the media/ATP serial dilution as compared to the media/ATP serial dilution with the addition of U-73 at 1000 times the concentration used in treating the cells. No difference in luminescence absorption was demonstrated. Data analysis. The concentrations necessary to produce a 50% decrease in cellular ATP (IC 50 = 0.5 surviving fraction = 0.5 fractional cellular ATP) were calculated for each cytotoxic agent, using the median effect plot of log (fa/fu) vs log C [fu = fraction unaffected = surviving fraction (SF); fa = fraction affected = 1 - SF; C = drug concentration [21]]. The t test was employed to calculate statistical significance. A P value ~0.05 was considered significant. RESULTS
Dose-response curves of all the cervical and ovarian specimens were performed for U-73 and DDP over a dose range of 0.1X to 5X PPC. Tables 1 and 2 display the IC 50s of the cervical and ovarian specimens, respectively, for U-73 and DDP. The IC 50s were calculated in rig/ml for purposes of comparison. The mean IC 50s for U-73 and DDP were 0.519 rig/ml (range of 0.005-1.627 rig/ml) and 2018 rig/ml (range of 215-4125 rig/ml), respectively, for the cervical carcinoma specimens and 0.324 rig/ml (range of 0.010-1.998 rig/ml) and 2649 rig/ml (range of 1008-4800 rig/ml), respectively, for the ovarian carcinoma specimens. Comparison of the mean IC 50s for U-
188
HIGHTOWER
TABLE 1 IC 50sof Fresh Cervical Carcinoma Specimenswhen Treated with U-73 and DDP
ET AL. *
u-73 126%
23391 !?a&91 !23&91
. . . . . .l
Patient ID
u-73
DDP”
233-91 235-91 236-91 242-91 243-91 246-91 247-91 259-91 267-91 278-91 279-91
0.186 0.221 0.005 0.534 0.459 0.696 0.406 0.384 0.221 1.627 0.972
2055 470 215 4125 2500 1615 760 3950 3000 3108 403
-
60%
.. ..
,&‘&91
243-91 24691
-
247-91
-
ZmSl
40%
287-91 -.-.-* _,_. m&91
20%
-
0%
0
12
3
4
5
ms1
6
DDP 126% 1
’ IC 50s measured in rig/ml.
73 and DDP over the dose range of 0.1X to 5X PPC on cervical and ovarian specimens demonstrated statistical significance using the test with P < 0.001, respectively. Figure 1 displays the dose-response curves for the eleven cervical carcinoma specimens. The curves are displayed as the curve resulting from the percentage survival at various drug concentrations. For descriptive purposes, an ATP surviving fraction of 50% or less at 0.5X is considered sensitive and greater than 50% as resistant. Using this criteria, eight of the cervical specimens were sensitive to U-73 and four were sensitive to DDP. Figure 2 displays the dose-response curves for the seven ovarian specimens. Of these seven specimens, five were sensitive to U-73 but none was sensitive to DDP. DISCUSSION U-73 represents an entirely new class of cytotoxic agents developed as an analog of the potent antitumor agent CC-1065. It was synthesized to eliminate the delayed fatal hepatoxocity that CC-1065 caused in healthy mice but maintain similar action and antitumor efficacy TABLE 2 IC 50sof Fresh Ovarian CarcinomaSpecimenswhen Treated with U-73 and DDP Patient ID
u-73
DDP”
226-91 227-91 229-91 249-91* 270-91’ 271-91 282-91
0.016 0.014 0.010 0.013 0.112 0.106 1.998
1498 4800 1943 1008 4450 2790 2053
a IC 50s measured in rig/ml. b Recurrent ovarian carcinoma.
100% 80%
0
12
3
4
5
6
FIG. 1. Dose-response curves of fresh cervical carcinoma specimens treated with U-73 and DDP. PPC represents the peak plasma concentration for DDP or the reported reference value for U-73.
[1,2,14]. Its spiral structure was found to promote preferential and selective binding to specific DNA sequences [12,13]. Because of these characteristics, this drug is effective at low doses [8-111. Comparing the IC 50s from Tables 1 and 2, U-73 was approximately 4000 times as cytotoxic per unit of mass as DDP on cervical carcinoma compared to over 8000 times for ovarian carcinoma. This comparison between cervical and ovarian carcinoma IC 50s was statistically significant. Of the 11 cervical specimens, 73% were sensitive to U-73 but only 36% were sensitive to DDP. This correlates with the overall response rate of 38% noted in the Gynecologic Oncology Group study [22]. Approximately 71% of the ovarian specimens were sensitive to U-73 in contrast to all of the specimens showing resistance to DDP. The dose-response curves of the ovarian specimens (Fig. 2) demonstrated two resistant tumors to U-73. DDP has remained one of the most commonly used chemotherapeutic agents for both cervical and ovarian carcinoma. The poor response of the ovarian carcinoma specimens may be a function of small sample size or the definition of sensitivity that was used. In this in vitro study, U-73 demonstrated superiority in cytotoxicity over DDP as a single agent on cervical and ovarian carcinoma tumor specimens. U-73 exhibits cy-
COMPARISON
OF U-73,975 AND CISPLATIN 3.
u-73
120%
-
22691
100%
-
297-91
-
999-91
-
24991 n&91
--
28291 271-91
3 %
80%
VI Y
60%
I!
$”
40% 20% 0% 0
1
2
3
4
5
6
DDP 120%
a.* wIi
1
100%
ON CERVIX
Chidester, C. G., Krueger, W. C., Mizsak, S. A., Duchamp, D. J., and Martin, D. G. The structure of CC-1065, a potent antitumor agent, and its binding to DNA, J. Am. Chem. Sot. 103(25), 76297635 (1981).
Hurley, L. H., Warpehoski, M. A., Lee, C. S., McGovern, .I. P., Scahill, T. A., Kelly, R. C., Mitchell, M. A., Wicnienski, N. A., Gebhard, I., Johnson, P. D., and Bradford, V. S. Sequence specificity of DNA alkylation by the unnatural enantiomer of CC-1065 and its synthetic analogs, J. Am. Chem. Sot. ll2(12), 4633-4649 (1990). 5 DeKoning, T. F., Kelly, R. C., Wallace, T. L., and Li, L. H. Antitumor activity and biochemical effect of three selected cyclopropapyrroloindole (CPI) analogs, Proc. Am. Assoc. Cancer Res. 30, 491 (1989). [Abstract 19531 6 DeKoning, T. F., Postmus, R. J., Wallace, T. L., Kelly, R. C., and Li, L. H. Therapeutic evaluation of three cyclopropapyrroloindole (CPI) analogs against human tumor xenografts, Proc. Am. 4
Assoc.
80%
189
AND OVARY
7
60%
Cancer
Res. 31, 348 (1990).
Hurley, L. H., and Needham-VanDevanter, D. R. Covalent binding of antitumor antibiotics in the minor groove of DNA. Mechanism of action of CC-1065 and the pyrrolo(l,4)benzodiazepines, Act.
Chem.
Rex
19(B),
203-237
(1986).
Hanka, L. J., Dietz, A., Gerpheide, S. A., Kuentzel, S. L., and Martin, D. G. CC-1065 (NSC 298223), a new antitumor antibiotic. Production, in vitro, biological activity microbiological assay, and taxonomy of the producing organism, J. Antibiot. 31, 1211 (1978). 9. Martin, D. G., Hanka, L. J., and Neil, G. L. Isolation, characterization, and preliminary antitumor evaluation of CC-1065, a potent new agent from fermentation, Proc. Am. Assoc. Cancer Res. 8.
0
1
2
3
4
5
6
FIG. 2. Dose-response curves of fresh ovarian carcinoma specimens treated with U-73 and DDP. PPC represents the peak plasma concentration for DDP or the reported reference value for U-73. Patient ID 249-91 and 270-91 represent recurrent ovarian carcinoma.
totoxicity in picogram amounts compared to microgram amounts for DDP. Because of the small sample size, especially of ovarian tissue, additional studies are necessary before any conclusions can be made. The arbitrary assignment of 50% as constituting an active versus an inactive response may have little bearing on the ultimate activity. The concentration of U-73 required for in vivo activity may not correlate well with in vitro data. In addition, in vitro data does not consider toxicity to normal tissues. U-73 is completing phase I trials which will address these issues. Only after these trials are performed will we know if the smaller amounts of drug needed to effect a response are obtainable considering the potential toxicity of the drug to the patient. In summary, U-73 is a potent antitumor agent in this in vitro study using the ATP-CSA for analysis of the cytotoxic effect and appears to be a promising chemotherapeutic agent. REFERENCES 1.
Warpehoski, M. A. CC-1065 analogs: Therapeutic potential of a sequence specific drug-DNA reaction, Proc. Am. Assoc. Cancer Res. 30, 668 (1989).
2.
Warpehoski, M. A. Stereoelectronic factors influencing the biological activity and DNA interaction of synthetic antitumor agents modeled on CC-1065. J. Med. Chem. 31. 3 C19881. \ ,
19, 99 (1978).
10. Martin, D. G., Chidester, C. G., Duchamp, D. J., and Misak, S. A. Structure of CC-1065 (NSC 298223), a new antitumor antibiotic, J. Antibiot. 33, 902 (1980). 11. Martin, D. G., Biles, C., Gerpheide, S. A., Hanka, L. J., and Krueger, W. C. CC-1065 (NSC 298223), a potent new antitumor agent: Improved production and isolation, characterization, and antitumor activity, J. Antibiot. 34, 1119 (1981). 12. Reynolds, V. L., McGovren, J. P., and Hurley, L. H. The chemistry, mechanism of action and biological properties of CC-1065, a potent antitumor antibiotic, Antibiotics 39, 319 (1986). 13. Wierenga, W., Bhuyan, B. K., Kelly, R. C., Krueger, W. C., and Li, L. H. Antitumor activity and biochemistry of novel analogs of the antibiotic CC-1065. Adv. Enzyme Regul. 25, 141 (1986). 14. McGovren, J. P., Clarke, G. L., Pratt, E. A., and DeKoning, T. F. Preliminary toxicity studies with the DNA-binding antibiotic, CC1065, J. Antibiot. 37(l), 63-70 (1984). 15. DeKoning, T. F., Krueger, W. C., Wallace, T. L., Prairie, M. D., Warpehoski, M. A., Kelly, R. C., and Li, L. H. Influence of structure and chirality on the antitumor activity and biochemical effects of CC-1065 and its analogs, Proc. Am. Assoc. Cancer Res. 28, 262 (1987). 16. Warpehoski, M. A., Kelly, R. C., McGovren, J. P., and Wierenga, W. N-2-substitutedtetrahydro-4-oxocyclopropa[c]pyrrolo-[3,2-e] indoles: Novel anticancer agents modeled on CC-1065, Proc. Am. Assoc. Cancer Res. 26, 221 (1985). [Abstract] 17. Deppe, G. Chemotherapy of gynecologic cancer, 2nd ed., WileyLiss, New York, p. 127 (1990). 18. Alberts, D., and Chen, G. Tabular summary of pharmacokinetic parameters relevant to in vitro drug assay, in cloning human tumor stem cells (S. Salmon, Ed.), Liss, New York, pp. 351-359 (1980). 19. Sevin, B. U., Peng, Z., Perras, J., Ganjei, P., Penalver, M., and
190
HIGHTOWER
Averette, H. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing, Gynecol. Oncol. 31, 191-204 (1988). 20. Kangas, L., Gronroos, M., and Nieminen, A. L. Bioluminescence of cellular ATP: A new method for evaluating cytotoxic agents in vitro. Med. Biol. 62, 338-343 (1984).
ET AL. 21. Chou, T., and Talaly, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul. 22, 27-55 (1984). 22. DiSaia, P. J., and Creaseman, W. T. Clinical gynecologic oncology, Mosby, St. Louis, 3rd ed., p. 115 (1989).
ONCOLOGY
47, 186-190 (1992)
Comparison of U-73,975 and Cisplatin Cytotoxicity in Fresh Cervical and Ovarian Carcinoma Specimens with the ATP-Chemosensitivity Assay’ RANDALL D. HIGHTOWER, M.D.,2 BERND-UWE UNTCH, M.D.,3 ROBERTOANGIOLI, M.D.,4
SEVIN, M.D., PH.D., JAMES P. PERRAS,PH.D., MICHAEL REINALD~ RAMOS, B.S., AND HERVY AVERETTE, M.D.
Divkion of Gynecologic Oncology, Department of Obstetrics and Gynecology, University of Miami School of Medicine, Miami, Florida 33101 Received January 22, 1992
U-73,975 (U-73), a closely related synthetic analogueof the antitumor agent CC-1065, acts by binding tightly in the minor grooveof DNA. A comparisonwasmadebetweenthe cytotoxicity of U-73 and cisplatin (DDP) on 11 fresh cervical and 7 fresh ovarian carcinoma specimens.The ATP-chemosensitivity assay as previously described(Sevin et al. Gynecd. Oncol. 31, 191204, 1988)was used to determinethe cytotoxic effect of U-73 and DDP. IC 50swerecalculatedusing regressionanalysis.The meanIC 50sfor U-73 and DDP were519pg/ml and 2918rig/ml, respectively,for the cervical carcinomaspecimensand 324pg/ml and 2649 rig/ml, respectively, for the ovarian carcinoma specimens. Signilkance comparing U-73 and DDP for cervical and ovarian tissuewas demonstratedwith P < 0.001. U-73 was4000 timesascytotoxic per unit of massas DDP on cervical carcinoma comparedto over 8000 times for ovarian carcinoma. Basedon thesein vitro data, U-73 appearsto bea very promisingantitumor agent for cervical and ovarian carcinoma. 0 1992 Academic press, hf.
INTRODUCTION u-73,975
antitumor organism,
(U-73) is a new synthetic analog of the potent agent CC-1065 [l-6]. Isolated from the soil Streptomyces zelensis, in 1978, CC-1065 has
Presented at the 23rd Annual Meeting of the Society of Gynecologic Oncologists, San Antonio, TX, March 15-18, 1992. ’ This article was written while Randall D. Hightower, lieutenant commander in the U.S. Navy, was a fellow at the University of Miami/Jackson Memorial Hospital training in Gynecologic Oncology. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. ’ To whom correspondence should be addressed at Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, (D52), University of Miami School of Medicine, P.O. Box 016960, Miami, FL, 33101. Fax: (305) 325-1469. 3 Supported in part by the Grimmke Foundation, Germany. 4 Supported in part by Consorzio Interuniversitario Nazionale per la Bio-Oncologia (CINBO), Italy.
been shown to inhibit DNA synthesis and exhibits nonintercalative binding in the minor groove of doublestranded DNA in the adenine-thymidine regions followed by covalent bonding with the N-3 adenine [l-4,7-11]. Its spiral structure was found to promote preferential and selective binding to specific DNA sequences [12,13]. The adjacent cyclopropyl group can then initiate the alkylating reactions and form covalent bonds to cellular DNA [1,2,12]. Given intravenously to healthy mice, it was discovered that CC-1065 caused a delayed fatal hepatotoxicity [1,2,14]. Subsequently, the segment of CC-1065 incorporating the alkylating conjugated cyclopropyl ring was synthesized [1,13-151. U-73 was thus developed which resulted in the elimination of the fatal hepatotoxicity while maintaining similar binding and alkylating action and antitumor efficacy [1,14-161. U-73 appears to only attack double-stranded DNA and specific adenines [l]. This compound was synthesized and studied by Upjohn of Kalamazoo, MI [2]. The efficacy of U-73 has been evaluated with a number of human solid tumor xenografts. When mice received a single intravenous injection after tumor implantation, U-73 was highly active against clear cell Cak.-1 carcinoma xenograft and yielded 97 to 100% inhibition of tumor growth measured by tumor weight (TGI). When mice received multiple intravenous injections, U-73 was also highly active against both colon Cx- and Lx-l tumor xenografts and produced a greater than 90% TGI [6]. U-73 is therapeutically highly active against a broad spectrum of tumors including L1210 leukemia, colon 38, and pancreas 02 tumors [5]. This structurally simplified analog showed activity superior to that of CC-1065 against a number of experimental tumors after a single intravenous injection [15]. Chemotherapy for carcinoma of the cervix has been generally reserved for patients with recurrent or advanced disease. Cisplatin (DDP) is the most extensively studied
186 00!%8258/!42
$4.00
Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
COMPARISON
OF U-73,975 AND CISPLATIN
single agent in squamous cell carcinoma of the cervix [21]. The standard treatment for ovarian malignancy includes cytoreductive surgery followed by adjuvant chemotherapy with one or a combination of several agents, commonly including DDP [17]. Thus, DDP was the single agent chosen for comparison in these gynecologic malignancies. The cytotoxicity of U-73 was compared to DDP on freshly obtained cervical and ovarian carcinoma specimen using the ATP-chemosensitivity assay in this in vitro study. MATERIALS
AND METHODS
Tumor tissue specimens were obtained from 17 patients who underwent either a radical hysterectomy or exploratory laparotomy for staging of cervical carcinoma or exploratory laparotomy with cytoreduction for ovarian carcinoma at the University of Miami/Jackson Memorial Medical Center between January and June 1991. Eleven of these specimens represented cervical carcinoma and seven ovarian carcinoma. Two of the ovarian carcinoma tissues obtained were from patients with recurrent disease. One cervical specimen represented a metastatic node in a patient with cervical carcinoma. The specimens were immediately brought to the lab for processing. On arrival at the laboratory the Tumor preparation. cervical tumor was pretreated for 20 min with 100 IU/ml penicillin (5 x ), streptomycin 100 pg/ml (5 x ), and fungizone (5 x ) to sterilize the surface of the tumor. The ovarian tumor specimens received no pretreatment since they had been obtained from a sterile environment. The tumors were then minced into fine pieces with iris scissors and the resulting suspension incubated overnight in McCoy’s media with 5% fetal bovine serum (FBS), penicillin (2 X ), streptomycin (2 X ), 0.2 mg/ml collagenase II (Worthington), 2.25 PUK/ml pronase (Calbiochem), and 1400 U/ml DNase (Calbiochem). After enzyme treatment the cell suspension was separated from the tissue fragments and then suspended in enriched CMRL media with 15% FBS. This suspension contained both single cells and small clusters of up to 30 cells. Approximate cell counts were obtained using trypan blue exclusion. The cells were then diluted to a cell concentration of 100,000 viable cells/ml for plating into 24-well plates. These cells were plated by distributing the suspension in 0.5-ml aliquots over an underlayer of 0.5% agar in McCoy’s media containing 15% FBS. Drugs. DDP was tested using the reported peak plasma concentration (PPC) as the reference value [18]: cisplatin (Bristol Myers) 2.5 pg/ml. The PPC for U-73 has not been determined as U-73 is currently undergoing phase I trials. The PPC was arbitrarily chosen as 1 rig/ml as determined by preliminary data and manufacturer’s suggestions. All agents were freshly dissolved in Hanks’
ON CERVIX
AND OVARY
187
balanced salt solution (HBSS). U-73 was kindly provided by the Upjohn Company. A TP chemosensitivity assay. Triplicate wells were used for each of the five drug concentrations for each drug tested (0.1X, 0.2X, 0.5X, 1X, 5X PPC). The cells were plated at 50,000 ceils per well followed by continuous drug exposure. A 6-day continuous exposure was used because of the difficulty in manipulating fresh tumor cells without affecting the viability of the cells in order to remove the drug. Twenty microliters of each drug was added to the appropriate wells at a concentration sufficient to obtain the proper final concentration. Dose-response curves were obtained for the fractional intracellular ATP, representing cell survival, relative to untreated controls on Day 6 by extracting the ATP from the cells in situ with 4% trichloroacetic acid. ATP luminescence was determined as previously described [19]. Cytostatic drugs do not influence the bioluminescence reaction at reasonable concentrations, but very high levels (mmol/liter) of colored drugs, such as doxorubicin, quench the light reaction [20]. U-73 is a colorless compound. To ensure that U-73 did not absorb the luminescence signal, two standard curves were obtained. The standard curves were obtained after serially diluting a known quantity of ATP with media and evaluating the bioluminescence of the media/ATP serial dilution as compared to the media/ATP serial dilution with the addition of U-73 at 1000 times the concentration used in treating the cells. No difference in luminescence absorption was demonstrated. Data analysis. The concentrations necessary to produce a 50% decrease in cellular ATP (IC 50 = 0.5 surviving fraction = 0.5 fractional cellular ATP) were calculated for each cytotoxic agent, using the median effect plot of log (fa/fu) vs log C [fu = fraction unaffected = surviving fraction (SF); fa = fraction affected = 1 - SF; C = drug concentration [21]]. The t test was employed to calculate statistical significance. A P value ~0.05 was considered significant. RESULTS
Dose-response curves of all the cervical and ovarian specimens were performed for U-73 and DDP over a dose range of 0.1X to 5X PPC. Tables 1 and 2 display the IC 50s of the cervical and ovarian specimens, respectively, for U-73 and DDP. The IC 50s were calculated in rig/ml for purposes of comparison. The mean IC 50s for U-73 and DDP were 0.519 rig/ml (range of 0.005-1.627 rig/ml) and 2018 rig/ml (range of 215-4125 rig/ml), respectively, for the cervical carcinoma specimens and 0.324 rig/ml (range of 0.010-1.998 rig/ml) and 2649 rig/ml (range of 1008-4800 rig/ml), respectively, for the ovarian carcinoma specimens. Comparison of the mean IC 50s for U-
188
HIGHTOWER
TABLE 1 IC 50sof Fresh Cervical Carcinoma Specimenswhen Treated with U-73 and DDP
ET AL. *
u-73 126%
23391 !?a&91 !23&91
. . . . . .l
Patient ID
u-73
DDP”
233-91 235-91 236-91 242-91 243-91 246-91 247-91 259-91 267-91 278-91 279-91
0.186 0.221 0.005 0.534 0.459 0.696 0.406 0.384 0.221 1.627 0.972
2055 470 215 4125 2500 1615 760 3950 3000 3108 403
-
60%
.. ..
,&‘&91
243-91 24691
-
247-91
-
ZmSl
40%
287-91 -.-.-* _,_. m&91
20%
-
0%
0
12
3
4
5
ms1
6
DDP 126% 1
’ IC 50s measured in rig/ml.
73 and DDP over the dose range of 0.1X to 5X PPC on cervical and ovarian specimens demonstrated statistical significance using the test with P < 0.001, respectively. Figure 1 displays the dose-response curves for the eleven cervical carcinoma specimens. The curves are displayed as the curve resulting from the percentage survival at various drug concentrations. For descriptive purposes, an ATP surviving fraction of 50% or less at 0.5X is considered sensitive and greater than 50% as resistant. Using this criteria, eight of the cervical specimens were sensitive to U-73 and four were sensitive to DDP. Figure 2 displays the dose-response curves for the seven ovarian specimens. Of these seven specimens, five were sensitive to U-73 but none was sensitive to DDP. DISCUSSION U-73 represents an entirely new class of cytotoxic agents developed as an analog of the potent antitumor agent CC-1065. It was synthesized to eliminate the delayed fatal hepatoxocity that CC-1065 caused in healthy mice but maintain similar action and antitumor efficacy TABLE 2 IC 50sof Fresh Ovarian CarcinomaSpecimenswhen Treated with U-73 and DDP Patient ID
u-73
DDP”
226-91 227-91 229-91 249-91* 270-91’ 271-91 282-91
0.016 0.014 0.010 0.013 0.112 0.106 1.998
1498 4800 1943 1008 4450 2790 2053
a IC 50s measured in rig/ml. b Recurrent ovarian carcinoma.
100% 80%
0
12
3
4
5
6
FIG. 1. Dose-response curves of fresh cervical carcinoma specimens treated with U-73 and DDP. PPC represents the peak plasma concentration for DDP or the reported reference value for U-73.
[1,2,14]. Its spiral structure was found to promote preferential and selective binding to specific DNA sequences [12,13]. Because of these characteristics, this drug is effective at low doses [8-111. Comparing the IC 50s from Tables 1 and 2, U-73 was approximately 4000 times as cytotoxic per unit of mass as DDP on cervical carcinoma compared to over 8000 times for ovarian carcinoma. This comparison between cervical and ovarian carcinoma IC 50s was statistically significant. Of the 11 cervical specimens, 73% were sensitive to U-73 but only 36% were sensitive to DDP. This correlates with the overall response rate of 38% noted in the Gynecologic Oncology Group study [22]. Approximately 71% of the ovarian specimens were sensitive to U-73 in contrast to all of the specimens showing resistance to DDP. The dose-response curves of the ovarian specimens (Fig. 2) demonstrated two resistant tumors to U-73. DDP has remained one of the most commonly used chemotherapeutic agents for both cervical and ovarian carcinoma. The poor response of the ovarian carcinoma specimens may be a function of small sample size or the definition of sensitivity that was used. In this in vitro study, U-73 demonstrated superiority in cytotoxicity over DDP as a single agent on cervical and ovarian carcinoma tumor specimens. U-73 exhibits cy-
COMPARISON
OF U-73,975 AND CISPLATIN 3.
u-73
120%
-
22691
100%
-
297-91
-
999-91
-
24991 n&91
--
28291 271-91
3 %
80%
VI Y
60%
I!
$”
40% 20% 0% 0
1
2
3
4
5
6
DDP 120%
a.* wIi
1
100%
ON CERVIX
Chidester, C. G., Krueger, W. C., Mizsak, S. A., Duchamp, D. J., and Martin, D. G. The structure of CC-1065, a potent antitumor agent, and its binding to DNA, J. Am. Chem. Sot. 103(25), 76297635 (1981).
Hurley, L. H., Warpehoski, M. A., Lee, C. S., McGovern, .I. P., Scahill, T. A., Kelly, R. C., Mitchell, M. A., Wicnienski, N. A., Gebhard, I., Johnson, P. D., and Bradford, V. S. Sequence specificity of DNA alkylation by the unnatural enantiomer of CC-1065 and its synthetic analogs, J. Am. Chem. Sot. ll2(12), 4633-4649 (1990). 5 DeKoning, T. F., Kelly, R. C., Wallace, T. L., and Li, L. H. Antitumor activity and biochemical effect of three selected cyclopropapyrroloindole (CPI) analogs, Proc. Am. Assoc. Cancer Res. 30, 491 (1989). [Abstract 19531 6 DeKoning, T. F., Postmus, R. J., Wallace, T. L., Kelly, R. C., and Li, L. H. Therapeutic evaluation of three cyclopropapyrroloindole (CPI) analogs against human tumor xenografts, Proc. Am. 4
Assoc.
80%
189
AND OVARY
7
60%
Cancer
Res. 31, 348 (1990).
Hurley, L. H., and Needham-VanDevanter, D. R. Covalent binding of antitumor antibiotics in the minor groove of DNA. Mechanism of action of CC-1065 and the pyrrolo(l,4)benzodiazepines, Act.
Chem.
Rex
19(B),
203-237
(1986).
Hanka, L. J., Dietz, A., Gerpheide, S. A., Kuentzel, S. L., and Martin, D. G. CC-1065 (NSC 298223), a new antitumor antibiotic. Production, in vitro, biological activity microbiological assay, and taxonomy of the producing organism, J. Antibiot. 31, 1211 (1978). 9. Martin, D. G., Hanka, L. J., and Neil, G. L. Isolation, characterization, and preliminary antitumor evaluation of CC-1065, a potent new agent from fermentation, Proc. Am. Assoc. Cancer Res. 8.
0
1
2
3
4
5
6
FIG. 2. Dose-response curves of fresh ovarian carcinoma specimens treated with U-73 and DDP. PPC represents the peak plasma concentration for DDP or the reported reference value for U-73. Patient ID 249-91 and 270-91 represent recurrent ovarian carcinoma.
totoxicity in picogram amounts compared to microgram amounts for DDP. Because of the small sample size, especially of ovarian tissue, additional studies are necessary before any conclusions can be made. The arbitrary assignment of 50% as constituting an active versus an inactive response may have little bearing on the ultimate activity. The concentration of U-73 required for in vivo activity may not correlate well with in vitro data. In addition, in vitro data does not consider toxicity to normal tissues. U-73 is completing phase I trials which will address these issues. Only after these trials are performed will we know if the smaller amounts of drug needed to effect a response are obtainable considering the potential toxicity of the drug to the patient. In summary, U-73 is a potent antitumor agent in this in vitro study using the ATP-CSA for analysis of the cytotoxic effect and appears to be a promising chemotherapeutic agent. REFERENCES 1.
Warpehoski, M. A. CC-1065 analogs: Therapeutic potential of a sequence specific drug-DNA reaction, Proc. Am. Assoc. Cancer Res. 30, 668 (1989).
2.
Warpehoski, M. A. Stereoelectronic factors influencing the biological activity and DNA interaction of synthetic antitumor agents modeled on CC-1065. J. Med. Chem. 31. 3 C19881. \ ,
19, 99 (1978).
10. Martin, D. G., Chidester, C. G., Duchamp, D. J., and Misak, S. A. Structure of CC-1065 (NSC 298223), a new antitumor antibiotic, J. Antibiot. 33, 902 (1980). 11. Martin, D. G., Biles, C., Gerpheide, S. A., Hanka, L. J., and Krueger, W. C. CC-1065 (NSC 298223), a potent new antitumor agent: Improved production and isolation, characterization, and antitumor activity, J. Antibiot. 34, 1119 (1981). 12. Reynolds, V. L., McGovren, J. P., and Hurley, L. H. The chemistry, mechanism of action and biological properties of CC-1065, a potent antitumor antibiotic, Antibiotics 39, 319 (1986). 13. Wierenga, W., Bhuyan, B. K., Kelly, R. C., Krueger, W. C., and Li, L. H. Antitumor activity and biochemistry of novel analogs of the antibiotic CC-1065. Adv. Enzyme Regul. 25, 141 (1986). 14. McGovren, J. P., Clarke, G. L., Pratt, E. A., and DeKoning, T. F. Preliminary toxicity studies with the DNA-binding antibiotic, CC1065, J. Antibiot. 37(l), 63-70 (1984). 15. DeKoning, T. F., Krueger, W. C., Wallace, T. L., Prairie, M. D., Warpehoski, M. A., Kelly, R. C., and Li, L. H. Influence of structure and chirality on the antitumor activity and biochemical effects of CC-1065 and its analogs, Proc. Am. Assoc. Cancer Res. 28, 262 (1987). 16. Warpehoski, M. A., Kelly, R. C., McGovren, J. P., and Wierenga, W. N-2-substitutedtetrahydro-4-oxocyclopropa[c]pyrrolo-[3,2-e] indoles: Novel anticancer agents modeled on CC-1065, Proc. Am. Assoc. Cancer Res. 26, 221 (1985). [Abstract] 17. Deppe, G. Chemotherapy of gynecologic cancer, 2nd ed., WileyLiss, New York, p. 127 (1990). 18. Alberts, D., and Chen, G. Tabular summary of pharmacokinetic parameters relevant to in vitro drug assay, in cloning human tumor stem cells (S. Salmon, Ed.), Liss, New York, pp. 351-359 (1980). 19. Sevin, B. U., Peng, Z., Perras, J., Ganjei, P., Penalver, M., and
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Averette, H. Application of an ATP-bioluminescence assay in human tumor chemosensitivity testing, Gynecol. Oncol. 31, 191-204 (1988). 20. Kangas, L., Gronroos, M., and Nieminen, A. L. Bioluminescence of cellular ATP: A new method for evaluating cytotoxic agents in vitro. Med. Biol. 62, 338-343 (1984).
ET AL. 21. Chou, T., and Talaly, P. Quantitative analysis of dose-effect relationships: The combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul. 22, 27-55 (1984). 22. DiSaia, P. J., and Creaseman, W. T. Clinical gynecologic oncology, Mosby, St. Louis, 3rd ed., p. 115 (1989).
