revealed the presence of viable transitional-cell carcinoma. The remaining five patients continued to respond to therapy at more than 4 months, 5 months, 5 months, 6 months, and 8 months, respectively. Each of the five patients continued to receive treatment.
288
on human salivary gland adenocarcinoma cell line in culture. Int J Oral Surg 13:35-44, 1984 (12) WADLER S, SCHWARTZ EL, GOLDMAN M, ET
AL: Fluorouracil and recombinant alfa-2a-interferon: An active regimen against advanced colorectal carcinoma. J Clin Oncol 7:1769— 1775,1989 (13) PAZDUR R, ABBRUZZESE J, FAINTUCH J, ET AL:
Phase II study of recombinant interferon alpha (rIFN) and 5-fluorouracil (5-FU) in patients (pts) with advanced colorectal carcinoma. Proc ASCO 9:455, 1990 (14) ELJAS L, SANDOVAL JM: Interferon effects
upon fluorouracil metabolism by HL-60 cells. Biochem Biophys Res Commun 163:867-874, 1989 (15) WADLER S, SCHWARTZ EL, WERSTO R, ET AL:
Interferon (IFN) modulates the activity of 5fluorouracil against two human colon cancer cell lines. Proc AACR 30:569, 1989 (16) MORIKAWA K, FAN D, DENKINS YM, ET AL:
Mechanisms of combined effects of gammainterferon and 5-fluorouracil on human colon cancers implanted into nude mice. Cancer Res 49:799-805,1989 (17) STOLFI
RL,
MARTIN
DS:
Modulation
of
chemotherapeutic drug activity with polyribonucleotides or with interferon. J Biol Response Mod 4:634-639, 1985
Studies With RP 56976 (Taxotere): A Semisynthetic Analogue of Taxol Israel Ringel, Susan Band Horwitz*
RP 56976 (taxotere), a new semisynthetic analogue of taxol, is a potentially important chemotherapeutic agent for the treatment of cancer. We report here that this drug is a potent inhibitor of cell replication and, like taxol, promotes the in vitro assembly of
Received August 22, 1990; revised November 15, 1990-, accepted November 29, 1990. Supported in pan by the Israel Cancer Association (I. Ringel) and by Public Health Service grant CA39821 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (S. B. Horwitz). I. Ringel, Department of Pharmacology, Hadassah Medical School, The Hebrew University, Jerusalem, Israel. S. B. Horwitz, Departments of Molecular Pharmacology and Cell Biology, Albert Einstein College of Medicine, Bronx, NY. We thank Prof Francois Lavelle of RhonePoulenc Santi for providing RP 56976. 'Correspondence to: Susan B. Horwitz, PhD, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Regina on December 12, 2016
ing the combination of 5-FU and IFN-a. The frequency of the neurologic side effects may be much higher than we report because only a small number of patients received repeated therapy in this study population. The 30% response rate achieved in these patients with highly advanced metastatic urothelial tumors and poor perDiscussion formance status suggests that the combination of 5-FU and IFN-a has The 30% response rate achieved in significant antitumor activity. If the these heavily pretreated patients with a results of this trial are confirmed, early poor performance status is encouraging. introduction of this combination of drugs The fact that some of these responses are for treatment of metastatic urothelial relatively durable adds to the significance tumors should be considered. Additional of these findings. We believe that these refinement of the combination of 5-FU data strongly suggest that the combinaand IFN-a will be necessary for the idention of 5-FU and IFN-a is synergistic. In tification of its minimum active dose and a recently completed phase II trial of the its subsequent inclusion in currently accombination of 5-FU and mitomycin at tive chemotherapy regimens. our institution, only 1 of 18 previously treated patients achieved a response of 5 months' duration. (Dexeus et al: un- References published data). In that trial, the dose of 5-FU was 1000 mg per m2 of BSA. The (/) CARTER SK, WASSERMAN TH: The chemotherapy of urologic cancer. Cancer increased response rate achieved with the 36:729-747,1975 combination of 5-FU and IFN-a is un- (2) PAVONE-MACALUSO M: Chemotherapy of vesilikely to be attributed to the increased cal and prostatic tumours. Br J Urol 43:701708,1971 dose intensity of 5-FU alone. Responding (3) PROW GR JR, BROSS IDJ, SLACK NH, ET AL: patients had evidence of tumor regression Carcinoma of the bladder, 5-fluorouracil and during the first course of therapy despite the critical role of a placebo. A cooperative group report. I. Cancer 22:926-931, 1968 a starting dose of 5-FU combined with a (4) GLENN JF, HUNT LD, LATHEM JE: 2 dose of IFN-a of only 750 mg per m of Chemotherapy of bladder carcinoma with 5BSA. In addition, the available clinical fluorouracil. Cancer Chemother Rep 27:67— 69,1963 data on the use of IFN-a as a single agent (5) YAOODA A: Chemotherapy of metastatic bladfor metastatic urothelial tumors suggest der cancer. Cancer 45(7 suppl): 1879-1888, that this agent is ineffective when used 1980 (6) NEVIN JE m , MELNICK I, BAOOERLY JT, ET AL: alone (8). Advanced carcinoma of bladder Treatment The mechanism of action of the comusing hypogastric artery infusion with 5fluorouracil, either as a single agent or in combination of 5-FU and IFN-a remains a bination with bleomycin or Adriamycin and matter of speculation. Although other supervoltage radiation. J Urol 112:752-758, mechanisms have been proposed, many 1974 (7) LOGOTHETIS CJ, SAMUELS ML, WALLACE S, ET investigators believe that IFN-a bioAL: Management of pelvic complications of chemically modulates the intracellular malignant urothelial tumors with combined metabolism of 5-FU (13-17). intra-arterial and iv chemotherapy. Cancer Treat Rep 66:1501-1507,1982 The toxic effects of this combination (S) NUJIMA T: Phase II study of interferon-a 2A are occasionally severe. The frequency (Sch 30500) for tumors of urogenital tract Jpn and severity of nonhematologic toxic efClin Med Pharmacol 1:395-406,1985 fects may be exaggerated in our study (9) WADLER S, SCHWARTZ EL, WERSTO R, ET AU Interferon (IFN) modulates the activity of 5population because of poor performance fluorouracil against two human colon cancer status and large volume of disease in the cell lines. Proc AACR 30:569, 1989 patients we treated. Although the muco- (10) EUAS L, CRISSMAN HA: Interferon effects upon the adenocarcinoma 38 and HL-60 cell sitis and neurologic side effects of this lines: Antiproliferative responses and synercombination were prominent features, the gistic interactions with halogenated pyrimidine antiroetabolites. Cancer Res 48:4868-4873, hematologic side effects were modest. A 1988 similar high frequency of neurologic ( / / ) SATO M, YOSHIDA H, URATA M, ET AU Effects toxic effects was encountered in similar of 5-fluorouracil and the combination of 5trials in patients with colon cancer receivfluorouracil and human leukocyte interferon
stable microtubules in the absence of guanosine triphosphate and induces microtubule-bundle formation in cells. Compared with taxol, RP 56976 is slightly more active as a promoter of tubulin polymerization. As an inhibitor of cell replication, RP 56976 is 2.5-fold more potent than taxol in J774.2 and P388 cells and at least 5-fold more potent in taxol-resistant cells. [J Natl Cancer Inst 83:288-291,1991]
Vol. 83, No. 4, February 20, 1991
0
/" II r"
Materials and Methods
\
\ ^ HO
:
Drugs Taxol was obtained from the National Cancer Institute, and RP 56976 was a gift from Rhone-Poulenc Santi, France. Each was dissolved in 100% Me,SO. The maximum final concentration of Me2SO was 1% in experiments measuring tubulin polymerization and 0.2% in assays determining cell cytotoxicity. These concentrations had no significant effect on tubulin polymerization, microtubule structure, or the replication of cells. Preparation of Microtubule Protein and Measurement of Tubulin Polymerization Calf brain microtubule protein was purified by two cycles of temperature-dependent assembly-disassembly by a procedure (13) modified from that of Shelanski et al (14). Microtubule assembly, in the presence and absence of drugs, was monitored spectrophotometrically at 35°C, and changes in turbidity (representative of polymer mass) were monitored at 350 nm (15). Samples for electron microscopy were placed on carbon-over-parlodion-coated grids (300 mesh), negatively stained with 2% uranyl acetate, and analyzed on a Philips 300 electron microscope at 80 kV.
o
yo
R1
R1
TAXOL
OCOCH)
wynt
OH
OQCHj),
Fig 1. Structural formulas of taxol and RP 56976. Downloaded from http://jnci.oxfordjournals.org/ at University of Regina on December 12, 2016
Taxol, a novel diterpenoid isolated from the bark of the Pacific yew, Taxus brevifolia, is an antimitotic agent and a potent inhibitor of cell replication. Incubation of cells with taxol results in the formation of highly organized bundles of microtubules (/). Unlike other plantderived antimicrotubule agents (eg, colchicine, podophyllotoxin, or the vinca alkaloids) that inhibit microtubule assembly, taxol promotes the assembly of tubulin and stabilizes the formed polymers against depolymerization by Ca" and low temperature (23). Taxol has demonstrated good activity against intraperitoneally implanted B16 melanoma and human MX-1 mammary tumor xenografts in mice (4) and has been tested in a number of human malignant neoplasms, with particularly encouraging results in advanced ovarian epithelial neoplasms (5-8). A major problem is the lack of sufficient quantities of the drug for adequate clinical trials. The Pacific yew is an extremely slow-growing evergreen species, and the isolation of taxol from its bark results in a low yield of drug and death of the tree. Therefore, major efforts are being directed toward total synthesis of taxol and the preparation of taxol analogues. Recently, a semisynthetic taxol derivative, RP 56976 (taxotere; A'-debenzoyl-A'-tert-butoxycarbonyl-10-deacetyl taxol), was prepared at the Institut de Chimie des Substances Naturelles of the Centre National de la Recherche Scientifique, France, and was found at Rhone-Poulenc Santd to have significant potential as a chemotherapeutic drug for the treatment of cancer (912). This analogue is of special interest not only because it is active against cancer but also because its precursor, 10deacetyl baccatin III, has been isolated from the leaves of Taxus baccata, which
can regenerate. We report here on some properties of this promising taxol analogue.
(courtesy of Dr A. Ramu, The Hebrew University-Hadassah Medical School, Jerusalem, Israel), a mouse macrophagelike cell line, J774.2, and its taxol-resistant variant, J7.TAX-50 (17). The latter was maintained in 45 \iM taxol and was washed before the determination of the ED^ for RP 56976. Immunofluorescence CHO cells were plated on sterile coverslips, and after 24 hours, fresh medium containing 10 \iM of drug was added for 6 hours. The cells were treated with a mouse antibody to tubulin (BioYeda, Israel) and a second antibody, fluorescein-conjugated rabbit antimouse antibody (N.L. Cappel Laboratories, Cochranville, Pa). The detailed procedure has been described (14).
Cytotoxicity of Taxol and RP 56976 The median effective dose (EDy,) for taxol and RP 56976 was determined in triplicate as previously described (16). Experiments were done with P388, a mouse lymphocytic leukemia cell line
Results and Discussion Structure/activity-relationship studies done with taxol and its derivatives have emphasized the importance of the C-13
Table 1. Properties of taxol and RP 56976
Drug Taxol RP 56976
Initial slope,'* AU/min(x 10 •
1.96 2.65
2
)
Solubilityt in water, UJW
J774.2
J7.TAX-5O
P388
35 47
0.05 0.02
45§ 8
0.01 0.004
•Initial assembly slopes from the experiment described in Fig 2. AU = absorbancc units. tAll measurements were performed on a reverse-phase high-pressure liquid chromatography column (RP18), using CH3OH and H2O mixed in a ratio of 60:40 as the carrier at 230 nm. tEDjo = drug concentration that inhibits cell division by 50% after 72 h. §Maximum solubility of drug in medium is between 45 and 50 \lM.
REPORTS
289
0.2
•
i
i
mix
I
o
in
J —
M
/* f
•P
288
on human salivary gland adenocarcinoma cell line in culture. Int J Oral Surg 13:35-44, 1984 (12) WADLER S, SCHWARTZ EL, GOLDMAN M, ET
AL: Fluorouracil and recombinant alfa-2a-interferon: An active regimen against advanced colorectal carcinoma. J Clin Oncol 7:1769— 1775,1989 (13) PAZDUR R, ABBRUZZESE J, FAINTUCH J, ET AL:
Phase II study of recombinant interferon alpha (rIFN) and 5-fluorouracil (5-FU) in patients (pts) with advanced colorectal carcinoma. Proc ASCO 9:455, 1990 (14) ELJAS L, SANDOVAL JM: Interferon effects
upon fluorouracil metabolism by HL-60 cells. Biochem Biophys Res Commun 163:867-874, 1989 (15) WADLER S, SCHWARTZ EL, WERSTO R, ET AL:
Interferon (IFN) modulates the activity of 5fluorouracil against two human colon cancer cell lines. Proc AACR 30:569, 1989 (16) MORIKAWA K, FAN D, DENKINS YM, ET AL:
Mechanisms of combined effects of gammainterferon and 5-fluorouracil on human colon cancers implanted into nude mice. Cancer Res 49:799-805,1989 (17) STOLFI
RL,
MARTIN
DS:
Modulation
of
chemotherapeutic drug activity with polyribonucleotides or with interferon. J Biol Response Mod 4:634-639, 1985
Studies With RP 56976 (Taxotere): A Semisynthetic Analogue of Taxol Israel Ringel, Susan Band Horwitz*
RP 56976 (taxotere), a new semisynthetic analogue of taxol, is a potentially important chemotherapeutic agent for the treatment of cancer. We report here that this drug is a potent inhibitor of cell replication and, like taxol, promotes the in vitro assembly of
Received August 22, 1990; revised November 15, 1990-, accepted November 29, 1990. Supported in pan by the Israel Cancer Association (I. Ringel) and by Public Health Service grant CA39821 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services (S. B. Horwitz). I. Ringel, Department of Pharmacology, Hadassah Medical School, The Hebrew University, Jerusalem, Israel. S. B. Horwitz, Departments of Molecular Pharmacology and Cell Biology, Albert Einstein College of Medicine, Bronx, NY. We thank Prof Francois Lavelle of RhonePoulenc Santi for providing RP 56976. 'Correspondence to: Susan B. Horwitz, PhD, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461.
Journal of the National Cancer Institute
Downloaded from http://jnci.oxfordjournals.org/ at University of Regina on December 12, 2016
ing the combination of 5-FU and IFN-a. The frequency of the neurologic side effects may be much higher than we report because only a small number of patients received repeated therapy in this study population. The 30% response rate achieved in these patients with highly advanced metastatic urothelial tumors and poor perDiscussion formance status suggests that the combination of 5-FU and IFN-a has The 30% response rate achieved in significant antitumor activity. If the these heavily pretreated patients with a results of this trial are confirmed, early poor performance status is encouraging. introduction of this combination of drugs The fact that some of these responses are for treatment of metastatic urothelial relatively durable adds to the significance tumors should be considered. Additional of these findings. We believe that these refinement of the combination of 5-FU data strongly suggest that the combinaand IFN-a will be necessary for the idention of 5-FU and IFN-a is synergistic. In tification of its minimum active dose and a recently completed phase II trial of the its subsequent inclusion in currently accombination of 5-FU and mitomycin at tive chemotherapy regimens. our institution, only 1 of 18 previously treated patients achieved a response of 5 months' duration. (Dexeus et al: un- References published data). In that trial, the dose of 5-FU was 1000 mg per m2 of BSA. The (/) CARTER SK, WASSERMAN TH: The chemotherapy of urologic cancer. Cancer increased response rate achieved with the 36:729-747,1975 combination of 5-FU and IFN-a is un- (2) PAVONE-MACALUSO M: Chemotherapy of vesilikely to be attributed to the increased cal and prostatic tumours. Br J Urol 43:701708,1971 dose intensity of 5-FU alone. Responding (3) PROW GR JR, BROSS IDJ, SLACK NH, ET AL: patients had evidence of tumor regression Carcinoma of the bladder, 5-fluorouracil and during the first course of therapy despite the critical role of a placebo. A cooperative group report. I. Cancer 22:926-931, 1968 a starting dose of 5-FU combined with a (4) GLENN JF, HUNT LD, LATHEM JE: 2 dose of IFN-a of only 750 mg per m of Chemotherapy of bladder carcinoma with 5BSA. In addition, the available clinical fluorouracil. Cancer Chemother Rep 27:67— 69,1963 data on the use of IFN-a as a single agent (5) YAOODA A: Chemotherapy of metastatic bladfor metastatic urothelial tumors suggest der cancer. Cancer 45(7 suppl): 1879-1888, that this agent is ineffective when used 1980 (6) NEVIN JE m , MELNICK I, BAOOERLY JT, ET AL: alone (8). Advanced carcinoma of bladder Treatment The mechanism of action of the comusing hypogastric artery infusion with 5fluorouracil, either as a single agent or in combination of 5-FU and IFN-a remains a bination with bleomycin or Adriamycin and matter of speculation. Although other supervoltage radiation. J Urol 112:752-758, mechanisms have been proposed, many 1974 (7) LOGOTHETIS CJ, SAMUELS ML, WALLACE S, ET investigators believe that IFN-a bioAL: Management of pelvic complications of chemically modulates the intracellular malignant urothelial tumors with combined metabolism of 5-FU (13-17). intra-arterial and iv chemotherapy. Cancer Treat Rep 66:1501-1507,1982 The toxic effects of this combination (S) NUJIMA T: Phase II study of interferon-a 2A are occasionally severe. The frequency (Sch 30500) for tumors of urogenital tract Jpn and severity of nonhematologic toxic efClin Med Pharmacol 1:395-406,1985 fects may be exaggerated in our study (9) WADLER S, SCHWARTZ EL, WERSTO R, ET AU Interferon (IFN) modulates the activity of 5population because of poor performance fluorouracil against two human colon cancer status and large volume of disease in the cell lines. Proc AACR 30:569, 1989 patients we treated. Although the muco- (10) EUAS L, CRISSMAN HA: Interferon effects upon the adenocarcinoma 38 and HL-60 cell sitis and neurologic side effects of this lines: Antiproliferative responses and synercombination were prominent features, the gistic interactions with halogenated pyrimidine antiroetabolites. Cancer Res 48:4868-4873, hematologic side effects were modest. A 1988 similar high frequency of neurologic ( / / ) SATO M, YOSHIDA H, URATA M, ET AU Effects toxic effects was encountered in similar of 5-fluorouracil and the combination of 5trials in patients with colon cancer receivfluorouracil and human leukocyte interferon
stable microtubules in the absence of guanosine triphosphate and induces microtubule-bundle formation in cells. Compared with taxol, RP 56976 is slightly more active as a promoter of tubulin polymerization. As an inhibitor of cell replication, RP 56976 is 2.5-fold more potent than taxol in J774.2 and P388 cells and at least 5-fold more potent in taxol-resistant cells. [J Natl Cancer Inst 83:288-291,1991]
Vol. 83, No. 4, February 20, 1991
0
/" II r"
Materials and Methods
\
\ ^ HO
:
Drugs Taxol was obtained from the National Cancer Institute, and RP 56976 was a gift from Rhone-Poulenc Santi, France. Each was dissolved in 100% Me,SO. The maximum final concentration of Me2SO was 1% in experiments measuring tubulin polymerization and 0.2% in assays determining cell cytotoxicity. These concentrations had no significant effect on tubulin polymerization, microtubule structure, or the replication of cells. Preparation of Microtubule Protein and Measurement of Tubulin Polymerization Calf brain microtubule protein was purified by two cycles of temperature-dependent assembly-disassembly by a procedure (13) modified from that of Shelanski et al (14). Microtubule assembly, in the presence and absence of drugs, was monitored spectrophotometrically at 35°C, and changes in turbidity (representative of polymer mass) were monitored at 350 nm (15). Samples for electron microscopy were placed on carbon-over-parlodion-coated grids (300 mesh), negatively stained with 2% uranyl acetate, and analyzed on a Philips 300 electron microscope at 80 kV.
o
yo
R1
R1
TAXOL
OCOCH)
wynt
OH
OQCHj),
Fig 1. Structural formulas of taxol and RP 56976. Downloaded from http://jnci.oxfordjournals.org/ at University of Regina on December 12, 2016
Taxol, a novel diterpenoid isolated from the bark of the Pacific yew, Taxus brevifolia, is an antimitotic agent and a potent inhibitor of cell replication. Incubation of cells with taxol results in the formation of highly organized bundles of microtubules (/). Unlike other plantderived antimicrotubule agents (eg, colchicine, podophyllotoxin, or the vinca alkaloids) that inhibit microtubule assembly, taxol promotes the assembly of tubulin and stabilizes the formed polymers against depolymerization by Ca" and low temperature (23). Taxol has demonstrated good activity against intraperitoneally implanted B16 melanoma and human MX-1 mammary tumor xenografts in mice (4) and has been tested in a number of human malignant neoplasms, with particularly encouraging results in advanced ovarian epithelial neoplasms (5-8). A major problem is the lack of sufficient quantities of the drug for adequate clinical trials. The Pacific yew is an extremely slow-growing evergreen species, and the isolation of taxol from its bark results in a low yield of drug and death of the tree. Therefore, major efforts are being directed toward total synthesis of taxol and the preparation of taxol analogues. Recently, a semisynthetic taxol derivative, RP 56976 (taxotere; A'-debenzoyl-A'-tert-butoxycarbonyl-10-deacetyl taxol), was prepared at the Institut de Chimie des Substances Naturelles of the Centre National de la Recherche Scientifique, France, and was found at Rhone-Poulenc Santd to have significant potential as a chemotherapeutic drug for the treatment of cancer (912). This analogue is of special interest not only because it is active against cancer but also because its precursor, 10deacetyl baccatin III, has been isolated from the leaves of Taxus baccata, which
can regenerate. We report here on some properties of this promising taxol analogue.
(courtesy of Dr A. Ramu, The Hebrew University-Hadassah Medical School, Jerusalem, Israel), a mouse macrophagelike cell line, J774.2, and its taxol-resistant variant, J7.TAX-50 (17). The latter was maintained in 45 \iM taxol and was washed before the determination of the ED^ for RP 56976. Immunofluorescence CHO cells were plated on sterile coverslips, and after 24 hours, fresh medium containing 10 \iM of drug was added for 6 hours. The cells were treated with a mouse antibody to tubulin (BioYeda, Israel) and a second antibody, fluorescein-conjugated rabbit antimouse antibody (N.L. Cappel Laboratories, Cochranville, Pa). The detailed procedure has been described (14).
Cytotoxicity of Taxol and RP 56976 The median effective dose (EDy,) for taxol and RP 56976 was determined in triplicate as previously described (16). Experiments were done with P388, a mouse lymphocytic leukemia cell line
Results and Discussion Structure/activity-relationship studies done with taxol and its derivatives have emphasized the importance of the C-13
Table 1. Properties of taxol and RP 56976
Drug Taxol RP 56976
Initial slope,'* AU/min(x 10 •
1.96 2.65
2
)
Solubilityt in water, UJW
J774.2
J7.TAX-5O
P388
35 47
0.05 0.02
45§ 8
0.01 0.004
•Initial assembly slopes from the experiment described in Fig 2. AU = absorbancc units. tAll measurements were performed on a reverse-phase high-pressure liquid chromatography column (RP18), using CH3OH and H2O mixed in a ratio of 60:40 as the carrier at 230 nm. tEDjo = drug concentration that inhibits cell division by 50% after 72 h. §Maximum solubility of drug in medium is between 45 and 50 \lM.
REPORTS
289
0.2
•
i
i
mix
I
o
in
J —
M
/* f
•P
